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Development of dihydromyricetin (DHM) as a novel therapy for alcoholic liver disease (ALD) and alcohol use disorder (AUD)

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Development of dihydromyricetin (DHM) as a novel therapy for alcoholic liver disease (ALD) and alcohol use disorder (AUD)

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Content i

Development of Dihydromyricetin (DHM) as a Novel Therapy for Alcoholic Liver Disease
(ALD) and Alcohol Use Disorder (AUD)

By

Joshua Silva

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSPHY
CLINICAL AND EXPERIMENTAL THERAPEUTICS

May 2021

ii

ACKNOWLEDGEMENTS
I have received a great deal of support, assistance, and training throughout my training and
writing of my dissertation.
I would first like to thank my advisor, Dr. Daryl L. Davies, whose expertise and guidance was
invaluable throughout my research training and professional development. The professional
opportunities that I have ahead of me are a result of your guidance and your challenging me to
become better. I would also like to thank my co-advisor, Dr. Jing Liang, for her guidance and
provided opportunities that allowed me to strengthen my scientific progress and idea
developments here at USC. Both Dr. Davies’ and Dr. Liang’s insightful feedback helped me
sharpen my thinking and encouraged me to become a better scientist and mentor.
I would also like to thank my committee members and co-advisors, Drs. Cadenas, Louie, and
Okamoto. My research endeavors and findings are the product of their expertise and mentorship
throughout my training. I sincerely thank you all for your time to advise me and assist me in
shaping my investigations. Otherwise, I would have been all over the place with my research.
I would also like to acknowledge my team of young researchers that I had the honor of
mentoring and training. Many of these accomplishments were made possible with your
involvement, and I look forward to seeing you excel as you all move forward.
In addition, I would like to thank my wife, family, and friends. My motivation to complete my
Ph.D. goals was strongly encouraged by your support. I certainly could not have done this
without the support from you all, as well as the many happy distractions that allowed me to
remain motivated throughout my endeavors.
All of this was possible because of the support of all of you, and I sincerely thank you all for
your role in encouraging me to excel.
iii

TABLE OF CONTENTS

Acknowledgments………………………………………………………………………..…...…..ii
List of Figures………………………………………………………………………………....…..v
Abstract……………………………………………………………………………..……...…....viii
Chapter 1: Introduction………………………………………………………………………..…..1
Alcohol Consumption Rates and Co-Morbidities……………………...……..……….1
AUD Development……………….……………………………………………..…….2
Current AUD Therapies………………….……………………………………..….….5
Alcoholic Liver Disease (ALD)…………………………………………………...…..7
Stages of ALD…………………………………………………………………...…….8
Treatment Options for ALD………………………………………………….………11
Ethanol Metabolism and Biochemical Responses in the Liver………………...……16
Dihydromyricetin (DHM) as a Natural Polyphenol………………………………….21
DHM Health Benefits………………………………………………………..………22
DHM as a Flavonoid Enhancer of Bioavailability……………………………..…….24
Mechanisms of DHM BA Enhancement……………………………………….……24
Purinergic (P2X) 4 Receptors as Novel Drug Targets of AUD………………..…….26

Chapter 2: Dihydromyricetin Protects the Liver via Changes in Lipid Metabolism and Enhanced
Ethanol Metabolism
Abstract………………………………………………………………………...…….31
Introduction……………………………………………………………………….….33
Methods…………………………………………………………………………...….35
Results………………………………………………………………………….…….43
Discussion………………………………………………………………..…….…….58

Chapter 3: Dihydromyricetin Improves Mitochondrial Outcomes in the Liver of Alcohol-Fed
Mice via the AMPK/Sirt-1/PGC-1α Signaling Axis
Abstract…………………………………………………………………………...….66
Introduction…………………………………………………………………….…….68
Methods…………………………………………………………………………...….71
Results…………………………………………………………………………….….75
Discussion………………………………………………………………………...….80

iv

Chapter 4: A Novel Pharmacotherapy Approach Using P-glycoprotein (Pgp/ABCB1) Efflux
Inhibitor Combined with Ivermectin to Reduce Alcohol Drinking and Preference in Mice
Abstract………………………………………………………………………...…….87
Introduction……………………………………………………………………….….89
Methods…………………………………………………………………………...….91
Results…………………………………………………………………………….….94
Discussion…………………………………………………………………….…….105

Chapter 5: A Novel Dual Drug Approach that Combines Ivermectin and Dihydromyricetin
(DHM), a Natural Hepatoprotective Product, to Reduce Alcohol Drinking and Preference in
Mice
Abstract………………………………………………………………………….….110
Introduction…………………………………………………………………...…….111
Results……………………………………………………………………...……….115
Discussion……………………………………………………………………….….125
Methods………………………………………………………………….………….130

Chapter 6
Conclusions…………………………………………………………...…………….135

Bibliography……………………………………………………………………………………140

v

List of Figures
Figure
1.1 ALD Spectrum and Progression ……………………………………………………….……11
1.2 Proposed strategy for the management of patients with late-stage ALD ………………..…13
1.3 Alcohol (Ethanol) Metabolism in the liver …………………………….……………………17
1.4 Ethanol metabolism and liver oxidant stress ………………………………………..………19
1.5 Chemical structure of flavonoids ……………………………………………………………22
2.1 DHM ameliorates ethanol (EtOH)‐induced pathomorphology and hepatic/serum triglyceride
levels in EtOH‐fed mice …………………………………………………………………………44
2.2 DHM directly reduces ethanol (EtOH)‐mediated mature SREBP‐1 expression and lipid
uptake in EtOH oxidizing (VL‐17A) and nonoxidizing (HepG2) cell lines……………..………46
2.3 DHM administration counteracts EtOH‐mediated inhibition of AMPK and downstream lipid
metabolic responses …………………………………………………………………………..…47
2.4 DHM significantly reduces hepatic enzyme release, exhibits dose‐dependent
antiinflammatory actions, and maintains serum BDNF levels …………………….……………50
2.5 DHM reduces serum ethanol (EtOH) and acetaldehyde (ACH) concentrations in mice
administered 3.5g/kg EtOH, reverses chronic EtOH‐mediated depletion of hepatic NAD
+
levels,
and induces hepatic ADH1/ALDH2 …………………….……………………………....………53
2.6 DHM reduces hepatic CYP2E1 expression and increases the hepatic expression of Nrf2 and
HO‐1 antioxidant pathways ……………………………………………………….…...……..…55
2.7 DHM increases the expression of catalase and suppresses ethanol (EtOH)‐mediated ROS
generation in vitro ………………………………………………………………………………57
vi

3.1 DHM administration significantly increased the expression of hepatic Sirt-1, pAMPK
(Thr172), and Sirt-3 compared to EtOH fed mice ………………..………………………….…76
3.2 DHM administration significantly increased Sirt-1 deacetylation of PGC-1α and overall Sirt
activity in the livers of EtOH-fed mice ………………………………….………….….….……77
3.3 DHM administration significantly reversed EtOH-mediated stress on hepatic mitochondrial
content and ATP concentrations …………………………………………………………..……79
4.1 IVM, combined with TQ, significantly reduced ethanol intake at lower doses. IVM (0.5 – 2.0
mg/kg) combined with TQ (10 mg/kg) significantly reduced ethanol intake in comparison to
baseline values and IVM dose controls ………………….………………………….………….98
4.2 IVM, combined with TQ, significantly reduced 10% ethanol (10E) preference at lower doses.
IVM (0.5 – 2.0 mg/kg) combined with TQ (10 mg/kg) significantly reduced 10E preference in
comparison to baseline and IVM dose controls……………………………………………..…101
4.3 TQ (10 mg/kg) combined with IVM significantly reduces the dosing for ethanol consumption
and 10E preference over a period of five days. A) IVM (0.5 – 2.5 mg/kg) combined with TQ (10
mg/kg) significantly reduces ethanol intake relative to baseline, with 0.5 – 2.0 mg/kg IVM and
TQ showing significant effects compared to IVM doses alone………………………….……104
5.1 Randomized within-subjects drug treatment layout for behavioral analysis……………....117
5.2 DHM (10 mg/kg) combined with IVM reduces the dosing necessary to significantly decrease
EtOH consumption and 10E preference in male C57BL/6J mice over a period of 24-h……….117
5.3 DHM (10 mg/kg) combined with IVM significantly reduces the dosing for EtOH consumption
and 10E preference in female C57BL/6J mice over a period of 24-h. A) IVM (1.0 – 2.5 mg/kg)
combined with DHM (10 mg/kg) significantly reduced EtOH intake relative to saline treatment
vii

(Ctl), with 1.0 – 2.0 mg/kg IVM and DHM showing significant effects compared to IVM doses
alone…………………………………………………………………………………..……..…121
5.4 DHM (10 mg/kg) combined with IVM significantly reduces the dosing for EtOH consumption
and 10E preference over 24-h in male and female C57BL/6J mice with no sex-specific
differences …………………………………………………………...................................……122
5.5 Chemical structures of taxifolin (a potent Pgp ATPase inhibitor) and DHM, depicting
variation only at the 5’ position of ring B ………………………..…………………..….…..…124
5.6 Lowest energy binding conformation of DHM (yellow) in NBD1 of human Pgp (PDB:6C0V)
………………………………………………………………….…………………………….…124

viii

ABSTRACT
Development of Dihydromyricetin (DHM) as a Novel Therapy for Alcoholic Liver Disease
(ALD) and Alcohol Use Disorder (AUD)
By
Joshua Silva
Alcohol use disorder (AUD) affects over 18 million people in the US. Unfortunately,
pharmacotherapies available for AUD have limited clinical success and are under prescribed.
Furthermore, excess alcohol (ethanol) consumption is a significant cause of chronic liver diseases,
accounting for nearly half of the United States' cirrhosis-associated deaths. Ethanol-induced liver
toxicity, resulting in the development of alcoholic liver disease (ALD), is linked to ethanol
metabolism and its associated increase in proinflammatory cytokines, oxidative stress, and the
subsequent activation of Kupffer cells. Dihydromyricetin (DHM), a bioflavonoid isolated from
Hovenia dulcis, can reduce intoxication and potentially protect against chemical-induced liver
injuries. But there remains a lack of information regarding the mechanisms of DHM on ethanol
metabolism and hepatoprotection. As such, the described investigations herein, tested the
hypothesis that DHM supplementation enhances ethanol metabolism and reduces ethanol-
mediated fatty liver, thus promoting hepatocellular health. Through investigations utilizing a
forced drinking model that induces ALD in C57BL/6J mice, I found that DHM supplementation
(both 5 and 10 mg/kg intraperitoneal [i.p.]) ameliorated the development of fatty liver and hepatic
inflammation. Ethanol-mediated lipid accumulation and DHM effects against lipid deposits were
determined using H&E stains, triglyceride measurements, and intracellular lipid assays. Likewise,
I identified that DHM reversed ethanol-mediated inhibition of key metabolic signaling pathways
(AMP activated protein Kinase [AMPK]) in the liver that is associated with ALD pathology,
ix

suggesting metabolic and anti-inflammatory benefits of DHM. To expand on these metabolic
benefits, I also evaluated the effects of DHM on hepatic energy levels and mitochondrial responses
to understand the diverse mechanisms of DHM and its potential to ameliorate mitochondrial-
related damage following chronic ethanol injury. Through these investigations, I found that DHM
maintained hepatic mitochondrial density and ATP and that these effects are partly due to the
DHM-mediated activation of AMPK and the Sirtuin metabolic regulators critical for antioxidant
and mitochondrial pathways. Furthermore, using a two-bottle choice study, I found that the
combination of a potent P-glycoprotein (Pgp) inhibitor, tariquidar (TQ; 10 mg/kg; i.p.) combined
with ivermectin (IVM; 0.5 – 2.5 mg/kg; i.p.) significantly enhanced the potency of IVM on EtOH
intake and preference. To expand on these combinatorial studies, I also tested the hypothesis that
DHM, a natural product suggested to inhibit P-glycoprotein (Pgp/ABCB1), can enhance IVM
potency as measured by changes in ethanol consumption. Overall, I found that DHM increased
IVM potency in reducing EtOH consumption, resulting in significant effects at the 1.0 mg/kg dose
of IVM. Collectively, my work added to the evidence supporting the feasibility of this novel
combinatorial approach in reducing EtOH consumption and illustrate the utility of DHM in a novel
combinatorial approach to target both AUD and ALD.

1

CHAPTER 1
INTRODUCTION
1.1 Alcohol Consumption Rates and Co-Morbidities
Excessive alcohol consumption is a global healthcare problem with significant social,
economic, and clinical consequences. Alcohol use and specifically high-risk drinking (e.g., binge
drinking), which often leads to alcohol use disorder (AUD), significantly contributes to the burden
of disease in the US and worldwide (Rehm and Monteiro, 2005; Rehm, 2011; Murray et al., 2012;
Roerecke and Rehm, 2013; Laramée et al., 2015; Gakidou et al., 2017). AUD and high-risk
drinking are significant risk factors for morbidity and mortality from fetal alcohol spectrum
disorders (Wilhelm and Guizzetti, 2016), hypertension (Taylor et al., 2009), cardiovascular
diseases (Roerecke and Rehm, 2010; Piano and Phillips, 2014), stroke (Patra et al., 2010), liver
cirrhosis (Rehm et al., 2010; Rehm and Roerecke, 2015), several types of cancer (Corrao et al.,
2004; Bagnardi et al., 2015; Praud et al., 2016) and infections (Matsumoto et al., 2005; Yadav and
Lowenfels, 2013; Samokhvalov et al., 2015), and various injuries (Taylor et al., 2010).
Furthermore, AUD and high-risk drinking greatly impact the quality of life (Hasin et al., 2007a;
Dawson et al., 2009), are associated with psychiatric comorbidities (Grant et al., 2004; Kessler et
al., 2005), impair productivity and functioning, and place psychological and financial burdens on
society as a whole.
Unfortunately, even with the seriousness of the various physical and psychiatric harms of
high risk-drinking and AUD, alcohol consumption in the United States has risen with a 50%
increase in AUD prevalence from 2001 to 2012 (Grant et al., 2017). Furthermore, high-risk
drinking increased by approximately 30%, from 9.7% to 12.6%, representing about 20.2 million
to 29.6 million Americans (Grant et al., 2017). In relation to these increases in high-risk drinking
2

behavior, the added psychological distress and economic impact associated with the COVID-19
pandemic have further exacerbated these outcomes in the United States population, in which
alcohol was used as a coping motive for many individuals affected by the pandemic (McPhee et
al., 2020). In total, it is estimated that AUD affects nearly 1 in 6 Americans, with a disproportionate
increase in specific populations. Of these populations, women were found to have an alarming rise
in rates of AUD relative to men, demonstrating a narrowing of the gender gap in the drinking
patterns and AUD observed between 2001 – 2002 (Keyes et al., 2008, 2011; Grant et al., 2017).
Older adults, a population more at risk for disability, morbidity, and mortality from alcohol-related
chronic diseases (Ryan et al., 2013), were also found to have a 65.2% increase in high-risk
drinking, a 106.7% increase in AUD, and a 22.4% overall increase in alcohol use (Grant et al.,
2017). Furthermore, increases in alcohol use, high-risk drinking, and AUD are much more
significant among minorities than white individuals, with complex factors such as adversities that
disproportionately affect racial/ethnic minorities and persistent socioeconomic disadvantages that
lead to stress and demoralization (Mulia et al., 2009; Chartier and Caetano, 2010; Caetano et al.,
2014; Grant et al., 2017).
1.2 AUD Development
According to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5;
American Psychiatric Association), an essential feature of substance abuse is a maladaptive pattern
of substance use manifested by recurrent and significant adverse consequences related to the
repeated use of those substances. This repeated use of substances, e.g., alcohol, may result in a
repeated failure to fulfill major role obligations, repeated use in hazardous situations, multiple
legal problems, or recurrent social and interpersonal issues. Therefore, a maladaptive pattern of
alcohol use is likely to result in several deleterious outcomes, with significant risk to the safety of
3

individuals due to engagements in risky behavior (Sher et al., 2005). Although further
investigations are necessary to elucidate the particular mechanisms involved in the development
of AUD, several factors (described next) have been implicated in the development and severity of
AUD.
Typically, alcohol problems can develop in late adolescence and early adulthood
(Nurnberger et al., 2004b). However, they can also manifest at any time during adult life.
Unhealthy drinking patterns at an early age is suggested to act as a strong predictor of developing
AUD (Deutsch et al., 2013). Likewise, a family history of alcohol dependence is known to increase
the risk of AUD by two-fold (Nurnberger et al., 2004a; Deak et al., 2019), with males more likely
to develop AUD than females (Hasin et al., 2007b; Delker et al., 2016). Beyond the family history
and age of exposure, there are frequent co-occurrences of other psychiatric disorders that likely
contribute to the development of AUD, with common documentation of other substance use
disorders (Carroll et al., 1993, 1998b; Burns and Teesson, 2002), mood (Burns and Teesson, 2002;
Brière et al., 2014), anxiety (Burns and Teesson, 2002; Schneier et al., 2010; Gilpin et al., 2015),
and schizophrenic disorders (Drake and Mueser, 2002; Koskinen et al., 2009). Unfortunately,
treatment settings where the patient has a “dual diagnosis” can be challenging to the clinician due
to potential lack of responses and specialized treatment option necessary in comparison to patients
with a single diagnosis (Kessler, 1997; Gentil et al., 2009; Manning et al., 2009). In addition, these
patients are more likely to have greater rates of relapse, readmission rates, and increased
manifestations of symptoms that are chronic, severe, and refractory in nature (Rosenthal, 2003).
Concerning these co-occurrences of AUD with other disorders, it is suggested that these pathways
may form from a common causal pathway, such as genetic factors (e.g., rapid alcohol metabolism
and/or CNS receptors) identified in behavioral genetic research (Luthar et al., 1993; Reich et al.,
4

1998; Heath et al., 1999; Henderson et al., 2000; Zuckerman and Kuhlman, 2000; Knopik et al.,
2004; Sher et al., 2005; Agrawal and Lynskey, 2008). Therefore, it is suggested that the co-
occurrence of disorders with AUD may be due to one condition contributing to the development
of the second, as observed in cases of alcohol self-medication to mask or reduce other psychiatric
symptoms and/or stressors (Kalodner et al., 1989; Bolton et al., 2009; Robinson et al., 2009). These
medicating effects are suggested to be the result of positive reinforcement via dopamine,
endocannabinoids, opioid peptides, and γ-aminobutyric acid (Koob and Le Moal, 2008), whereas
the negative reinforcement involves the increased recruitment of corticotropin-releasing factor
(CRF), glutamatergic systems, and the down-regulation of γ-aminobutyric acid transmission
(Koob and Le Moal, 2008; Longo et al., 2016). Therefore, alcohol has been found to act as a
medication relief for negative effects (e.g., stress and anxiety) that results in higher rates of self-
medication and an increased risk for relying on alcohol (Sher et al., 2005), a phenomenon referred
to as the “tension-reduction hypothesis” (Cappell and Herman, 1972; Greeley and Oei, 1999;
Butler et al., 2010; Hasking et al., 2011). However, further studies regarding self-medicating
negative effects and/or the development of a secondary disorder are necessary to identify the
precise mechanisms involved in the development of AUD, as identifying loci that contribute to
alcohol dependence has proven problematic (Dick and Foroud, 2003; Treutlein et al., 2009; Bierut
et al., 2010). Furthermore, negative affect regulation from drinking is likely conditional upon
intraindividual and situational factors (Greeley and Oei, 1999; Sher et al., 2005). In total, when
considering the patient, the reasons and mechanisms for the development of AUD may differ
between individuals, with the causes contributed by several factors, including genetics,
environmental stressors, and/or the co-occurrence of other psychiatric disorders.

5

1.3 Current AUD Therapies
Pharmacotherapies
With the increasing rates of AUD and alcohol use in the United States, there is an
essential need for therapeutic interventions to reduce alcohol consumption in patients suffering
from AUD. This is especially critical when considering the subsequent alcohol-related organ
damage (AROD) that occurs with chronic alcohol use and AUD. Currently, there are limited
pharmacological options approved by the FDA to assist in the treatment of AUD and alcohol
abstinence. Three medications are currently approved for the treatment of AUD and are often
administered in combination with behavioral therapies to reduce alcohol dependence. Disulfiram,
approved in 1951 by the FDA for alcoholism, functions in blocking key metabolic enzymes of
alcohol (ethanol), resulting in higher concentrations of the toxic metabolite, acetaldehyde, that
contribute to unpleasant symptoms when consuming alcohol (Carpenter et al., 2018). Oral and
injectable naltrexone, approved by the FDA in 1963, assists in reducing craving for alcohol by
reducing the reward circuitry that contributes to alcohol addiction (Carpenter et al., 2018).
Acamprosate, approved in 2004, assists patients that have initiated cessation of alcohol by
reducing some of the symptoms that often contribute to alcohol relapse (Carpenter et al., 2018).
Unfortunately, the success rates for abstinence with these therapies remain low due to
underutilization, patient relapse, low patient demand, and lack of health care provider confidence
in efficiency (Harris et al., 2013). Considering the low success rates of approved therapies for
alcohol abuse and the potential for these therapies to induce drug-related liver damage (Liu and
Wang, 2019), it is clear that many challenges remain to develop more effective medications for
AUD. Therefore, the development of novel medications that effectively target AUD is critical to
address the alcohol abuse that consequently leads to the development of ALD.
6

Psychosocial and Behavioral Treatments
Beyond the use of pharmacotherapies, there are multiple forms of psychosocial and
behavioral therapies that can be conducted either alone or in combination with
pharmacotherapies. Brief interventions, a counseling strategy delivered by health care providers,
aim at educating the patients about problematic drinking, motivation to changes in behavior, and
reinforcement of skills to adequately address problematic drinking (Fleming et al., 1999). Studies
support the use of these brief interventions to reduce drinking within the primary care setting.
Notably, these beneficial responses are apparent when the intervention is repeated over multiple
visits with follow-up consultations (Fleming et al., 1999; Bertholet et al., 2005; O’Donnell et al.,
2014). Although this type of intervention has been found to reinforce other therapeutic
approaches, brief interventions are not sufficient on their own in patients with heavy alcohol use
or dependence (O’Connor et al., 1997; Saitz, 2010).
Other psychosocial and behavioral therapies for AUD include the 12-step facilitation,
cognitive behavioral therapy (CBT), and motivational enhancement therapy (MET) (Leggio and
Lee, 2017a). The twelve-step facilitation, a program grounded in acceptance, spirituality, and
moral inventories (Longabaugh et al., 1998), is abstinence-based and involves patient
participation in Alcoholics Anonymous meetings. CBT focuses on identifying patient triggers
and maladaptive behaviors that induce relapse by encouraging coping mechanisms that replace
alcohol (Larimer et al., 2003). MET seeks to frame the decision to stop or modify drinking in
terms of a dilemma and helps the patient work through the issue by adapting their behavior to
change (Miller et al., 1993). A more recent development is the use of mobile phone applications
that are aimed at supporting the reduction of high-risk drinking through easy-to-use and easily
accessible support systems provided to the user (Gustafson et al., 2014). In regards to the
7

efficacy of each of these programs, there is no established superior cognitive therapy as these
cognitive interventions have been found to be equivalent in patient success as indicated by the
Matching Alcoholism Treatments to Client Heterogeneity (MATCH) trial (Carroll et al., 1998a;
Leggio and Lee, 2017b). Furthermore, these programs and interventions can be used alongside
the administration of pharmacotherapies and have often been reported to have higher rates of
success when combined than when provided alone.
1.4 Alcoholic Liver Disease (ALD)
With persistent uncontrolled alcohol use resulting from limited and ineffective therapies
(e.g., therapeutics and/or abstinence counseling), many patients suffering from AUD have an
increased risk of developing AROD. A major form of AROD is alcohol liver disease (ALD) or
alternatively referred to as alcohol-related liver damage (ALRD). Notably, ALD is one of the
most prevalent and growing liver diseases in the United States, resulting from chronic heavy
ethanol consumption and rampant alcohol use. Rates of ALD continue to increase alongside the
rising rates of AUD and chronic alcohol consumption (Seitz et al., 2018), with ALD being the
second leading indication for liver transplantation in the United States (Goldberg et al., 2017).
Chronic, heavy alcohol consumption, classified as the consumption of over 40g of pure ethanol
per day over a sustained period of time (years), leads to the highest risk of ALD (Rehm et al.,
2010). However, evidence now suggests that chronic consumption of 12-24g of ethanol per day
leads to an increased risk of cirrhosis (late-stage ALD) (Rehm et al., 2010). Although excessive
drinking over a long period damages nearly every organ in the body, it is the liver that sustains
the earliest and greatest degree of injury due to its primary role in metabolizing ethanol (Lieber,
2000). Although much of the damage is due to continuous ethanol consumption, other factors
likely to influence the progression and development of ALD include sex, genetics, obesity,
8

smoking, and other environmental and genetic factors (Seitz et al., 2018). Therefore, continuous
liver damage induced by chronic ethanol consumption can be exacerbated by environmental and
genetic factors.
An additional factor suggested to contribute to the development of ALD is binge
drinking. Binge drinking is defined as rapid drinking patterns that bring blood alcohol
concentration (BAC) levels to 0.08 g/dL or greater in less than two hours (National Institute of
Alcohol Abuse and Alcoholism [NIAAA]). Due to the rapid increase in ethanol concentrations
with binge drinking, the metabolism of ethanol by the liver becomes saturated, thereby reducing
the efficiency of ethanol clearance and added stress to the liver and other organs of the body
(O’Shea et al., 2010; Gao and Bataller, 2011). Additionally, binge drinking is associated with a
greater risk of developing adverse health consequences and/or legal trouble (Jean-Bernard;
Wechsler et al., 1994; Gowin et al., 2017). However, data regarding binge drinking and the
increased risks of developing ALD is currently nonexistent and requires further elucidation,
especially when considering that specific alcohol-dependent patient populations may frequently
engage in chronic binge drinking (Beseler et al., 2010, 2012). Therefore, in combination with
chronic ethanol use, it is unquestionable that the development of ALD and cirrhosis correlates to
the length of time over which ethanol has been consumed, with the possibility of binge drinking
exacerbating ALD development.
1.5 Stages of ALD
ALD is comprised of a spectrum of injury to the liver with continued ethanol
consumption (Fig 1). This spectrum begins with alcoholic fatty liver, characterized by hepatic
steatosis (an accumulation of triglycerides in hepatocytes). Simple steatosis, or fatty liver, can
occur in up to 90% of heavy drinkers and can appear within 3 to 7 days of initial heavy alcohol
9

consumption (Fleming and McGee, 1984; Chacko and Reinus, 2016). This condition is
commonly asymptomatic, with the patient displaying normal or mildly increased serum
aminotransferase levels, including alanine aminotransferase (ALT) and aspartate
aminotransferase (AST), and gamma-glutamyltransferase (GGT), hepatic enzymes found to be
elevated in the blood following liver injury (Cabezas et al., 2016). Characteristic histological
findings of simple steatosis include large-droplet steatosis without significant inflammation or
necrosis in the centrilobular zone of the liver, a region surrounding the central vein that is most
sensitive to ischemia due to poor oxygenation and metabolic toxins, including ethanol,
metabolized by the high density of cytochrome P450 enzymes (Lieber, 1997; Theise, 2013;
Chacko and Reinus, 2016; Mak and Png, 2019). However, in severe cases of ethanol-induced
simple steatosis, there may be involvement of the entire lobule. The presence of simple steatosis
is no longer considered to be a benign condition, as it has been found that subjects with alcoholic
fatty liver and continued alcohol consumption progressed to fibrosis or cirrhosis over a long
period of continued drinking (Teli et al., 1995; Chacko and Reinus, 2016).
With continued ethanol consumption, there is an increased chance that these individuals
will progress and develop hepatic inflammation, hepatocyte injury, and hepatocyte enlargement
due to lipid accumulation. The presence of hepatic inflammation and enlargement (ballooning) is
histologically defined as alcoholic steatohepatitis (ASH), which may progress slowly with
continued ethanol consumption (Seitz et al., 2018). ASH is estimated to occur in 10% - 35% of
heavy drinkers and has a poor prognosis (O’Shea et al., 2010). Individuals with ASH may
present mild asymptomatic disease markers manifested by hepatomegaly (enlargement of the
liver) and increased serum aminotransferase levels, with the AST level being doubled the amount
or greater than ALT levels (Sass and Shaikh, 2006). Severe ASH is associated with jaundice,
10

fever, discomfort, hepatomegaly, and malnutrition (Chacko and Reinus, 2016). Pathological
features of severe ASH include Mallory hyaline, also referred to as Mallory bodies or
“alcoholic” hyalines, enlarged hepatocytes, and neutrophil-predominant inflammation (Fleming
and McGee, 1984; Theise, 2013; Chacko and Reinus, 2016). Unfortunately, the short-term
mortality, defined as mortality occurring less than 90 days after presentation to a hospital, may
be as high as 50% in patients with severe ASH. Therefore, prognostic tools such as the Maddrey
Discriminant Function, Model for End-stage Liver Disease (MELD) score, the Lille Model, and
the Glasgow Alcoholic Hepatitis Score are essential to guide the care for these individuals (Dunn
et al., 2005; Forrest et al., 2005; Chacko and Reinus, 2016).
Individuals with ASH have an increased risk of developing cirrhosis of the liver. Notably,
cirrhosis of the liver is the 12
th
most common cause of death in the United States (Yoon and Yi,
2012), in which 27% of patients with mild ASH and 68% of those with severe ASH developed
cirrhosis (Bird and Williams, 1988). Furthermore, individuals that ceased ethanol consumption
were found to have persistent ASH, with 18% of these individuals developing cirrhosis of the
liver (Galambos, 1972). Determination of the precise mechanisms of the disease is challenging,
as several factors may contribute to this development. Although it is well understood that ethanol
is hepatotoxic and that excessive consumption increases the risk of developing cirrhosis, there is
no clear dose-dependent relationship between drinking and the development of alcoholic
cirrhosis (Chacko and Reinus, 2016). Additionally, other factors are likely to increase the risk of
cirrhosis with ethanol consumption, such as hepatitis C virus infection and obesity (Corrao and
Arico, 1998; Hart et al., 2010; Seitz et al., 2018). Regardless, this continuous cycle of injury,
exacerbated by other stressors and repair in the liver, results in collagen deposition between
11

portal areas and central veins that entrap hepatocytes, resulting in alcoholic cirrhosis (Theise,
2013).

Fig 1. ALD Spectrum and Progression. Chronic heavy (40g or more of ethanol/day) ethanol
consumption over months and years will result in 90 – 100% of individuals developing ALD. 10-
35% of individuals with alcoholic fatty liver that continue chronic ethanol consumption will
develop alcoholic steatohepatitis. Additionally, 8 -20% of chronic heavy drinkers will develop
alcoholic liver cirrhosis, with further development into hepatocellular cancer in about 2% of
patients that continue drinking.
1.6 Treatment Options for ALD
Due to the excessive ethanol consumption contributing to ALD development, these
disease states are reversible and preventable with timely treatments. Unfortunately, due to ALD
often being asymptomatic in the early stages, it is challenging for the patient to identify potential
concerns to report to their physician, and identification of these early stages is often reliant on
laboratory findings. Screening and treatment for AUDs and/or patterns of ethanol abuse is often
the first approach for treating ALD (Mathurin et al., 2012). The treatment options for ALD are
dependent on the disease stage, in which detection at early stages can be treated with behavioral
12

therapies to reduce/cease ethanol consumption and potentially reverse early-stage ALD (Saberi
et al., 2016). However, as ALD progresses and the patient continues to struggle with ethanol
consumption, there are treatment options available for the management of ALD that often rely on
corticosteroid therapies for the suppression of inflammation (Fig. 2) (O’Shea et al., 2010;
Mathurin et al., 2012; Saberi et al., 2016). These mechanisms are believed to be caused by
suppression of transcription of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-
α and interleukin (IL)-8, in which these suppressive effects may provide a survival benefit in
patients with late-stage ALD (Morgan, 1996; Forrest et al., 2013). However, more research is
necessary as there have been conflicting results to corticosteroid survival benefits in patients
with late-stage ALD (Mathurin et al., 2002; Rambaldi et al., 2008). Furthermore, proper
screening of the patient must be conducted beforehand to determine the benefits of corticosteroid
therapy for chronic use. Unfortunately, there are additional challenges and significant risks with
the use of chronic steroid therapies, such as the increased potential for infections, especially in
sick populations. These treatment options are of further concern when considering that
approximately 40% of patients suffering from late-stage ALD are unresponsive to corticosteroid
treatment. Finally, there remains a lack of knowledge of the benefits and risks of long-term
steroid use in patients suffering from ALD. Collectively, these shortcomings demonstrate the
important need for new treatments for patients dealing with the various stages of ALD.

13

Fig 2. Proposed strategy for the management of patients with late-stage ALD. Adapted from
Saberi et al., 2016.

Abstinence
Abstinence is the most obvious direct treatment option for patients suffering from ALD,
as continued drinking can further lead to late-stage ALD (Borowsky et al., 1981; Pessione et al.,
2003). Abstinence from ethanol consumption has been found to improve the survival and
prognosis of patients with ALD and prevents progression to late-stage ALD through histologic
improvement and reduction in portal pressure (Luca et al., 1997; Veldt et al., 2002). Early-stage
ALD and ASH can be reversed in humans after abstinence for several weeks (Teli et al., 1995).
14

These findings are supported by animal models of ALD, in which a reversal in hepatic fatty acid
metabolism and fatty liver is observed in male Wistar rats withdrawn from chronic alcohol
consumption (Thomes et al., 2019). Furthermore, AUD pharmacotherapies (naltrexone,
disulfiram, and acamprosate) can be prescribed alongside cognitive therapies to further benefit
the patient's success of withdrawing abstaining from alcohol consumption and abuse. However,
limited success and underutilization of these therapies have been observed in patients suffering
from AUD, as many individuals often relapse with treatment due to social factors, stress,
psychological distress, traumatic experiences, and/or personality disorders (Sliedrecht et al.,
2019). Furthermore, challenges of defining recovery from AUD require further clarification as
many individuals may be opposed to complete abstinence from alcohol consumption in
comparison to taking control of alcohol-consuming behaviors (Witkiewitz and Tucker, 2020).
Nutritional Therapy
Malnutrition is a major complication of ALD, in which the hepatotoxicity of ethanol can
contribute to malnutrition and is also enhanced by malnutrition (Patek, 1979; Chao et al., 2016).
Malnutrition, therefore, worsens the clinical outcomes of ALD, and nutritional support improves
the nutritional status and may likely improve clinical outcomes. The mechanism contributing to
malnutrition are multifactorial. Factors such as inadequate caloric intake, anorexia, vomiting,
mal-digestion, metabolic disturbances, or mal-absorption can cause malnutrition in patients with
ALD (Singal and Charlton, 2012; Suk et al., 2014). Furthermore, the severity of ALD and
development of serious complications are generally correlated with the severity of malnutrition
(Mendenhall et al., 1993; Suk et al., 2014).
Nutritional therapy at early stages of ALD can alleviate symptoms, improve treatment
response, and improve the quality of life (Campillo et al., 2003). The goals for nutrition therapy
15

include the provision of adequate calories and nutrients that support the regeneration of
hepatocytes (Hirsch et al., 1999). An inadequate protein source is one factor that has been
commonly observed in patients suffering from ALD, in which incorporation of branched-chain
amino acids at 34g/d has been shown to decrease the number of hospitalizations due to
complications involving liver cirrhosis (Marchesini et al., 1990; Hirsch et al., 1993).
Additionally, nutritional therapies that increase the amount of protein between 1.2 – 1.5 g/kg/day
along with 35 – 40 kcal/kg/day and with medical treatment can benefit ALD depending on its
stage (Plauth et al., 2006). Therefore, supplementation of depleted vitamins and minerals, along
with proper nutrition, can improve clinical outcomes in patients suffering from ALD.
Liver Transplantation
In North America, ALD is one of the most common indications for liver transplantation,
with survival rates being comparable with other causes (Suk et al., 2014). Liver transplantation is
often the last option for patients with late-stage ALD that have demonstrated a lack of response
to therapies that treat ALD symptoms. The indications for transplantation in patients with ALD
are similar to other end-stage liver diseases. For ALD, patients with severe ASH that do not
respond to corticosteroid therapies and other alternative therapies have a mortality rate of 50% -
75% at six months and are therefore recommended for liver transplantation. Unfortunately, it is
recommended that a patient with ALD abstain from ethanol use for at least six months prior to
liver transplantation, which is a challenge for those that continue to struggle with AUD and
ethanol abuse (Yang et al., 2008; Lucey et al., 2009). Therefore, many North American liver
transplantation centers do not consider patients with severe ASH as candidates for liver
transplants because they are unable to meet the criterion of 6-month abstinence (Suk et al.,
2014). Due to this reason, along with the lack of effective therapies for ALD, studies are
16

currently investigating the potential to reduce that 6-month timeline of abstinence in anticipation
of a life-extending liver transplant (Lee and Terrault, 2018). Ultimately, the need for late-stage
ALD patients to rely on liver transplantation due to unresponsiveness to current therapies for
ALD symptoms highlights a significant need for novel therapies to prevent/reduce the severity of
ALD and reduce ethanol abuse in patients suffering from AUD.
1.7 Ethanol Metabolism and Biochemical Responses in the Liver
1.7.1 Ethanol Metabolism in the Liver
Although a small percentage of ethanol metabolism occurs in non-liver tissues, such as
the gastric mucosa or central nervous system [CNS] (Caballeria et al., 1989; Lieber, 1994), the
liver is the main organ responsible for metabolizing ingested ethanol. As illustrated in Fig. 3,
once an individual consumes ethanol, it will be absorbed mainly from the small intestine into the
veins that collect blood from the stomach and bowels and from the hepatic portal vein, which
carries the blood to the liver (Zakhari, 2006). Upon entering the liver, the blood ethanol is then
exposed to several key metabolizing enzymes. Of these enzymes, alcohol dehydrogenase (ADH)
is mainly responsible for the oxidative metabolism of ethanol, via the reduction of nicotinamide
adenine dinucleotide (NAD
+
), to its metabolite, acetaldehyde in the cytosol (Dey and
Cederbaum, 2006; Zakhari, 2006; Purohit et al., 2009)(Fig 3). Acetaldehyde is a highly reactive,
toxic, and carcinogenic compound that the liver effectively oxidizes via acetaldehyde
dehydrogenase (ALDH) enzymes, particularly ALDH2 of the mitochondria, to acetate for rapid
clearance and removal from the liver (Lieber, 1994; Mandal et al., 2017; Teschke, 2018).
Depending on the concentrations of ethanol in the blood (blood ethanol concentrations [BECs]),
ADH can be rapidly saturated by ethanol, thereby increasing the metabolism of ethanol by
cytochrome P450 enzymes of the microsomal ethanol oxidizing system (MEOS) (Mandal et al.,
17

2017). Furthermore, catalase enzymes of the peroxisome will play a minor role in the oxidation
of ethanol (Fig 3). This pathway oxidizes ethanol in the presence of an H2O2-generating system
(Mandal et al., 2017). Although non-oxidative metabolism of ethanol is minimal and is not as
well elucidated as the oxidative mechanisms, two major pathways are known to contribute to this
process in the liver (Laposata and Lange, 1986; Hamamoto et al., 1990). Non-oxidative
metabolism involves the esterification of ethanol. The major enzymes involved in this pathway
are the fatty acid ethyl ester (FAEE) synthase, which forms ester products from ethanol. A
second product that has been identified is the phosphatidylethanol product catalyzed by
phosphatidylcholine-specific phospholipase D (Zakhari, 2006; Mandal et al., 2017). Overall, the
oxidative and nonoxidative pathways of ethanol metabolism are interrelated. Inhibition of the
oxidative metabolism of ethanol results in an increase of the nonoxidative pathway, thereby
increasing the production of FAEEs in the liver and pancreas (Werner et al., 2002).

Fig 3. Alcohol (Ethanol) Metabolism in the liver. Following ethanol consumption, most of the
ethanol is absorbed from the stomach and small intestine. Blood ethanol will quickly reach the
liver, where it is primarily metabolized by an oxidative and non-oxidative pathway. The
18

oxidative pathway mainly consists of ADH, CYP2E1 in the endoplasmic reticulum, and minor
metabolism by catalase in the peroxisomes. Oxidative metabolism of ethanol results in the
production of acetaldehyde, which is rapidly transported to the mitochondria for conversion to
acetate via acetaldehyde dehydrogenase (ALDH)2. Non-oxidative metabolism takes place in the
presence of fatty acids where fatty acid ethyl ester (FAEE) synthase metabolized ethanol to a
FAEE. Likewise, phospholipase D is involved in the non-oxidative metabolism of ethanol,
thereby producing phosphatidylethanol from ethanol (Adapted from Mandal et al., 2017).
1.7.2 Ethanol-Mediated Biochemical Responses
The primary oxidation of ethanol in the liver contributes to various biochemical reactions
that result from the activity of ethanol, the production of ethanol metabolites, production of
reactive oxygen species (ROS) via oxidation, and the consumption of energy pools, including
NAD
+
, utilized for ethanol and acetaldehyde metabolism. Furthermore, the highly reactive and
toxic acetaldehyde products produced from ethanol metabolism result in additional biochemical
responses or protein adducts that contribute to injury in the liver (Barry and McGivan, 1985;
Lieber, 1988; Tuma, 2002; Setshedi et al., 2010). As ethanol concentrations increase, ethanol
clearance will be reduced due to ADH saturation, resulting in the depletion of NAD
+
energy
pools and subsequent increases in the concentration of toxic acetaldehyde due to reduced ALDH
activity. The increased concentrations of acetaldehyde in the liver also increases the risk for
tissue damage via protein adducts and increases the potential for carcinogenesis. Furthermore,
the molecular mechanisms attributed to the combination of increased oxidative metabolites
produced by the metabolism of ethanol, the inflammatory response, gut microbiota, the increase
in lipopolysaccharide, and the innate immune system consequentially result in the progress of
ethanol-mediated injury to the liver (Dhanda et al., 2012). Altogether, these combinations of
19

effects can result in several biochemical responses in the liver that further contribute to ethanol-
mediated injury.
At a cellular level, ethanol metabolism often induces injury to the liver via the production
of reactive oxygen species (ROS), which can be exacerbated by hypoxia, bacterial translocation,
and the release of proinflammatory cytokines (Cederbaum, 2006; Louvet and Mathurin, 2015).
The major sources of ROS resulting from ethanol metabolism are the mitochondria via
respiration, CYP2E1 in the endoplasmic reticulum, and Kupffer cells of the liver. These ROS are
mainly responsible for the lipid peroxidation of cell membranes (Fig 4) frequently observed with
ethanol consumption and exposure (Louvet and Mathurin, 2015; Yang et al., 2019).

Fig 4. Ethanol Metabolism and hepatic oxidant stress. Ethanol is oxidized principally by ADH,
CYP2E1, and catalase to acetaldehyde (Ach). Ach is a highly reactive intermediate that
covalently bands to proteins or can undergo secondary reactions to form MAA. CYP2E1 is
induced by ethanol and yields free radicals (ROS) to form malondialdehyde (MDA) and 4-
hydroxynonenal (4HNE) (Adapted from Yang et al., 2019).

The metabolism of ethanol and ROS production via ADH and CYP2E1 results in cytotoxic
effects leading to cell death, in which evidence suggests both apoptosis and necrosis in ALD
20

(Luedde et al., 2014). Additional factors following cell death, mainly necrosis, further promote
macrophage and neutrophil activation, fibrogenesis, and hepatic regeneration via cellular release
of damage-associated molecular patterns (DAMPS) (Dolganiuc et al., 2012; Brenner et al.,
2013). Chronic exposure to ethanol also increases hepatocyte sensitivity to oxidative stress via
the depletion of key antioxidant enzymes. Ethanol has been found to deplete glutathione, a
critical antioxidant enzyme that function in ROS protection when in the reduced state, and heme
oxygenase (HO)-1, an inhibitor of CYP2E1 and its subsequent ROS generation (Fernandez-
Checa et al., 1989; Fernández-Checa et al., 1991, 1993; Hirano et al., 1992; Gong et al., 2004).
These effects, combined with the highly reactive and toxic effects of acetaldehyde, promote
hepatocyte sensitivity to stressors and increase hepatocellular death. Furthermore, oxidative
stress of the hepatic endoplasmic reticulum, amplified by increased levels of homocysteine
levels, participates in factors of inflammation and tissue damage. Therefore, the ROS byproducts
of ethanol metabolism combined with ethanol-mediated inflammatory responses are critical
damaging factors involved in the pathogenesis of ALD.
1.7.3 Mechanisms of ethanol-induced steatosis
Chronic ethanol consumption promotes steatosis by disrupting hepatocellular lipid
metabolism. AMP-activated protein kinase (AMPK) is a multisubunit protein kinase that acts as
a key metabolic “master switch” that activates lipid catabolism and inhibits lipid synthesis in
response to low energy levels (Hardie et al., 1998; Winder and Hardie, 1999). Of these AMPK
downstream targets, acetyl-CoA carboxylase (ACC) is generally regarded as the rate-limiting
enzyme in fatty acid biosynthesis in the liver and other tissues (Abu-Elheiga et al., 2001).
Malonyl-CoA, the product of ACC, is a precursor for fatty acid synthesis and is also an inhibitor
of mitochondrial fatty acid oxidation via carnitine palmitoyltransferase (CPT)-1 (You et al.,
21

2004). AMPK is a critical step in this pathway, in which AMPK activation results in the
inhibition of ACC, resulting in increased mitochondrial beta-oxidation of lipids and reduced lipid
biosynthesis. Chronic ethanol exposure leads to inhibition of AMPK, thereby disrupting lipid
metabolism pathways via sterol regulatory element-binding proteins (SREBPs) and peroxisome
proliferation-activating receptor (PPAR)-α (Louvet and Mathurin, 2015). In addition to the
changes in ACC and CPT1, ethanol-mediated inhibition of AMPK leads to increased SREBP-1
activity, resulting in further fatty acid synthesis in the liver (You et al., 2002). Additionally, the
expression of SREBP1 is increased with acetaldehyde and the pro-inflammatory marker TNF-α,
an inflammatory marker observed from ethanol-induced liver damage (Endo et al., 2007).
Therefore, ethanol-mediated responses in the liver inhibit the activity of AMPK resulting in an
overall lipid accumulation (steatosis) due to reduced breakdown of lipids and continued lipid
biosynthesis. The fat accumulation that results from excess ethanol metabolism can be both
macrovesicular (having one large fat droplet per hepatocyte) or microvesicular (having many
small fat droplets per hepatocytes) and are often an early indicator of ALD severity (Ishak et al.,
1991; Day and James, 1998; You and Arteel, 2019).
1.8 Dihydromyricetin (DHM) as Natural Polyphenol
Natural polyphenols are secondary metabolites of plants that are generally involved in
defense against ultraviolet radiation or pathogens. These natural compounds are becoming
widely recognized as potential agents for the prevention and treatment of several disorders, such
as cancer, cardiovascular disease, diabetes, aging, and neurodegeneration (Li et al., 2014).
Polyphenols comprise a large group of secondary plant metabolites ranging from small
molecules to highly polymerized compounds, having at least one aromatic ring with one or more
hydroxyl functional groups (Manach et al., 2004). Depending on their chemical structures,
22

polyphenols can be chemically divided into several classes, such as flavonoids, phenolic acids,
lignans, and other classifications (Manach et al., 2004; Zhou et al., 2016). In particular, the
flavonoids share common structures of 2 aromatic rings that are bound together by 3 carbon
atoms that form an oxygenated heterocycle (Fig 4A). With this general structure, flavonoids can
be further divided into 6 subclasses as a function of the type of heterocycle involved: flavonols,
flavones, isoflavones, flavanones, anthocyanidins, and flavanols (Fig 4B).

Fig 5. Chemical structure of flavonoids. (Adapted from Manach et al., 2004)

1.9 DHM Health Benefits
Flavonoids tend to have activities on multiple targets and are suggested to have several
health benefits. Of these benefits, polyphenols have been found to have many pharmacological
effects that range from mediating oxidative stress, modifying lipid metabolism and insulin
resistance, and mitigating inflammatory damage (Li et al., 2014; Domitrović and Potočnjak,
2016). Building evidence suggests that dihydromyricetin (DHM), a bioactive flavonol extracted
23

from Hovenia dulcis, also has a broad range of benefits, including antioxidant (Okuma et al.,
1995), antitumor, and free radical scavenging capacities can aid in the reduction of lipid
peroxidation (Jiang et al., 2014; Hou et al., 2015). Furthermore, there is building evidence
supporting the use of DHM for the treatment of alcohol use disorder (AUD) and the possible
amelioration of ALD in animal models. For example, Shen and colleagues found that DHM
potentiates GABAA receptors and reduces the effects of ethanol on the same receptors (Shen et
al., 2012). This activity resulted in a DHM-mediated reduction of ethanol intoxication and
alcohol withdrawal syndrome in rat models (Shen et al., 2012). DHM administration has also
been found to reduce ethanol-dependent lipid accumulation in the liver via mechanisms of
activated autophagy and reducing inflammation, further supporting the beneficial effects of
DHM on ethanol and chemical-induced outcomes (Fang et al., 2007; Qiu et al., 2017, 2019).
The potential hepatoprotective effects of DHM may be linked to its ability to protect cells
against inflammatory responses and oxidative species, key signaling factors involved in the
pathogenesis of ALD (Hou et al., 2015; Liang et al., 2015). In human umbilical vein endothelial
cells (HUVEC) and HepG2 hepatoblastoma (HB) cells, DHM has been found to suppress ROS
and oxidative stress via its ability to scavenge radicals and induced regulatory mechanisms (Hou
et al., 2015; Xie et al., 2016). DHM also protects HUVECs from oxidative damage by altering
mitochondrial apoptotic pathways involving Bax, Bcl-2, and the activation of caspase-9/caspase-
3, meanwhile reducing lipid accumulation and lipogenesis in vitro (Hou et al., 2015; Liang et al.,
2015; Xie et al., 2016). The proposed protective effects of DHM have also been attributed to its
electrophilic properties and involvement in the dissociation of NRF2 from Keap1, thereby
activating cellular antioxidant mechanisms (Qiu et al., 2017; Chu et al., 2018). Interestingly, these
data support the development of DHM as a dietary flavonol to help reduce the consequences of
24

oxidative stress and lipid metabolism to promote liver health. Therefore, one major goal of my
dissertation is to elucidate the liver-protective benefits of DHM against ethanol-induced liver
damage and develop this therapy for the treatment of ALD.
1.10 DHM as a Flavonoid Enhancer of Bioavailability
In addition to the anti-inflammatory and anti-oxidative properties of DHM, this dietary
flavonoid has also been reported to increase the bioavailability (BA) and pharmacokinetic (PK)
profiles of other co-administered therapies. Similar to other flavonoid classes, DHM is
metabolized by various Phase-I and Phase-II metabolic enzymes. Primarily, the metabolic
enzymes of cytochrome P450 (CYP)3A4, CYP2E1, and CYP2D6 have been illustrated to play a
role in the metabolism of DHM. Human CYP3A4 is one of the most abundant drug-metabolizing
CYP isoforms in the human organs, including the liver and intestines, and plays a significant role
in the oxidation of xenobiotics and contributes to the biotransformation of approximately 60% of
current therapeutics (Pandit et al., 2011), including DHM (Liu et al., 2017). In addition to the
activity of CYP3A4 on DHM metabolism, CYP2D6 and CYP2E1 have been found to contribute
to the metabolism of DHM, with additional findings suggesting inhibition of these enzymes by
DHM when using human liver microsomes (Liu et al., 2017). Furthermore, these effects have
been observed in animal models, in which DHM was found to improve the PK profile of co-
administered therapies that rely on these metabolic enzymes, thereby enhancing the BA and PK
profile for added therapeutic benefits (Wong et al., 2015; Zhu et al., 2015; Deng et al., 2020).
1.11 Mechanisms of DHM BA Enhancement
Beyond the prevalent drug-metabolizing enzymes, an essential mediator of drug BA and
PK properties is the expression and activity of drug efflux transporter, P-glycoprotein
(Pgp/ABCB1). Pgp is a 170 kDa transmembrane glycoprotein ATP-ase transporter belonging to
25

the ABC (ATP Binding Cassette) family (Dean et al., 2001; Ayrton and Morgan, 2008; Leopoldo
et al., 2019). The Pgp transporter is localized in several barriers including the blood-brain barrier
(BBB), blood-cerebrospinal fluid (B-CSF), and blood-testis barrier (BTB), where it functions to
regulate the absorption and excretion of xenobiotics (Giacomini et al., 2010). Additionally, the
Pgp efflux transporter is highly expressed in tissues of the kidney, liver, placenta, and villus tip
of enterocytes in the gut, where it plays a crucial role in the metabolism and elimination of drugs
(Leopoldo et al., 2019). Therefore, the expression and activity of Pgp are crucial for the organs
and tissues to eliminate xenobiotics. However, the activity of this efflux transporter also affects
the PK profile and BA of many therapeutic interventions.
Due to the activity and role of Pgp in the efflux of therapeutics, the inhibition of the Pgp
efflux pump is often investigated in order to improve the delivery of therapeutic agents (Amin,
2013). For instance, the use of Pgp inhibitors (first, second, or third-generation inhibitors) are
currently under investigation for use to enhance the BA of anti-cancer therapeutics (Kruijtzer et
al., 2002; Sandler et al., 2004; D’Cunha et al., 2016; Brings et al., 2019), alongside
investigational improvements in the inhibiting properties and safety of the Pgp inhibitor.
Therefore, inhibitors or modulators of Pgp efflux have been found to benefit the BA and PK
profile of therapeutics that are otherwise diminished with Pgp efflux. Furthermore, many of these
investigations have led to the development of the current 3
rd
generation of Pgp inhibitors that are
reported to have higher specificity, binding, and improved safety outcomes when compared to
the 1
st
generation Pgp inhibitors.
Importantly, several flavonoids have also been identified to have Pgp inhibiting
properties that benefit the PK profile of therapeutic compounds targeting Pgp expressing tissues
(Limtrakul et al., 2005; Brand et al., 2006; Srinivas, 2019). Of these flavonoids, quercetin and
26

taxifolin, flavonoids of the same family as DHM, share structures similar to DHM that have been
demonstrated to aid in its inhibition of Pgp efflux. More recently, DHM has also been
investigated in its ability to inhibit Pgp, where current data suggests that it acts as a Pgp inhibitor
that can enhance the BA of xenobiotic substrates (Wong et al., 2015; Sun et al., 2018; Deng et
al., 2020). Although the mechanism(s) associated with DHM’s ability to inhibit Pgp is unclear,
recent structure-activity relationship (SAR) studies of flavonols and Pgp activity indicate that
structural components of DHM are consistent with potent non-competitive Pgp inhibition of ATP
hydrolysis (Xia et al., 2019). For example, the 3’-OH, 4’-OH, and 2,3-saturation of DHM are
associated with enhanced non-competitive Pgp inhibition (Di Pietro et al., 2002; Wong et al.,
2015; Cui et al., 2018). Supporting these findings, taxifolin, a flavonol with a similar structure to
DHM, was found to inhibit Pgp ATPase at concentrations as low as 100 nM through interactions
with the Pgp nucleotide-binding domain (NBD). These findings suggest a novel function of
DHM, in which it can act as a non-competitive Pgp ATPase inhibitor. However, further
clarification of the mechanism of Pgp inhibition is necessary. Collectively, DHM is suggested to
be a flavonoid that also acts as a Pgp inhibitor and may be utilized in combination with other
therapeutic compounds to benefit their BA and PK profile. Therefore, DHM can potentially be
used to enhance the BA and PK profile of therapeutics limited by their PK profiles, as has been
observed with therapeutic candidates for the treatment of CNS disorders, including AUD.
1.12 Purinergic (P2X) 4 Receptors as Novel Drug Targets of AUD
P2X4 Receptor Involvement in Alcohol Consumption
The development of ALD is reliant on the frequent use and dependence on alcohol. Of course,
in developing therapies for ALD, compounds that can reduce alcohol consumption should have
the potential to significantly reduce or help support alcohol abstinence which would lead to
27

improvement in liver outcomes. Given the limited success of available AUD pharmacotherapies
and cognitive therapies, novel targets of AUD are critical for developing strategies to assist patients
with strong alcohol dependence. One novel target for AUD that is currently under consideration is
the P2X4 receptor. Accumulating evidence suggests that P2X receptors, including P2X4 receptors,
are broadly distributed throughout neurons and microglia and are essential for the action of ethanol
(Li et al., 1993, 1994, 1998; Kidd et al., 1995; Weight et al., 1999; Kanjhan et al., 1999; Xiong et
al., 2000; Franke et al., 2001; Davies et al., 2002; Asatryan et al., 2008, 2015; Xiao et al., 2008;
Popova et al., 2010, 2020; Wyatt et al., 2013; Khoja et al., 2018b). Therefore, our laboratory has
been investigating P2X4 receptors as a novel target for the treatment of AUD (Yardley et al., 2012;
Wyatt et al., 2014; Khoja et al., 2018b, 2018a; Huynh et al., 2019a, 2019b; Popova et al., 2020).
In correlation with the investigations of ethanol’s activity on P2X4 receptors, an inverse
relationship was identified between P2X4 receptor expression and innate rodent ethanol
consumption and preference. Further evidence linking P2X4Rs with the action of ethanol comes
from findings where lower levels of whole-brain expression of p2x4r mRNA in inbred rats were
associated with a high ethanol-drinking phenotype (i.e., inverse relationship) compared to those
with a lower ethanol-drinking phenotype (Tabakoff et al., 2009). Surprisingly, opposite findings
were reported for HAD-1 vs. LAD-1 rats and alcohol-preferring vs. non-alcohol preferring rats
(McBride et al., 2012). The common theme from these investigations is that manipulation of p2x4r
expression is associated with changes in ethanol consumption. In agreement with this hypothesis,
our laboratory has demonstrated that male P2X4KO mice (i.e., p2rx4 deleted) and WT mice with
reduced expression of P2X4 receptors consumed significantly more ethanol than wildtype (WT)
controls (Wyatt et al., 2014; Khoja et al., 2018a), with no difference in saccharin intake (Wyatt et
al., 2014).
28

P2X4Rs as a Novel Target for AUD Therapies
In connection with our investigations of the role of P2X4 receptors on ethanol intake, our
lab has found that ivermectin (IVM), an FDA-approved semi-synthetic macrocyclic lactone, acts
as a positive allosteric modulator (PAM) of P2X4 that reduces/eliminates the inhibitory effects
of ethanol on P2X4 receptors (Li et al., 1993; Kidd et al., 1995; Asatryan et al., 2008, 2010;
Franklin et al., 2014). This activity of IVM on P2X4 receptors suggests that IVM, and other
members of the avermectin family, can act as a new class of pharmacotherapies to prevent and/or
treat AUD. In support of this hypothesis, we found that IVM significantly reduced ethanol intake
in mice and that this effect likely reflected IVM’s ability to modulate ligand-gated ion channels
(LGICs) (Asatryan et al., 2010, 2014; Yardley et al., 2012, 2015). Based on this work, we
hypothesized that structural modifications that enhance IVM’s effects on key receptors and/or
increase its brain concentration should improve its anti-alcohol efficacy. Our lab began testing
this hypothesis by comparing GABAAR and P2X4 receptor activity and CNS exposure levels of
three avermectins, IVM, abamectin (ABM), and selamectin (SEL), with in vivo efficacy at
reducing ethanol intake in mice. Notably, IVM and ABM, but not SEL potentiated P2X4
receptor function and antagonized the inhibitory effects of ethanol on P2X4 receptor function
expressed in Xenopus oocytes. These findings were further supported with our demonstration of
moxidectin (MOX) acting as a P2X4 receptor modulator that significantly reduced ethanol intake
in animal models using a two-bottle choice model and a drinking in the dark (DID) model of
binge-like ethanol consumption (Huynh et al., 2017, 2019b; Khoja et al., 2018b). In agreement
with the role of P2X4 receptor activity on ethanol intake, IVM and ABM were found to
significantly reduce ethanol intake in mice due to their actions on P2X4 receptors. Overall, this
work suggests that P2X4 receptors are involved in the modulation of ethanol activity and that
29

pharmacotherapies that target P2X4 receptors can potentially modify ethanol drinking behaviors
and alcohol abuse in patients suffering from AUD.
PK Limitations of Identified P2X4 Modulators (PAMs) Targeting AUD
With the identification of P2X4 receptor PAMs (e.g., IVM & MOX) potentially acting as
new therapeutic agents for AUD, there are limitations to these compounds that diminish the CNS
retention and must be considered. For instance, the pharmacological activity of MOX resulting in
the reduction of alcohol intake has been found to occur more rapidly than that of IVM (Huynh et
al., 2017; Khoja et al., 2018b). Both compounds are similar in their overall macrocyclic lactone
structure. Yet, differences in key chemical and structural properties significantly affect the CNS
response time and retention, including the higher lipophilicity of MOX that allows for enhanced
brain penetration (Perez et al., 2009; Ménez et al., 2012; Huynh et al., 2017, 2019b).
Furthermore, although both IVM and MOX are hydrophobic and permeable to the CNS, IVM is
further limited in its responses in the brain due to its higher affinity for the Pgp efflux transporter
(Kemper et al., 2004; Potschka, 2010; Amin, 2013). Notably, the high affinity of IVM for Pgp,
relative to MOX, thereby leads to its rapid removal from the blood-brain barrier (BBB), likely
contributing to its slower onset of reducing alcohol consumption (e.g., 9 hours [IVM] vs. 4 hours
[MOX]) (Huynh et al., 2017). Collectively, these data suggest that the limitations of the IVM PK
profile are likely contributed by its elimination from Pgp transporters and reduced retention
within the CNS. In support of these findings, Pgp KO has been shown to increase the retention
time and concentrations of IVM in the CNS due to reduced elimination at the BBB (GEYER et
al., 2009), suggesting that Pgp is greatly involved in limiting the availability of IVM in the CNS
and delaying the onset of its pharmacological response in the brain. Therefore, strategies to
enhance the retention and BA of these potential AUD therapeutics can significantly benefit the
30

repurposing of candidate P2X4 receptor PAMs, and potentially enhance the potency of these
compounds as AUD therapeutics.
Collectively, these findings and gaps in our knowledge helped in the formation of my
dissertation proposal in which I tested the hypothesis that DHM acts as a hepatoprotective
flavonoid with Pgp inhibiting properties that can benefit the treatment of both AUD and the
subsequent development of ALD. This will be accomplished by investigating the utility of DHM
for the prevention/reduction of ALD following ethanol abuse, and the potential of using this
hepatoprotective flavonoid to benefit the potency of IVM as a Pgp inhibitor in treating AUD.

31

CHAPTER 2
Dihydromyricetin protects the liver via changes in lipid metabolism and enhanced ethanol
metabolism

Joshua Silva
a
, Xin Yu
a
, Renita Moradian
a
Carson Folk
a
, Maximilian H. Spatz
a
, Phoebe Kim
a
,
Adil A. Bhatti
a
, Daryl L. Davies
a
and Jing Liang
a
*
a
Titus Family Department of Clinical Pharmacy, School of Pharmacy, University of
Southern California, Los Angeles, USA
*
Corresponding author
Jing Liang, Titus Family Department of Clinical Pharmacy, School of Pharmacy,
University of Southern California, Los Angeles, USA
Tel. 323.442.1427
E-mail address: jliang1@usc.edu

Abstract
Introduction
Excess alcohol (ethanol) consumption is a significant cause of chronic liver disease, accounting
for nearly half of the cirrhosis-associated deaths in the United States. Ethanol-induced liver
toxicity is linked to ethanol metabolism and its associated increase in proinflammatory cytokines,
oxidative stress, and the subsequent activation of Kupffer cells. Dihydromyricetin (DHM), a
bioflavonoid isolated from Hovenia dulcis, can reduce ethanol intoxication and potentially protect
against chemical-induced liver injuries. But there remains a paucity of information regarding the
effects of DHM on ethanol metabolism and liver protection. As such, the current study tests the
32

hypothesis that DHM supplementation enhances ethanol metabolism and reduces ethanol-
mediated lipid dysregulation, thus promoting hepatocellular health.
Methods
The hepatoprotective effect of DHM (5 and 10 mg/kg; intraperitoneal injection) was evaluated
using male C57BL/6J mice and a forced drinking ad libitum ethanol feeding model and
HepG2/VL-17A Hepatoblastoma cell models. Ethanol-mediated lipid accumulation and DHM
effects against lipid deposits were determined via H&E stains, triglyceride measurements, and
intracellular lipid dyes. Protein expression of phosphorylated/total proteins and serum and hepatic
cytokines were determined via Western blot and protein array. Total NAD
+
/NADH Assay of liver
homogenates was used to detect NAD+ levels.
Results
DHM reduced liver steatosis, liver triglycerides, and liver injury markers in mice chronically fed
ethanol. DHM treatment resulted in increased activation of AMPK and downstream targets,
carnitine palmitoyltransferase (CPT)-1a, and acetyl CoA carboxylase (ACC)-1. DHM induced
expression of ethanol metabolizing enzymes and reduces ethanol and acetaldehyde concentrations,
effects that may be partly explained by changes in NAD
+
. Furthermore, DHM reduced the
expression of pro-inflammatory cytokines and chemokines in sera and cell models.
Conclusion
In total, these findings support the utility of DHM as a dietary supplement to reduce ethanol-
induced liver injury via changes in lipid metabolism, enhancement of ethanol metabolism and
suppressing inflammation responses to promote liver health.
Keywords: Ethanol, Dihydromyricetin, Alcohol Liver Damage, Steatosis
33

Abbreviations: ethanol (EtOH), acetaldehyde (ACH), alcoholic liver disease (ALD), reactive
oxygen species (ROS), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH)
Introduction
Alcoholic liver disease (ALD) is primarily due to ethanol-mediated injury of the liver that
leads to the accumulation of fats, inflammation, and reactive oxygen species (ROS). ALD
constitutes a significant public health concern in the United States, where it is estimated to affect
over 14 million people (Orman et al., 2013). Alcohol (ethanol)-induced fatty liver generally begins
as hepatic steatosis, which is characterized by an excessive buildup of lipid droplets in hepatocytes.
Overtime, continued consumption of high levels of ethanol can lead to steatohepatitis and cirrhosis.
The molecular mechanisms underlying the progression of ethanol-mediated disease are thought to
be attributed to the combination of increased oxidative metabolites produced by the metabolism
of ethanol, the inflammatory response, gut microbiota, and the increase in lipopolysaccharide, and
the innate immune system (Dhanda et al., 2012). Interestingly, polyphenols, the most abundant
antioxidants in our daily diet, may have protective effects against ethanol-induced liver injury,
possibly via anti-inflammatory and antioxidant activity. Previous investigations suggest that the
chemical and biological properties of polyphenols are involved in activating mechanisms of
hepato-protection against ethanol-induced oxidative damage (Ko et al., 2011; Tian et al., 2012).
Additionally, several phenolic compounds have been reported to act as anti-inflammatory agents
that have indirect antioxidative activity via mechanisms of upregulating antioxidant enzymes that
respond to oxidative stress (Chen and Kong, 2004).
Building evidence suggests that dihydromyricetin (DHM), a bioactive flavonoid extracted
from Hovenia dulcis, has a broad range of beneficial properties, including antioxidant activity
(Okuma et al., 1995), antitumor activity, and free radical scavenging capacities which can aid in
34

the reduction of lipid peroxidation (Jiang et al., 2014; Hou et al., 2015). Furthermore, there is
cumulating evidence supporting the use of DHM for the treatment of alcohol use disorder (AUD)
and the possible reduction/prevention of ALD in animal models. For example, Shen and colleagues
found that DHM potentiates GABAA receptors and reduces the effects of ethanol on the same
receptors. This activity resulted in a DHM reduction of ethanol intoxication as well as a reduction
of alcohol withdrawal syndrome in rats (Shen et al., 2012). DHM administration has also been
found to reduce ethanol-dependent lipid accumulation in the liver via mechanisms of increased
autophagy and reducing inflammatory responses, further supporting the beneficial effects of DHM
on ethanol and chemical-induced outcomes (Fang et al., 2007; Qiu et al., 2017, 2019).
The hepatoprotective effects of DHM may be linked to its ability to protect cells against
inflammatory responses and oxidative species (Hou et al., 2015; Liang et al., 2015). In human
umbilical vein endothelial cells (HUVEC) and HepG2 hepatoblastoma (HB) cells, DHM has been
shown to reduce ROS and oxidative stress via regulatory mechanisms and its ability to scavenge
radicals (Hou et al., 2015; Xie et al., 2016). DHM has also been shown to protect HUVECs from
oxidative stress damage by altering mitochondrial apoptotic pathways involving Bcl-2, Bax, and
the activation of caspase-9/caspase-3, meanwhile inducing autophagy and reducing lipid
accumulation and lipogenesis in vitro (Hou et al., 2015; Liang et al., 2015; Xie et al., 2016). The
proposed protective effects of DHM have also been attributed to its electrophilic properties and
dissociation of Nrf2 from Keap1, thereby promoting the expression and activity of antioxidant
mechanisms (Qiu et al., 2017; Chu et al., 2018). Collectively, these data support the development
of DHM as a dietary supplement to help reduce the consequences of oxidative stress and lipid
metabolism and to promote liver health. However, the liver-protective mechanisms of DHM are
still not well understood. In addition, much of the earlier findings were based on findings from
35

HepG2 HB cell lines that lack enzymes capable of oxidatively metabolizing ethanol. The current
study tests the hypothesis that DHM supplementation enhances ethanol metabolism and reduces
ethanol-mediated lipid dysregulation, thus promoting hepatocellular health. This was
accomplished by investigating the molecular mechanisms related to DHM activity using ethanol-
exposed HB cells capable of oxidatively metabolizing ethanol, and an in vivo forced drinking
mouse model to evaluate the intracellular mechanisms that contribute to hepatoprotection.
Materials and Methods
Chemicals and Reagents
DHM: [(2R, 3R)-3, 5, 7-trihydroxy-2-(3, 4, 5-trihydroxyphenyl)-2,3-dihydrochromen-4-
one], MW 320.25, HPLC grade, >98% (Master Herbs Inc., Pomona, CA) was used in this study.
Penicillin-streptomycin, fetal bovine serum (FBS), minimum essential medium (MEM),
phosphate-buffered saline (PBS), and Dulbecco’s Phosphate-Buffered Saline and Tween 20 were
purchased from ThermoFisher (Life Technologies, Foster City, CA). Dimethyl sulfoxide (DMSO)
and 200 proof pure ethyl alcohol were purchased from Sigma-Aldrich (St. Louis, MO).
Animals and Experimental Design
Thirty-two 6-week old male C57Bl/6J mice were purchased from Jackson Laboratories
(Bar Harbor, ME). Mice were housed in temperature, light, and humidity-controlled conditions
with a 12-h light/dark cycle. The mice were randomized into four groups and acclimated by single
housing for one week prior to one week of daily injections of DHM (5 mg/kg or 10 mg/kg) or
saline before the start of the experiment. Doses of DHM (5 and 10 mg/kg) were selected based on
previous studies that identified beneficial effects of 10 mg/kg (i.p.) against dopaminergic injury,
with the addition of 5 mg/kg to evaluate the potential for a lower dose in hepatoprotection (Ren et
al., 2016). The groups were organized as follows: 1) Water-fed + daily saline intraperitoneal (i.p).
36

injections (n=6), 2) EtOH-fed + daily saline i.p. injections (n=6), 3) EtOH-fed + daily DHM i.p.
injections (5 mg/kg; n=10), and 4) EtOH-fed + daily DHM i.p. injections (10 mg/kg; n=10).
Following a forced drinking ad libitum feeding protocol (Tsukamoto et al., 1990; Keegan et al.,
1995; Brandon-Warner et al., 2012),mice were provided single bottle access to ethanol, gradually
increasing the percentage of ethanol from 5 – 10%, then 20% every 4 days until reaching 30%
ethanol (day 13 thru day 56). Mice were maintained on 30% ethanol single bottle access every day
for 6 weeks after the two-week period of gradually increasing the ethanol concentration provided.
Throughout the study, mice were administered DHM or saline 5 days a week via i.p. injection.
Mice in the DHM group received either 5 mg/kg or 10 mg/kg of DHM throughout the entire study.
Simultaneously, mice in the control groups were provided equivalent volumes of saline control via
i.p. injection and provided either water (control groups) or the equivalent concentrations of ethanol
throughout the study. All ethanol containing bottles were replaced with fresh ethanol every day to
ensure high concentrations of ethanol by volume. All experimental procedures were approved by
the USC IACUC committee, and all methods were carried out in accordance with relevant
guidelines and regulations. At the end of the experimental period, the mice were euthanized via
CO2 and cervical dislocation. All organs of all mice were immediately weighed after euthanization
and organ harvesting. The serum was prepared by centrifugation at 1000 x g at 4°C for 10 min and
was measured immediately or stored at -20°C for subsequent biochemical detection. The livers
were immediately dissected and then fixed in 10% neutral buffered formalin for histopathological
examination. The remainder of the fresh liver tissue was snap-frozen in liquid nitrogen, followed
by preservation at -80°C until utilized.
Determination of serum biomarkers (AST, ALT, Triglycerides, and BDNF)
37

The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in
the serum samples were measured using the Sigma ALT and AST Activity Assay Kit (St. Louis,
MO) and read using the BioTek Synergy H1 Hybrid Multi-Mode Reader plate reader (BioTek,
Winooski, VT). Serum triglyceride content was measured using the Cayman Chemical
Triglyceride Colorimetric Assay kit (Cayman Chemical, Ann Arbor, MI). Serum BDNF was
measured using the R&D Systems (Minneapolis, MN, USA) Total BDNF Quantikine ELISA Kit
and following the kit protocol and guidelines for serum tissue analysis.
Determination of hepatic triglycerides
Snap frozen liver tissues were prepared according to the Cayman Chemical Triglyceride
Colorimetric Assay kit protocol. In short, snap-frozen liver samples were weighed and cut out to
measure a 350 mg portion. The liver sample was minced in 2 mL of the kit provided diluted NP40
substitute assay reagent containing protease inhibitor cocktail. The samples were then centrifuged
at 10,000 x g for 10 minutes at 4°C, and the supernatant was stored on ice or at -80°C for longer
storage. The samples were further diluted using the NP40 substitute assay reagent before running
samples.
Hepatic histopathological evaluation and examination of biomarkers
Liver sections were fixed in 10% neutral buffered formalin solution for a minimum of 24h,
embedded in paraffin wax, sectioned at 5-μm thickness, stained with hematoxylin-eosin (H&E)
and digitally photographed with a light microscope at a total magnification of 200 X. Anti-
CYP2E1 polyclonal antibody was purchased from Abcam (Cambridge, MA) and detected using
an Alexa Fluor 594 secondary anti-rabbit antibody and coverslipped using Vectashield DAPI (4′6-
diamidino-2-phenylindole 2HCl, Vector Labs) DAPI mounting media for IHC detection.
Cell culture and sample treatment
38

The HepG2 human HB cell line was kindly provided by Dr. Bangyan L. Stiles (USC School
of Pharmacy, Los Angeles, CA). VL-17A cells were kindly provided by Dr. Dahn L Clemens
(University of Nebraska Medical Center and Veterans Affairs Medical Center, Nebraska USA).
HepG2 cells were cultured in MEM medium with 10% FBS, 5% penicillin-streptomycin, and
grown in an atmosphere containing 5% CO2 at 37°C. VL-17A cells were cultured in MEM
medium with 10% FBS, 5% penicillin-streptomycin, 400 µg/ml zeocin, 400 µg/ml G418, and
grown in an atmosphere containing 5% CO2 at 37°C. HepG2 cells and VL-17A cells, expressing
both ADH and CYP2E1, were incubated with vehicle (0.02% DMSO), ethanol (50 mM – 200 mM
EtOH), DHM (100 nM – 50 µM, dissolved in DMSO) for 2 – 72 hours or co-treated with ethanol
(50 mM – 200 mM) and DHM (100 nM - 50 µM) for 2-72 hours. All cell plates cultured in ethanol
conditions were replaced with fresh ethanol-containing medium daily and wrapped with parafilm
to reduce ethanol and acetaldehyde evaporation and stabilize conditions of acute exposure.
Similarly, naïve cells utilized as controls for these ethanol studies were also wrapped with parafilm
to normalize cell plating and treatment conditions. All acetaldehyde assays and experiments were
conducted at 4ºC to reduce acetaldehyde evaporation and reduce variability between samples. We
proceeded with subsequent experiments under this condition. The maximal concentrations of
ethanol that we tested were based on previous work that identified concentrations of 50 – 100 mM
ethanol as causing minor effects on HepG2 viability, and concentrations of 200 mM ethanol and
above causing significant damage to cell viability (Castaneda and Kinne, 2004; Liu et al., 2014;
Xie et al., 2016). The maximal in vitro concentration of DHM tested in this experiment was 50
μM, as DHM concentrations above 50 μM induces cell death in HepG2 hepatoblastoma cell lines
(Liu et al., 2014).

39

Serum ethanol and acetaldehyde measurements
Eighteen 14-week old C57BL/6J male mice (Jackson Laboratories) housed in temperature,
light, and humidity-controlled conditions with a 12-h light/dark cycle were separated into three
groups and administered a single injection as follows: 1) 3.5 g/kg ethanol i.p, injection 2) 3.5 g/kg
ethanol + DHM (5 mg/kg) i.p. injection, and 3) 3.5 g/kg ethanol + DHM ( 10 mg/kg) i.p. injection
(6 mice per group). Mice were administered ethanol via i.p. injection to ensure constant
concentrations of ethanol in all mice for accurate comparisons between groups. All mice were
euthanized via CO2 and cervical dislocation 45 minutes post-injection, and whole blood samples
were collected, stored at room temperature for thirty minutes, and separated by refrigerated
centrifugation for 10 minutes at 2,000 x g. All samples were stored on ice immediately after
separation. Serum samples were analyzed immediately after separation using the Ethanol and
Acetaldehyde Assay Kit (Megazyme, Bray, Ireland) and H1 Hybrid Multi-Mode Reader Plate
(BioTek, Winooski, VT) according to the manufacturer’s guidelines. All acetaldehyde
measurements were conducted on ice to keep samples cold and preserve acetaldehyde
concentrations in solution.
Hepatic NAD
+
/NADH Measurements
Total NAD
+
and NADH concentrations were measured using a BioVision NAD
+
/NADH
Quantification Kit. Briefly, 20 mg of liver tissue was weighed, washed in cold 1X PBS,
homogenized in 400 μL of NADH/NAD extraction buffer, and centrifuged at 14000 rpm for 5 min
and extracted NADH/NAD was transferred to a new tube. To decompose NAD, and measure the
total NADH, an aliquot of the extracted NAD/NADH samples was heated at 60°C for 30 minutes
using a water bath, and then kept on ice for immediate evaluating following the protocol
guidelines.
40

Ethanol and Acetaldehyde Measurements
Ethanol concentration and acetaldehyde production were measured using an Ethanol and
Acetaldehyde Enzyme Assay Kit (Megazyme, Bray, Ireland) in 96-well plates. Acetaldehyde
ammonia trimer and ethanol were used as the standard according to the manufacturer’s instructions
for the acetaldehyde assay and ethanol assay, respectively. VL-17A cells and HepG2 cells were
incubated with 50 mM ethanol and either 100 nM – 50 µM DHM, 0.2% DMSO, or untreated for
two hours before measurements using the BioTek Synergy H1 Hybrid Multi-Mode Reader plate
reader (BioTek, Winooski, VT).
Measurement of intracellular reactive oxygen species (ROS) and Cytoxicity
Intracellular ROS generation was evaluated using the cell-permeant ThermoFisher
CellROX Deep Red Reagent fluorogenic probe kit. HepG2 and VL-17A cells (10 x 10
3
cells/well)
were seeded in 96-well plates and incubated with 50 – 100 mM ethanol, DHM, and ethanol with
DHM for 24 hours. After the incubation, CellROX Reagent was added to a final concentration of
5 µM to the cells and incubated for 30 minutes at 37°C. After incubation with CellROX, medium
and reagent were removed, and cells were washed three times with 1X PBS and measured
fluorometrically using a BioTek Synergy H1 Hybrid Multi-Mode Reader plate reader. Similar to
the design of the ROS measurement assay, cytotoxicity was evaluated using the Promega
Mitochondrial ToxGlo Assay kit (Southampton, UK), a cell-based assay that measures cytotoxicity
via a fluorogenic peptide substrate (bis-AAF-R110) and evaluated for fluorescence according to
the manufacturer’s protocol 24 hours after treatment conditions.
Measurement of Intracellular Lipid Accumulation
Intracellular lipid accumulation was assessed using Cayman’s Steatosis Colorimetric
Assay Kit with dye extraction, according to the manufacturer's protocol. HepG2 and VL-17A cells
41

(10 x 10
3
cells/well) were seeded in 96-well plates and incubated with various concentrations of
ethanol, DHM, and ethanol with DHM for 72 hours. Lipid accumulation was quantified using the
BioTek Synergy H1 Hybrid Multi-Mode Reader plate reader.
Immunodetection of Serum and Hepatic Cytokines
Relative levels of detected cytokines in the serum and liver of mice were measured using
a cytokine array kit. Briefly, sera were collected and centrifuged (2000 x g) for 10 min. The
supernatant was subjected to a cytokine array that measures 40 different mouse cytokines,
chemokines, and acute-phase proteins following the manufacturer’s instructions (R&D Systems;
Minneapolis, MN, USA). For liver analyses, small pieces of frozen liver samples were rinsed in
ice-cold 10 mM Tris-HCL (pH 7.4) and homogenized with RIPA lysis buffer containing fresh
cocktail protein inhibitors. The mixture was centrifuged at 12,000 x g for 15 min, and the
supernatant was subjected to cytokine array following manufacturer guidelines. For each array,
supernatants from four individual animals were pooled, and three arrays were performed per
condition. Finished membranes were exposed for 10 minutes to chemiluminescent detection
reagents, and the chemiluminescent signal was captured using a ChemiDoc (Bio-Rad) imaging
device. Densities were measured using ImageJ, with a fixed circular area placed over the grid-
identified location for each cytokine and chemokine. The p-value for the fold differences had to
be ≤ 0.05. Heat maps were generated after normalization of the data of the measured densities. The
normalized data were then expressed as fold changes of the control value and thus, for each
cytokine, with a color gradient from low to high concentrations (green to red).
Mitochondrial Isolation
Mitochondrial from liver tissue was isolated using the Abcam Mitochondria Isolation Kit
for Tissue and following the manufacturer guidelines and protocol. Briefly, liver tissue was washed
42

with washing buffer, homogenized in isolation buffer, and centrifuged at 1000 x g for 10 min. The
supernatant was centrifuged again at 12,000 x g for 15 min. Pellets (mitochondrial fraction) were
washed with isolation buffer containing protease inhibitor cocktail (Calbiochem) twice and
resuspended with isolation buffer with protease inhibitor cocktail. Mitochondria were then
quantified by BCA assay and for protein expression using Western blot.
Protein extraction and Western blot analysis
Small pieces of frozen liver samples were rinsed in ice-cold 10 mM Tris-HCL (pH 7.4)
and homogenized with RIPA lysis buffer containing fresh cocktail protein inhibitors. The mixture
was centrifuged at 12,000 x g for 15 min, and the supernatant was kept as the total protein
extractant at -80 °C. HepG2 and VL-17A cells (9 x 10
5
cells/dish) were seeded in 100 mm dishes
and treated with either the indicated concentrations of ethanol, DHM, or both ethanol and DHM
for 24 hours. Cell lysates were prepared using a 1% Triton-X 100 lysis buffer containing protease
and phosphatase inhibitors (Calbiochem). Cell extracts were quantified using the BCA Protein
Assay kit (Pierce Biotechnology, Rockford, IL) according to the manufacturer’s instructions. 50
µg of proteins were separated on a 4 – 20% sodium dodecyl sulfate polyacrylamide gel
electrophoresis and transferred to PVDF membranes for Western blot analysis (Bio-Rad
Laboratories, Hercules, CA). Transferred membrane was blocked with blocking buffer containing
5% skim milk (Bio-Rad) in 1X Tris-buffered saline with Tween 20 (Thermofisher) for 1 hour and
then incubated with primary antibodies (p-AMPK, AMPK, ADH1, ALDH1A1, ALDH2 Nrf2,
HO-1,p-ACC1, total ACC1, CPT1a, SREBP1, TNF-α, 4-HNE, VDAC, and IL-8) at appropriate
dilutions in 1X TBST overnight at 4°C. The membrane washed three times with 1X TBST for 10
minutes and incubated with secondary antibody in 1X TBST for 1 hour, and the images were
visualized with enhanced chemiluminescence detection reagent and Chemi-Doc (Bio-Rad)
43

imaging device. Anti-IL8 and anti-SREBP1 monoclonal antibodies were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). All other primary and secondary antibodies were purchased
from Cell Signaling (Beverly, MA). All trials were repeated in triplicates to confirm changes in
protein expression. Densitometry analysis was performed using the ImageJ Gel Analysis Tool and
normalized against untreated controls.
Data Analysis
All cellular experiments were performed in triplicates. Animal biochemical analyses were
conducted using 6 – 8 separate samples from mice groups, or 4 separate samples for control groups.
The data are presented as mean ± standard deviation. Statistical analysis included 2-way analysis
of variance followed by Bonferroni multiple comparison test using Prism 6 (GraphPad Software,
Inc., La Jolla, CA). Differences among groups were stated to be statistically significant when p ≤
0.05.
Results
DHM reduces ethanol-induced liver steatosis and triglyceride accumulation in the liver
DHM Attenuates Ethanol-Induced Liver Steatosis and Triglyceride Accumulation in the Liver
All ethanol-fed mice consumed an average of 39.43 g/kg of ethanol a day (Data S1A), with
no significant differences in ethanol intake, total fluid intake, food intake, or bodyweight between
all groups (Data S1). H&E staining results of mice chronically fed ethanol showed that ethanol-
induced hepatic lipid dysregulation based on the observation of swollen hepatocytes, hepatic
microvascular congestion, and macrosteatosis (Fig 1A). DHM was found to significantly reduce
liver histopathological changes induced by chronic ethanol consumption (Fig 1A; 5 and 10 mg/kg
DHM). Additionally, the administration of DHM (10 mg/kg) significantly reduced the ethanol-
induced changes in liver mass (Fig 1B; p<0.01; n=8) and hepatic triglyceride content (Fig 1C;
44

p<0.01; n=8). Furthermore, DHM administration significantly reduced triglyceride levels found in
serum (Fig 1D; 5 mg/kg p<0.05 and 10 mg/kg p<0.01; n=8).

Fig 1. DHM ameliorates ethanol (EtOH)‐induced pathomorphology and hepatic/serum
triglyceride levels in EtOH‐fed mice. (A) H&E (hematoxylin and eosin) staining confirmed that
DHM remarkably alleviated EtOH‐induced lipid deposition (white arrows). (B) EtOH‐fed mice
had a significantly larger liver mass compared against control (**p < 0.01; n = 8; 2‐way
ANOVA). DHM administration at 10 mg/kg significantly reduced the EtOH‐mediated hepatic
mass increase (**p < 0.01; n.s. between DHM groups and between DHM and control; n = 8; 2‐
way ANOVA), and (C) both 5 and 10 mg/kg significantly reduced triglyceride levels in the liver
(**p < 0.01; n = 6/group). (D) Serum triglycerides were significantly elevated in EtOH‐fed mice
45

and significantly reduced in mice administered both 5 and 10 mg/kg of DHM (**p < 0.01; no
significant difference between 10 mg/kg DHM [E + 10D] and control; n = 7; 2‐way ANOVA).
Data represented as mean ± SEM. ** p < 0.01 compared with corresponding EtOH controls;
n.s. = no significance; E = EtOH; D5 = 5 mg/kg DHM; D10 = 10 mg/kg DHM.
DHM reduces ethanol-induced intracellular lipid accumulation and mature SREBP-1
expression in vitro
DHM reduces the expression of the lipogenic transcription factor, sterol regulatory element-
binding protein (SREBP)-1.
HepG2 and VL-17A cells cultured in 50 – 200 mM ethanol for 24 hours resulted in a
marked increase in the expression of mature SREBP-1 relative to the untreated samples. With the
treatment of DHM, there was a significant reduction in the expression of the mature SREBP-1
protein during the 24-hour ethanol treatment period in HepG2 and VL17-A cells (Fig 2A & Data
S2A; **p<0.001; n=3).
DHM significantly reduces lipid accumulation in HepG2 and VL-17A in vitro models
HepG2 and VL-17A cells cultured in either 50 or 100 mM ethanol for 72 hours resulted in a
significant increase in intracellular lipids (Data S2B; **p<0.01). Treatment with DHM
significantly decreased intracellular lipid accumulation in both HepG2 and VL-17A cells (Data
S2B; *p<0.05 and **p<0.01, respectively). To further assess these changes, Nile Red staining of
HepG2 and VL-17A cells in either 50 or 100 mM ethanol and DHM were imaged. As presented,
50 and 100 mM ethanol resulted in much higher lipid stained droplets (red) than the DHM (5 μM)
co-treated counterpart in both HepG2 and VL-17A (Data S2C).
Furthermore, HepG2 and VL-17A cells showed significant accumulation of lipids in 50 and 100
mM ethanol co-treated with free fatty acids (FFAs) when compared against controls treated with
46

FFA but no ethanol (Fig 7B; **p<0.01). With 5 µM DHM co-treatment in 50 and 100 mM ethanol,
both cell lines showed a significant reduction in lipid accumulation when incubated with FFAs
(Fig 7B; **p<0.01) with a dose-dependent effect observed in HepG2 cells.

Fig 2. DHM directly reduces ethanol (EtOH)‐mediated mature SREBP‐1 expression and lipid
uptake in EtOH oxidizing (VL‐17A) and nonoxidizing (HepG2) cell lines. (A) Representative
WB image of HepG2 and VL‐17A cells cultured in EtOH and treated with 5 µM DHM or
untreated for 24 hours and immunoblotted with anti‐SREBP1 mAb (See Fig. S2A for ImageJ
quantification of triplicates). (B) HepG2 and VL‐17A cells were cultured in 50 or 100 mM
EtOH + 4 mM free fatty acids (2:1 Oleic to Palmitic acid) and either untreated or treated with
either 2.5 or 5 µM DHM for 72 hours before photometric detection of intracellular lipid
accumulation. Data represented as mean ± SEM. *p < 0.05 and **p < 0.01 compared with EtOH
controls. †p < 0.05 versus untreated control; n = 3. A.U., arbitrary units.
47

DHM administration results in the activation of AMPK and the inhibition of downstream lipid
processes
To identify DHM pharmacological mechanisms, we evaluated the activation state of
adenosine monophosphate-activated protein kinase (AMPK), via phosphorylation at threonine
(Thr)-172, and its downstream pathway involved in inhibiting FFA synthesis and activating lipid
transport. In male C57BL/6J mice administered DHM for nine weeks, we found that the
phosphorylation of AMPK at Thr172 was significantly increased relative to total AMPK
expression in the liver (Fig 3 & Data S3; *p<0.05; n=3/group). Direct phosphorylation of acetyl
CoA carboxylase 1 (ACC1) at the serine 79 (Ser79) residue by activated AMPK was also
significantly increased with DHM administration, suggesting that the increased activation of
AMPK results in the direct inhibition of ACC1, thereby resulting in reduced FFA synthesis (p-
ACC1; *p<0.05 and **p<0.01; n=3). Furthermore, the administration of DHM resulted in
significantly higher expression of carnitine palmitoyltransferase-1 (CPT1a), a mitochondrial outer
membrane protein regulated by AMPK that translocates long-chain fatty acids across the
membrane (Fig 3 & Data S3; **p<0.01; n=3). Collectively, these data suggest that DHM activates
AMPK via phosphorylation at the Thr172 site and results in lipid metabolic responses that inhibit
FFA synthesis and increases fatty acid translocation to the mitochondria for lipid oxidation.

Fig 3. DHM administration counteracts EtOH‐mediated inhibition of AMPK and downstream
lipid metabolic responses. Representative WB of phosphorylated AMPK (p‐AMPK [Thr172]),
48

total AMPK, phosphorylated ACC1 (p‐ACC1 [Ser79]), total ACC1, total CPT1a, and β‐actin
loading control. EtOH‐fed mice showed a significant reduction in p‐AMPK (Thr172) and
increase in p‐ACC1 (Ser79) relative to water‐fed controls (Ctl). DHM administration at both 5
and 10 mg/kg resulted in a significant increase in p‐AMPK (Thr172) and CPT1a expression, and
a significant reduction in p‐ACC1 (Ser79) relative to EtOH‐fed mice and water‐fed mice (Ctl).
The Western blot images are representative of Western blots obtained from 3 different biological
experiments. n = 3/group. See Fig. S3 for ImageJ quantification of triplicates.
DHM attenuated the ethanol-induced hepatic enzyme release and expression of pro-
inflammatory cytokines
Serum Markers of Liver Injury and Circulating Cytokines
Mice chronically fed ethanol had a significantly higher level of AST and ALT activity in
serum relative to water controls, suggesting liver injury (Fig 4A; **p<0.01; n=6/group).
Administration of DHM at 5 and 10 mg/kg significantly reduced the measured activity of serum
ALT and AST in ethanol-fed mice (Fig 4A; **p<0.01; n=6/group).
To assess the extent of ethanol-mediated injury, we evaluated the expression of cytokines
circulating in the sera of C57BL/6J mice. Interestingly, we found that markers of pro-inflammatory
cytokines TNF-α, interleukin (IL)-1α, interferon (IFN)-γ, TIMP metallopeptidase inhibitor 1
(TIMP1), and macrophage colony-stimulating factor (M-CSF) were reduced dose-dependently
with DHM treatment (Fig 4B; *p<0.05; n=4). Likewise, a reduction in chemokines such as
CXCL2, CCL9, CCL1, and levels of the circulating endothelial cell/leukocyte adhesion molecule,
intercellular adhesion molecule-1 (ICAM-1)/CD54 were reduced with treatment of DHM.
Therefore, the reduction in circulatory cytokines and chemokines involved in the activation of pro-
49

inflammatory activation are reduced with administration of DHM in a dose-dependent response
(Fig 4B; *p<0.05; n=4/group).
Hepatic Markers of Liver Injury and Inflammation
Protein expression of TNF-α was significantly elevated in mice chronically fed ethanol for
8 weeks relative to water controls (Fig 4C & Data S4A; **p<0.01; n = 3). However, when ethanol-
fed mice were administered DHM at both 5 and 10 mg/kg, we observed a significant reduction in
the protein expression of hepatic TNF- α (Fig 4C & Data S4C; **p<0.01; n=3).
Using a cytokine protein array, we found that ethanol-fed mice showed significant elevations of
hepatic CCL21, CCL6, ICAM, endostatin, IGFBP-2 complement component 5a (C5a), C-reactive
protein (CRP), and dipeptidyl peptidase (DPP)-4 (Fig 4D). Similar to our findings in serum, we
observed a DHM-dependent decrease in hepatic cytokines, chemokines, and proinflammatory
mediators, including IFN-γ, CCL21, DPP-4, CRP, and C5a.
DHM Ameliorates Ethanol-Mediated Reductions of serum BDNF
To further understand the extent of DHM-mediated anti-inflammatory benefits (Fig 4 &
Data S2), we investigated the changes in BDNF levels in mice sera. As illustrated in Fig. 4E,
chronic ethanol feeding resulted in a significant reduction of serum BDNF (*p<0.05; n = 6/group).
Furthermore, we found that DHM administration at both 5 and 10 mg/kg reversed the ethanol-
mediated reduction of serum BDNF (Fig 4E; *p<0.05; n = 6/group).
50

Fig 4. DHM significantly reduces hepatic enzyme release, exhibits dose‐dependent
antiinflammatory actions, and maintains serum BDNF levels. (A) DHM administration at both 5
and 10 mg/kg significantly inhibited the activities of serum ALT and AST comparable to control
values (n = 6/group). (B) DHM dose‐dependently decreased serum cytokine markers compared
to untreated ethanol (EtOH)‐fed mice serum (n = 4/group). (C) DHM administration, at both 5
and 10 mg/kg, significantly reduced EtOH‐mediated hepatic TNF‐α expression relative to EtOH‐
only controls (n = 3/group; see Fig. S4A for ImageJ quantification of triplicates). (D) Hepatic
51

cytokine analysis of mice chronically fed EtOH and either treated with DHM (5 or 10 mg/kg) or
untreated (n = 4/group). E) DHM administration at both 5 and 10 mg/kg significantly reversed
EtOH‐mediated reductions in serum BDNF concentrations (n = 6/group). Color key and
histogram illustrate the intensity of red being associated with larger fold increases in expression
relative to normalized control values (white), and intense greens are associated with fold
decreases (0.5‐fold the lowest) relative to normalized control values. Blue solid lines indicate
average fold values relative to control (dotted blue line). Data represented as mean ± SEM.
*p < 0.05 compared with corresponding EtOH controls. *p < 0.05 and **p < 0.01 compared with
corresponding EtOH controls. E = EtOH; D5 = 5 mg/kg DHM; D10 = 10 mg/kg DHM.
DHM Directly Suppresses the Ethanol Mediated Intracellular Expression of Inflammatory
Markers, Activated Caspase-3, and cytotoxicity using in vitro models
Through a series of in vitro investigations, we analyzed the isolated effects of DHM
treatment on ethanol-mediated hepatocellular expression of proinflammatory cytokines,
interleukin (IL)-8, and TNF – α, by Western blot. Additionally, the pro-apoptotic marker, cleaved
caspase-3, was evaluated in both cell lines. As illustrated in Data S4B, we found an ethanol-
dependent increase in TNF-α, IL-8, and a dose-dependent increase in the protein expression of
cleaved caspase-3 (Data S4B). DHM treatment (5 μM) resulted in significant reductions in the
expression of these markers. Furthermore, we found a significantly higher magnitude of cell death
associated with 100 mM ethanol in comparison to 50 mM (Data S4C; *p<0.05), and these effects
were ameliorated with DHM treatment.
DHM significantly enhanced the activity and expression of alcohol dehydrogenase (ADH) and
aldehyde dehydrogenase (ALDH) in isolated HB cell models and in vivo
52

DHM effects on ethanol metabolism were measured in both HepG2 and VL-17A cells
(Data S5). DHM was tested at concentrations ranging from 0.1 µM to a maximum of 50 µM. We
found a significant reduction in both ethanol and acetaldehyde (Data S5 A-C; *p<0.05)
concentrations in both cell lines when incubated with 50 mM ethanol and DHM for two hours.
Notably, 1 – 10 µM DHM significantly enhanced ethanol and acetaldehyde metabolism resulting
in a reduction of measured extracellular concentrations of ethanol (Data S5B) and acetaldehyde
(Data S5C; **p<0.01). Furthermore, 10 µM DHM significantly increased the production of acetic
acid in VL-17A cells relative to ethanol only controls and higher concentrations of DHM (Data
S5D; *p<0.05).
DHM Reduced Blood Ethanol and Acetaldehyde Concentrations in C57BL/6J Mice Serum and
Reversed Ethanol-Mediated Depletion of Nicotinamide Adenine Dinucleotide (NAD
+
)
A follow-up study was conducted in 14-week old C57BL/6J mice (Fig 5A & B). Mice
administered DHM simultaneously with ethanol exhibited significantly reduced ethanol and
acetaldehyde concentrations relative to ethanol controls 45 minutes post injections (Fig 5A & B;
*p<0.05 and **p<0.01; 2-way ANOVA).
We assessed the levels of NAD
+
relative to NADH in the liver (Fig 5C & D) in an effort to identify
mechanistic information regarding DHM effects on metabolic activity. Mice administered 5 and
10 mg/kg DHM with chronic ethanol feeding showed higher NAD
+
concentrations compared to
ethanol-fed and water-fed controls (Fig 5C; *p<0.05 5 and 10 mg/kg; 2-way ANOVA). Likewise,
an elevated concentration of NAD
+
to NADH (NAD
+
/NADH) ratio was observed in both doses of
DHM, with 10 mg/kg showing a significant increase in NAD
+
/NADH ratios (Fig 5D; *p<0.05 10
mg/kg; 2-way ANOVA).

53

DHM increases ethanol metabolizing enzyme expression in vitro and in vivo
Evaluation of DHM on the protein expression of ADH1, ALDH2, and ALDH1A1 ethanol
metabolizing enzymes in vitro are displayed in Data S6. Interestingly, treatment of HepG2 cells
with 5 µM DHM and the corresponding ethanol concentrations induced a higher expression level
of both ADH1 and ALDH1A1, with ALDH1A1 expression being significantly higher (Data S6A;
**p<0.01). Similarly, the treatment of DHM with ethanol incubation in VL-17A cells resulted in
a significant dose-dependent expression of ALDH2 (Data S6B; *p<0.05).
We next assessed the protein expression of both ADH1 and ALDH2 ethanol metabolizing enzymes
in the livers of ethanol-fed mice in comparison to those treated with DHM (Fig 5E). Similar to in
vitro findings (Data S6), we found that DHM administration resulted in the significant elevation
of both ADH1 and mitochondrial ALDH2 enzymes in the liver (Fig 5E; **p<0.01; n=4).

54

Fig 5. DHM reduces serum ethanol (EtOH) and acetaldehyde (ACH) concentrations in mice
administered 3.5g/kg EtOH, reverses chronic EtOH‐mediated depletion of hepatic NAD
+
levels,
and induces hepatic ADH1/ALDH2. Serum (A) EtOH and (B) ACH concentration differences
measured in 16‐week‐old mice injected with 3.5 g/kg EtOH and DHM (5 or 10 mg/kg)
45 minutes after injections (*p < 0.05 and **p < 0.01; n = 6/group, 2‐way ANOVA). (C) Mice
chronically fed EtOH for 8 weeks show significantly less NAD
+
concentrations in the liver than
water‐fed controls (*p < 0.05; n = 6/group; 2‐way ANOVA). Mice administered DHM at both 5
and 10 mg/kg showed elevated NAD
+
concentrations relative to EtOH‐fed controls and water‐fed
controls (*p < 0.05, compared to EtOH controls and †, *p < 0.05, compared to water‐fed
controls). (D) Hepatic NAD
+
/NADH ratio showing a significant increase in the ratio of mice
treated with 10 mg/kg DHM (*p < 0.05). (E) Representative Western blot images of hepatic
expression of ADH1 and ALDH2 in C57BL/6J mice chronically fed EtOH and either treated
with or without DHM. Data represented as mean ± SEM. E = EtOH, D5 = 5 mg/kg DHM, and
D10 = 10 mg/kg DHM.
DHM reduces the hepatic expression of CYP2E1 in mice chronically-fed ethanol and increases
the expression of Nrf2 and HO-1 antioxidant systems
DHM provided daily via i.p. injections significantly reduced the protein expression of
CYP2E1 (green) throughout the liver of ethanol-fed mice relative to no treatment (Fig 6A).
Furthermore, both doses of DHM administered via i.p. injection resulted in significant reductions
of CYP2E1 expression (Fig 6B; *p<0.05 E + 5D; **p<0.01 E + 10D; n=4). Our data also shows
that chronic ethanol feeding also resulted in a significant increase in the protein expression of Nrf2
(Fig 6C; *p<0.05; n=3). However, the administration of DHM resulted in higher protein expression
of Nrf2 compared to ethanol controls (Fig 6C; **p<0.01; n=3), suggesting induction of Nrf2. When
55

evaluating the protein expression of heme oxygenase (HO)-1, an antioxidant product of Nrf2
activation, we found that the expression was significantly reduced in the livers of mice chronically
fed ethanol (Fig 6D; **p<0.01; n=3). In contrast, administration of DHM resulted in a significant
dose-dependent increase in HO-1 production (Fig 6D; **p<0.01; n=3). Furthermore, DHM
administration significantly reduced the expression of 4-HNE (Fig 6E; n=3; **p<0.01 for 5mg/kg
and *p<0.05 for 10 mg/kg).

Fig 6. DHM reduces hepatic CYP2E1 expression and increases the hepatic expression of Nrf2
and HO‐1 antioxidant pathways. (A) Immunohistochemistry results of CYP2E1 in livers suggest
that DHM inhibited the expression of CYP2E1 in ethanol (EtOH)‐fed mice relative to EtOH only
(green = CYP2E1, blue = nuclei; n = 6/group). (B) Representative Western blot illustrating that
DHM significantly reduces the hepatic expression of CYP2E1 relative to untreated EtOH‐fed
mice (n = 3/group; *p < 0.05 5 mg/kg DHM and **p < 0.01 10 mg/kg DHM). (C) EtOH‐fed
56

mice showed a significant increase in Nrf2 relative to water controls (n = 3/group; *p < 0.05).
DHM (5 and 10 mg/kg) significantly increased the expression of Nrf2 in EtOH‐fed mice livers
relative to EtOH controls (n = 3/group; **p < 0.01). (D) EtOH‐fed mice displayed a significant
reduction in HO‐1 protein expression relative to water controls (n = 3/group; **p < 0.01). 5 and
10 mg/kg DHM significantly increased the expression of HO‐1 protein expression relative to
EtOH only (n = 3/group; **p < 0.01; significant difference between 5 and 10 mg/kg DHM,
*p < 0.05). (E) EtOH‐fed mice showed a significant increase in the hepatic expression of 4‐HNE
in comparison with water‐fed controls (n = 3/group; †p < 0.05). DHM (5 and 10 mg/kg)
significantly reduced the expression of 4‐HNE in the liver (n = 3/group; **p < 0.01 and
*p < 0.05, respectively). Bar graphs were generated by quantifying blots from 3 independent
experiments using ImageJ and normalized against intensity of the untreated lane. Data
represented as mean ± SEM. *p < 0.05 and **p < 0.01 compared with corresponding EtOH
controls; †, p < 0.05 versus water‐fed control. n = 3/group.
DHM suppresses ethanol-induced ROS generation in vitro
DHM Increases Expression of catalase, an antioxidant enzyme, and Reduces intracellular ROS
generation.
HepG2 and VL-17A cells were evaluated for changes in catalase expression when cultured
in ethanol and treated with DHM. Interestingly, we found no significant difference in catalase
expression when tested with 50 and 100 mM ethanol (Fig 7A). However, exposing the cell lines
to 5 µM DHM while being incubated in ethanol resulted in a significant increase in the cytosolic
expression of catalase (*p<0.05).
To assess changes in ROS, we exposed HepG2 and VL-17A cells to ethanol and treated
them with DHM (Fig 7B). ROS intensity was significantly increased in ethanol-treated cells
57

suggesting a significant increase in ethanol-induced ROS generation, with CYP2E1 expressing
VL-17A cells having higher ROS levels (Fig 7B; **p<0.01). Treatment with DHM for 24 hours
resulted in a significant decrease in ethanol-induced ROS generation (Fig 7B; *p<0.05 and
**p<0.01, respectively). At 100 mM ethanol, the 5 µM DHM treatment showed a greater effect in
reducing ethanol-induced ROS levels in both cell lines relative to 2.5 µM (Fig 7B; **p<0.01).

Fig 7. DHM increases the expression of catalase and suppresses ethanol (EtOH)‐mediated ROS
generation in vitro. (A) Representative Western blot image of HepG2 and VL‐17A cells cultured
in EtOH and treated with 5 µM DHM or untreated for 24 hours and immunoblotted with anti‐
Catalase mAb. (B) HepG2 and VL‐17A cells were cultured in 50 to 100 mM EtOH and treated
with 2.5 to 5 µM DHM or untreated for 24 hours before fluorometric analysis of intracellular
ROS levels. Bar graphs were generated by quantifying blots from 3 independent experiments
using ImageJ and normalized against intensity of the untreated lane. Data represented as
58

mean ± SEM. *p < 0.05 and **p < 0.01 compared with corresponding EtOH controls and
normalized with untreated controls; †p < 0.05 versus untreated control. n = 3/group. ROS,
reactive oxygen species. A.U., arbitrary units.
Discussion
Findings from this study support the hypothesis that DHM supplementation enhances
ethanol metabolism and reduces ethanol-mediated lipid dysregulation. The effects of DHM were
found to pharmacologically impact several key enzymatic functions involved in lipid metabolism,
anti-inflammation, ROS suppression, and ethanol metabolism. Furthermore, we unmasked several
intracellular mechanisms that resulted in the hepatoprotection observed in the livers of male
C57BL/6J mice chronically fed ethanol using a forced drinking ad libitum model. These findings
were further supported by a series of in vitro experiments that demonstrate that HB cell models of
human HepG2 and human VL-17A ethanol metabolizing cells are useful models for the study of
DHM mechanisms. This latter work also provided insights regarding the beneficial effects of DHM
in alleviating non-oxidative, HepG2, and oxidative, VL-17A, ethanol-mediated stress.
From our forced drinking rodent study, we found that all groups of male C57BL/6J mice consumed
equal amounts of ethanol (Data S1A & B) with no significant changes in fluid intake, food intake,
or % B.W. These levels of ethanol consumption were found to induce ethanol-mediated increases
in steatosis, hepatic microvascular congestion, and triglyceride levels in the liver (Fig 1A & B). In
contrast, daily administration of DHM significantly reversed lipid dysregulation. Therefore, the
changes in lipid metabolism observed in DHM treated mice could not be attributed to a reduction
in daily or weekly ethanol (g/kg) intake.
Using HepG2 and VL-17A cells, we found supporting evidence of DHM reducing ethanol-
induced lipid accumulations and identified a significant reduction in the expression of SREBP-1,
59

a transcription factor involved in fatty acid and lipid production, and FFA uptake (Fig 2). To better
elucidate this activity in vivo, we expanded our investigations on lipid metabolic signaling factors
in the liver of ethanol-fed mice that results in the inhibition of AMPK, an energy-sensing activator
of pathways that induce lipid breakdown and inhibition of lipid synthesis (García-Villafranca et
al., 2008; Galligan et al., 2012; Zeng et al., 2019). Similar to other polyphenolic compounds (Zang
et al., 2006), we found that DHM administration with ethanol feeding increased phosphorylation
at the Thr172 site of AMPK, thereby counteracting ethanol-related inhibition of AMPK and its
direct downstream products (Fig 3; Niu et al., 2012). These findings demonstrate that DHM
increases the activity of AMPK and its signaling/mediated effects on downstream lipid metabolic
pathways that are typically suppressed with chronic ethanol feeding. In total, this work indicates
that DHM administration reverses ethanol-mediated inhibition of hepatic AMPK and downstream
signaling mechanisms that results in lipid accumulation and stress. This action of DHM partly
explains the phenotypic reduction of lipids in the liver, and our in vitro findings of reduced
SREBP-1 expression.
Activation of AMPK, and the associated reduction in lipid accumulation/stress, is one
mechanism linked to decreases in hepatocellular stress and pro-inflammatory responses (Qiang et
al., 2016). In agreement with this finding, we found that DHM significantly reduced the levels of
serum ALT and AST in ethanol-fed mice, markers commonly associated with liver damage. The
extent of liver injury can also be evaluated by increased levels of pro-inflammatory markers such
as IL-8, TNF-α, and an associated increase in susceptibility to stress over different periods of
ethanol feeding or treatment (Hoek and Pastorino, 2002). We found that DHM significantly
reduced ethanol-mediated inflammatory responses via reductions in circulatory cytokines and
chemokines measured in serum (Fig 4B). Of these inflammatory markers, we found significant
60

dose-dependent reductions of IFN-γ, and TIMP1, IL-1 α, CXCL9, CCL2, IL-7, which have all
been associated with ALD, systemic inflammation, and fibrotic development in the liver (Das and
Vasudevan, 2007; Degré et al., 2012; Berres et al., 2015; Chen et al., 2015; Yilmaz and Eren,
2019). Furthermore, TNF - α, CCL-1, IL-13, and M-CSF were also found to be significantly
reduced with DHM administration.
Likewise, a reduction of cytokines and chemokines were observed in the liver, thereby
suggesting a significant effect of DHM mediating liver inflammation and alleviating ethanol-
induced inflammation (Figs 4 C & D and Data S4A). For instance, the significant dose-dependent
reduction of CRP in the liver suggests the hepatoprotective ability of DHM against a marker
proposed for liver damage and increased risks of liver cancer (Chen et al., 2015). Therefore, DHM
administration effectively reduces the early stages of ALD and the potential progression to
cirrhosis. Interestingly, we also found that chronic ethanol feeding induced a significant increase
in the hepatic expression of DPP-4, which has been reported to promote insulin resistance and
correlate with NAFLD (Baumeier et al., 2017; Zheng et al., 2017). Reductions in the ethanol-
mediated hyperexpression of DPP-4 illustrates the ability of DHM to mediate insulin resistance
associated with elevated hepatic DPP-4 and non-alcoholic fatty liver disease (NAFLD; Baumeier
et al., 2017). However, further evaluation is necessary to correlate this effect with ALD and
chronic ethanol feeding. In support of these findings, we found that DHM reduces cytotoxicity and
the expression of pro-inflammatory markers and the pro-apoptotic marker, caspase-3, in vitro
(Data S4). Furthermore, we assessed the serum levels of BDNF to expand our analysis of systemic
benefits in relation to the anti-inflammatory properties of DHM. Serum BDNF is reduced under
conditions of acute/chronic stress, including chronic alcoholic intake and chronic inflammation,
and is associated with alcohol withdrawal severity and depression alcoholic patients (John
61

MacLennan et al., 1995; Hensler et al., 2003; Huang et al., 2008, 2011; Shi et al., 2010; Xu et al.,
2010; Hilburn et al., 2011). Here, we report a reversed outcome of reduced serum BDNF with
chronic ethanol feeding (Fig 4E). These findings illustrate that the effects of DHM result in
systemic benefits against ethanol injury. This outcome warrants further investigation into the
effects of DHM on alcohol withdrawal and depression, as reduced serum BDNF levels are
associated with severe alcohol withdrawal and relapse. Furthermore, the observed changes in
serum BDNF may also play a role in the reported reduction of alcohol dependence (Shen et al.,
2012). Collectively, these findings of DHM hepatoprotection against ethanol-induced injury can
open new avenues of research on mechanisms of DHM that might protect the liver and other organs
against chemical stressors and diseases.
The observed hepatoprotection of DHM is likely to be contributed by several mechanisms
throughout the liver tissue and cellular responses. Therefore, demonstration that DHM enhances
ethanol metabolism via increased activity of ADH and ALDH sets the stage for future
investigations for the development of DHM for liver protection in humans as well as a tool for the
reduction of blood alcohol concentrations (BACs) that has been observed with DHM treatment in
ethanol consuming murine models (Shen et al., 2012; Sung et al., 2012). To test this hypothesis
and evaluate changes in ethanol metabolism, ethanol and acetaldehyde concentrations were
measured in cell media and mice serum with simultaneous treatment of DHM and ethanol. Our
current work demonstrated that DHM significantly increased ethanol and acetaldehyde
metabolism in both HepG2 and VL-17A cell lines (Data S5) and that this activity may contribute
to the reduced ethanol and acetaldehyde concentrations found in the sera of C57BL/6J mice
administered equal doses of ethanol (Fig 5A & B). To begin to investigate mechanistic
explanations for DHM’s ability to metabolize ethanol, we investigated the concentrations of the
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NAD
+
coenzyme in the liver. The elevated levels of NAD
+
relative to control and ethanol-fed mice
(Fig 5C & D), suggests that DHM modified hepatocellular bioenergetics that potentially plays a
role in enhancing NAD
+
-dependent ethanol metabolism and other NAD
+
dependent pathways.
Although the elevated NAD
+
concentrations partially provide evidence for this DHM-mediated
mechanism, future investigations need to evaluate DHM activity on other enzymatic systems such
as ADH/ALDH enzymatic activity, and other critical NAD
+
dependent enzymes.
In combination with ethanol metabolizing activity, DHM administration was also found to induce
ethanol/acetaldehyde metabolizing enzymes (Fig 5E & Data S6). Although evidence suggests that
DHM plays a role in enhancing ethanol metabolism, the mechanisms supporting a reduction of
ethanol intoxication and withdrawal behavior remain unclear. Therefore, the reported anti-alcohol
effects of DHM on GABAARs (Shen et al., 2012) and our findings on DHM enhancing ethanol
metabolism may partly contribute to the reduced intoxication behavior. However, the DHM-
mediated activity on enzyme activity and induction warrant further investigation to elucidate the
isolated or combination effects on ethanol metabolism and behavioral responses. Regardless, the
increased activity of ADH and ALDH, and induced expression, provides a novel mechanism of
DHM that may contribute to systemic protection against ethanol-mediated toxicities and
behavioral responses.
To investigate another potential mechanism of DHM hepatoprotection against ethanol injury, we
examined the role of DHM treatment on the ROS-producing CYP2E1 enzyme, and Nrf2 induction
of antioxidant enzymes in response to ROS stress and ethanol metabolism (Gong and Cederbaum,
2006; Osna and Donohue, 2007). Chronic ethanol intake and feeding are associated with an
increase in CYP2E1 expression and metabolism of ethanol, resulting in elevated ROS generation
and liver injury (Leung and Nieto, 2013). We found that DHM significantly reduced the expression
63

of CYP2E1 throughout the liver of ethanol-fed mice, and increased the expression of Nrf2 and its
downstream product, HO-1, supporting a previous investigation of DHM administered orally at 75
and 150 mg/kg (Fig 6; Qiu et al., 2017). Furthermore, these effects were found to reduce 4-HNE
expression in the liver, confirming reduced ROS stress in the liver (Fig 6E), data that was validated
in vitro by the increased expression of catalase, another product of Nrf2, and reduced ROS levels
(Fig 7). Therefore, this benefit of DHM administration is consistent with lower doses of DHM and
provides an additional mechanism illustrating the utility of DHM in counteracting ethanol injury
to the liver. Furthermore, the dual activation of both AMPK and Nrf2 antioxidant inducing activity
may explain the observed anti-inflammatory effects in hepatic tissue and in vitro.
In the present study, we delivered DHM via i.p. injections to increase its bioavailability rather than
using gavage administration or other oral delivery methods. We recognize that i.p. delivery of
DHM is not ideal, but it allowed for us to draw our first conclusions without the confound of
bioflavonoid bioavailability issues. Future studies will work on enhancing methods to deliver
DHM orally with the goal to maintain the beneficial effects of DHM, as identified in the present
study. Consequently, recent investigations have started focusing on improving the bioavailability
of DHM, but issues remain (Wang et al., 2016; Xiang et al., 2017; Zhao et al., 2019). As such, our
initial studies using i.p. delivery set the stage and benchmarks that can be used in future studies as
we continue to work to advance DHM to the clinic. Additionally, due to the limitations of
identifying direct effects of DHM on alcohol metabolic enzymes, future investigations should
determine whether DHM is directly influencing metabolic activity or whether this is an indirect
effect of DHM on cellular bioenergetics of DHM.
Collectively, this extensive line of research suggests that DHM acts on multiple pathways to
promote liver health and counteract ethanol injury. This work supports the use of DHM in
64

preventing/reducing ethanol-mediated damage to the liver and the subsequent development of
ALD. Overall, these findings support the hypothesis and demonstrate the potential for developing
DHM as a novel treatment to help mitigate the consequences of ethanol-induced oxidative stress
and lipid metabolism and to promote liver health.

65

CHAPTER 3
Dihydromyricetin Improves Mitochondrial Outcomes in the Liver of Alcohol-Fed Mice via
the AMPK/Sirt-1/PGC-1α Signaling Axis
Joshua Silva, Maximilian H. Spatz, Carson Folk, Arnold Chang, Enrique Cadenas, Jing Liang,
and Daryl L. Davies*
Titus Family Department of Clinical Pharmacy, University of Southern California School of
Pharmacy, Los Angeles, California, United States

*Address correspondence to:
Daryl L. Davies, Ph.D.
Professor, Titus Family Department of Clinical Pharmacy
University of Southern California School of Pharmacy
1985 Zonal Ave.
Los Angeles, California 90033
United States
Telephone: +1 323 442 1427
Email: ddavies@usc.edu

66

Abstract
Alcoholic liver disease (ALD), due to the multifactorial damage associated with alcohol
(ethanol) consumption and metabolism, is one of the most prevalent liver diseases in the United
States. The liver is the primary site of ethanol metabolism and is subsequently injured due to the
production of reactive oxygen species (ROS), acetaldehyde, and metabolic stress. Building
evidence suggests that dihydromyricetin (DHM), a bioactive flavonoid isolated from Hovenia
dulcis, provides hepatoprotection by enhancing ethanol metabolism in the liver by maintaining
hepatocellular bioenergetics, reductions of oxidative stress, and activating lipid oxidation
pathways. The present study investigates the utility of DHM on hepatic mitochondrial biogenesis
via activation of the AMP-activated protein kinase (AMPK)/Sirtuin (Sirt)-1/PPARG coactivator
1 (PGC)-1α signaling pathway. We utilized a forced drinking ad libitum study that chronically
fed 30% ethanol to male C57BL/6J mice over 8 weeks and induced ALD pathology. We found
that chronic ethanol feeding resulted in the suppression of AMPK activation and cytoplasmic
Sirt-1 and mitochondrial Sirt-3 expression, effects that were reversed with daily DHM
administration (5 mg/kg; intraperitoneally [i.p.]). Chronic ethanol feeding also resulted in hepatic
hyperacetylation of PGC-1α, which was improved with DHM administration and its mediated
increase of Sirt-1 activity. Furthermore, ethanol-fed mice were found to have increased
expression of mitochondrial transcription factor A (TFAM), reduced mitochondrial content as
assessed by mitochondrial DNA to nuclear DNA ratios, and significantly lower levels of hepatic
ATP. In contrast, DHM administration significantly increased TFAM expression, hepatic ATP
concentrations, and induced mitochondrial expression of respiratory complex III and V. In total,
this work demonstrates a novel mechanism of DHM that improves hepatic bioenergetics,
67

metabolic signaling, and mitochondrial viability, thus adding to the evidence supporting the use
of DHM for treatment of ALD and other metabolic disorders.
Highlights
• Dihydromyricetin (DHM), a bioactive flavonoid, improved mitochondrial outcomes in the
liver of male C57BL/6J mice after chronic alcohol feeding.
• Alcohol inhibition of key metabolic enzymes, AMPK and Sirtuins, and mitochondrial injury
are reversed with DHM administration.
• These findings support the utility of DHM, a dietary supplement, as a novel therapeutic for
the reduction/prevention of alcohol-related mitochondrial injury in the liver.
• This dietary flavonoid provides key metabolic responses that support its use for other
mitochondria-related disorders.
Keywords
alcoholic liver disease (ALD)
dihydromyricetin
ethanol
mitochondria
PGC-1α
sirtuin
Abbreviations
ALD, alcoholic liver disease
AMPK, AMP-activated protein kinase
DHM, dihydromyricetin
68

EtOH, ethanol
NAD, nicotinamide adenine dinucleotide
Nrf, nuclear respiratory factor
PGC-1α, PPARG coactivator-1α
ROS, reactive oxygen species
Sirt, Sirtuin
Introduction
Alcoholic liver disease (ALD) is one of the most prevalent liver diseases in the United
States, and it includes a spectrum of diseases ranging from reversible fatty liver to alcoholic
hepatitis, and cirrhosis (Mellinger et al., 2018). The majority of patients diagnosed with ALD are
also suffering from alcohol use disorder (AUD), where this latter condition affects over 17
million people in the United States. A concerning trend is the increased incidences of younger
adults being diagnosed with ALD due to increasing rates of alcohol (ethanol) abuse. The
mechanisms involved in the development of ALD are multifaceted, and it is becoming evident
that the disease spectrum and subsequent progress to late-stage ALD with prolonged ethanol
abuse results from the multifactorial injurious responses that occur throughout the body (Rehm,
Samokhvalov, & Shield, 2013; Rehm et al., 2010; Seitz et al., 2018). Ongoing cycles of high
levels of ethanol abuse and ethanol metabolism represent a primary mechanism of organ damage
and is one major contributing factor resulting in the pathology of ALD and subsequent
development of late-stage ALD (Han et al., 2012).
Ethanol is primarily metabolized in the liver by cytosolic alcohol dehydrogenase (ADH)
and the inducible cytochrome P450 2E1 (CYP2E1) in mitochondria and endoplasmic reticulum
(Lieber, Rubin, & DeCarli, 1970; Seitz et al., 2018). The metabolism of ethanol by ADH1 and
69

CYP2E1 results in the oxidation of ethanol to acetaldehyde (ACH), a highly reactive and toxic
metabolite, which is then further metabolized to acetate by aldehyde dehydrogenase (ALDH2)
located in the mitochondria in the liver. Throughout this metabolic process, reactive oxygen
species (ROS) formation occurs, with CYP2E1 oxidation contributing to much of the ROS stress
in the liver (Leung & Nieto, 2013; Lieber, 1997; Neve & Ingelman-Sundberg, 2000). The
combination of high rates of ethanol metabolism, increased ROS, and production of ACH results
in multiple responses in the liver that dysregulate energy signaling and lipid metabolic pathways,
alongside the induction of inflammatory responses (Ceni, Mello, & Galli, 2014; Seitz et al.,
2018). Additionally, ACH and ROS alter mitochondrial structure and activity, thereby leading to
functional impairment, including decreased oxidative phosphorylation (ATP generation), ROS
exacerbation, and a decrease in ALDH2 activity, resulting in elevated ACH (O’Shea et al.,
2010). Furthermore, the nicotinamide adenine dinucleotide (NAD)-dependent metabolism of
ethanol/ACH, which results in the depletion of hepatic NAD
+
, contributes to additional
metabolic stressors (French, 2016; Wang et al., 2018). In an effort to adapt to the ongoing
hepatic injury from high levels of ethanol exposure, mitochondria undergo various responses,
such as biogenesis, to maintain mitochondrial integrity and prevent further damage resulting
from dysfunctional metabolic responses (Degli Esposti et al., 2012; Pessayre et al., 2012;
Serviddio, Bellanti, Sastre, Vendemiale, & Altomare, 2010). Collectively, this work suggests that
pharmacological agents that target these aspects of ethanol-mediated stress on mitochondria
could be critical in protecting hepatocytes from energy impairments that result from
mitochondrial-related stress in the liver.
One key regulator of mitochondrial biogenesis and cellular energy metabolism is the
peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α), which belongs
70

to the family of PGC-1 transcription coactivators (Handschin, 2009; Scarpulla, 2011). PGC-1α is
present at low but inducible levels in the liver, where it also regulates most metabolic pathways,
such as fatty acid β-oxidation, gluconeogenesis, and ketogenesis (Puigserver et al., 2003; Rhee et
al., 2003; Yoon et al., 2001). Under normal physiological stress conditions (e.g., energy
deprivation, fasting, and/or low temperatures), PGC-1α is activated via cyclic AMP response
element-binding protein (CREB) and post-translationally via adenosine monophosphate-
activated protein kinase (AMPK) phosphorylation and NAD
+
-dependent sirtuin (SIRT)-1
deacetylation (Scarpulla, 2011). Notably, ethanol metabolism inhibits the AMPK-dependent
phosphorylation of PGC-1α necessary for activation, and depletes NAD
+
concentrations critical
for Sirt-1-driven deacetylation, thereby inhibiting PGC-1α activation via ROS stress and energy
depletion (Chaung, Jacob, Ji, & Wang, 2008; French, 2016; You, Jogasuria, Taylor, & Wu,
2015). This inhibition of mitochondrial biogenesis, in addition to mitochondrial stress and
damage, results in the accumulation of dysfunctional mitochondria and reduced hepatic ability to
selectively remove mitochondria by mitophagy (Eid, Ito, Maemura, & Otsuki, 2013; Williams,
Ni, Ding, & Ding, 2015).
Dihydromyricetin (DHM), an active bioflavonoid isolated from Hovenia dulcis, has been
used in Chinese traditional medicines for centuries, and has been shown to protect the liver
against chemically induced liver damage via increased Sirt-1 signaling (Ma et al., 2019). In
support of this hypothesis, we and others have shown that DHM can have multiple medicinal
benefits, including anti-inflammatory, antioxidant, hepatoprotective, and anti-alcohol properties
(Chen et al., 2015; Martínez-Coria, Mendoza-Rojas, Arrieta-Cruz, & López-Valdés, 2019; Qi et
al., 2012; Shen et al., 2012; Silva et al., 2020). As part of this later effort, we found that DHM
enhances ethanol metabolism in the liver, in part due to increased NAD
+
concentrations.
71

Moreover, we found that DHM can reduce hepatic lipid accumulation via AMPK activation in
ethanol-fed male C57BL/6J mice (Silva et al., 2020). However, much remains to be elucidated
regarding the mechanism by which these metabolic changes in response to ethanol occur. The
current study addresses this issue by investigating the hepatoprotective role of DHM in the
AMPK/Sirt-1/PGC-1α signaling axis and subsequent mitochondrial viability that is otherwise
altered by chronic ethanol feeding.
Methods
Animals and experimental design
A forced drinking ad libitum study was conducted as previously described using 8-week-
old male C57BL/6J mice and the provision of a single bottle of ethanol [ethanol-fed], starting at
5% and gradually increasing to 30% ethanol, or tap water [water-fed controls], for a total of 8
weeks (Brandon-Warner, Schrum, Schmidt, & McKillop, 2012; Keegan, Martini, & Batey, 1995;
Silva et al., 2020). Mice were grouped as follows: 1) water-fed + saline intraperitoneal (i.p.)
injections (n = 6), 2) ethanol-fed + saline i.p. injections (n = 6), and 3) ethanol-fed + DHM i.p.
injections (5 mg/kg; n = 10). Mice were administered DHM or saline 5 days a week via i.p.
injection due to studies showing that i.p. administration of DHM results in hepatoprotection
(Silva et al., 2020) and studies from other laboratories that have found benefits in reducing
inflammatory responses (Hou et al., 2015) and anti-depressant effects (Ren et al., 2018). All
experimental procedures were approved by the USC IACUC committee, and all methods were
carried out in accordance with relevant guidelines and regulations. At the end of the
experimental period, the mice were euthanized via CO2 and cervical dislocation. Fresh liver
tissue was snap-frozen in liquid nitrogen, followed by preservation at −80 °C until utilized.
72

Immunoprecipitation
Protein concentration of hepatic extracts was determined by Bradford Assay. A sufficient
amount of PGC-1α (Cell Signaling) was added into 200 μg of protein and gently rotated
overnight at 4 °C. The immunocomplex was captured by adding 120 μL protein Dynabeads
Protein G (ThermoFisher Scientific; San Jose, California, United States) and gently rotating at 4
°C for 3 hours and centrifuging at 1500 × g for 5 minutes. The precipitate was washed three
times with ice-cold RIPA buffer, resuspended in 3X sample buffer, eluted with Pierce IgE
Elution Buffer (Thermo Fisher Scientific), and subjected to Western blot.
Sirt-1 deacetylase and Sirt family deacetylase assays
Sirt-1 deacetylase activity was assayed using liver extracts from male C57BL/6J mice
used in the forced drinking study. The activity of Sirt-1 was measured using a Sirt-1 deacetylase
fluorometric assay kit (Abcam; ab156065; Cambridge, Massachusetts, United States) and
assessed according to the manufacturer’s protocol. Sirt-1 deacetylase activity was normalized by
protein content. Similarly, extracts were used to evaluate the deacetylase activity of all Sirt
enzymes using the fluorometric BioVision Sirtuin Activity Assay Kit. Determination of total Sirt
enzyme activity was conducted following the manufacturer protocol, and activity was
normalized by protein content.
Mitochondrial isolation, protein extraction, and Western blot analysis
Mitochondrial isolation, protein extraction, and Western blot analyses were conducted as
previously published (Silva et al., 2020). All primary and secondary antibodies were purchased
from Cell Signaling (Beverly, Massachusetts). Complex III (Ubiquinol-Cytochrome C Reductase
Core Protein 2; UQCRC2) and complex V (ATP Synthase F1 Subunit Alpha; ATP5A)
73

mitochondrial proteins were immunoblotted using antibodies purchased from Abcam. All trials
were repeated in triplicate to confirm changes in protein expression. Densitometry analysis was
performed using the ImageJ Gel Analysis Tool and normalized against untreated controls.
NovaQuant mtDNA/nDNA ratio
Real-time PCR analysis was performed with NovaQUANT Mouse Mitochondrial to
Nuclear DNA Ratio Kit (EMD Millipore; Billerica, Massachusetts) according to the
manufacturer’s instructions. DNA extractions were performed on frozen mouse liver tissue using
a Qiagen Dneasy Blood & Tissue Kit (QIAGEN; Chatsworth, Virginia, United States). A set of
four optimized PCR primer pairs targeting two mitochondrial genes (trLEV and 12s RNA) and
two nuclear genes (BECN1 and NEB) were pre-aliquoted in an Applied Biosystems MicroAmp
Fast Optical 96-well reaction plate. A fast real-time qPCR system (Applied Biosystems; Foster
City, California) was used to measure the ratio of mtDNA to nuclear DNA, the relative mtDNA
copy number, reflecting the relative mtDNA per cell. The results of the qPCR reactions were
analyzed with the 2-ΔCT method and normalized to WT control.
Hepatic NAD
+
/NADH measurements
Total NAD
+
and NADH concentrations were measured from C57BL/6J mice liver tissue
using the BioVision NAD
+
/NADH Quantification Kit and manufacturer protocol as previously
described (Silva et al., 2020). In short, 20 mg of liver tissue was weighed, washed in cold 1X
PBS, and homogenized in NADH/NAD extraction buffer for extraction of NADH/NAD. NAD
was then decomposed to measure total NADH by heating an aliquot of extracted NAD/NADH
samples at 60 °C for 30 minutes, then immediately placed on ice to assess NADH values
according to the manufacturer guidelines.
74

Hepatic ATP measurements
Livers were homogenized in pre-cooled Tris-EDTA extractant (0.1 M Tris-acetate buffer
+ 2 mM EDTA, pH 7.75) using a Branson Digital Sonifier 150 ultrasonic tissue disrupter-
homogenizer (Emerson; St. Louis, Missouri, United States). The homogenate was centrifuged at
10,000 × g for 10 minutes in a refrigerated centrifuge at 4 °C, and supernatant was collected.
Aliquots of supernatant were re-adjusted to pH 7.8 (according to required pH for assay
guidelines) with 320 µL of 2.5 M KOH, and precipitate was removed by a second centrifugation
(10,000 × g for 10 minutes). Aliquots of supernatant were transferred to a fresh tube, on ice, and
240 µL of Tris-HCL/EDTA (pH 7.75) was added. For supernatant ATP levels to be accurately
assayed using the Sigma ATP Bioluminescent Kit, final supernatant pH levels were re-adjusted
to pH 7.8 (Sigma-Aldrich; St. Louis, Missouri). ATP levels of hepatic tissue (n = 6 for each
group) were measured by using 100 μL of supernatant with an ATP luciferin bioluminescence
assay kit, according to the manufacturer’s guidelines. ATP assay mix was diluted with 5 mL of
sterile water, remained on ice, and protected from light for 1 hour to assure complete dissolution.
100 µL of ATP Assay Mix was added to each well and incubated at room temperature for 3
minutes to allow for hydrolysis of endogenous ATP. Immediately after adding 100 μL of tissue
homogenates, standards, or water controls with ATP Assay Mix, the sample was measured for
luciferase light production. Luminescence was measured using the BioTek Synergy H1 Hybrid
Multi-Mode Reader plate reader (BioTek; Winooski, Vermont, United States). Relative
luminescent units from six measurements were averaged for calculations.
Data analysis
Animal biochemical analyses were conducted using six separate samples from mice
groups, and three separate samples for Western blot analysis. The data are presented as mean ±
75

standard error of the mean (SEM). Statistical analysis included one-way analysis of variance
followed by Bonferroni multiple comparison tests using Prism (GraphPad Software, Inc.; La
Jolla, California). Differences among groups were stated to be statistically significant when p ≤
0.05.
Results
DHM reverses ethanol-mediated inhibition of hepatic AMPK and increases Sirt-1 and
mitochondrial Sirt-3 expression
We evaluated the expression of cytoplasmic Sirt-1, activated pAMPK (Thr
172
) relative to
total AMPK, mitochondrial Sirt-3, and voltage-dependent anion channel 1 (VDAC1) in the liver
of chronic ethanol-fed and water-fed mice. Ethanol feeding significantly reduced Sirt-1 and Sirt-
3 expression by 37% and 31%, respectively (Fig 1). Additionally, chronic ethanol feeding
inhibited the activation of AMPK (phosphorylation at Thr
172
) by 43% compared to water-fed
controls (†p < 0.05 vs. water-fed; Fig 1B). DHM administration (5 mg/kg; i.p.) significantly
reversed these ethanol-mediated responses in the liver (*p < 0.05; Fig 1B). No significant
differences were observed in VDAC 1 expression between all three groups.
76

Fig 1. DHM administration significantly increased the expression of hepatic Sirt-1, pAMPK
(Thr172), and Sirt-3 compared to EtOH fed mice. A) Western blot images of Sirt-1, β-actin
loading control, pAMPK (Thr172), total AMPK, Sirt-3, and VDAC1. B) ImageJ quantification
of designated triplicate blots.
DHM increases Sirt-1-mediated deacetylation of PGC-1α in ethanol-fed mouse livers
Recently, we found that DHM administration can significantly increase NAD
+

concentrations in the livers of ethanol-fed mice (Silva et al., 2020). To replicate these findings,
we assessed the NAD
+
/NADH ratios and found significant reductions with ethanol feeding (Fig
2A, †p < 0.05). In support of our previous results, we identified a reversal of ethanol-mediated
NAD
+
depletion with DHM administration. To assess DHM modifications of NAD-dependent
Sirt-1 activity in response to increased NAD
+
concentrations, we measured the deacetylation
extent of hepatic PGC-1α in ethanol-fed mice (Fig 2B). Chronic ethanol feeding significantly
77

increased PGC-1α hyperacetylation (Fig. 2B; ††p < 0.01 vs. water-fed control), suggesting
reduced Sirt-1-driven deacetylation. DHM administration reversed this outcome and resulted in
enhanced deacetylation of PGC-1α (Fig. 2B; **p < 0.01 vs. ethanol-fed control). Likewise, the
activity of Sirt-1 and other hepatic Sirt enzymes was found to be significantly reduced with
chronic ethanol feeding (Figs. 2C & D, †p < 0.05). Importantly, DHM administration reversed
these effects and significantly elevated Sirt-1 (Fig 2C) and total Sirt deacetylase activity (Fig 2D)
in the liver, correlating with the elevated Sirt-1 expression (Fig 1) and increased NAD
+

concentrations (Fig. 2A).

Fig 2. DHM administration significantly increased Sirt-1 deacetylation of PGC-1α and overall
Sirt activity in the livers of EtOH-fed mice. A) Mice chronically fed EtOH for 8 weeks show
significant depletions in NAD+/NADH ratios. Administration of DHM significantly reversed this
78

effect (n=4/group; 2-way ANOVA). B) Liver extracts were first immunoprecipitated with anti-
PGC1 antibody, and the immunoprecipitates were analyzed by Western blot with acetyl-lysine. C)
Sirt-1 and D) Total Sirt family deacetylation significantly increased in EtOH-fed mice treated with
DHM relative to EtOH-fed controls. ††p<0.01 and †p<0.05 vs. water-fed control (Ctl), *p<0.05
and **p<0.01 vs. EtOH-fed control.
DHM increases mitochondrial content and hepatic ATP levels in ethanol-fed mice
Due to the activation of AMPK (pAMPK Thr
172
, Fig. 1) and increased PGC-1α
deacetylation (Fig. 2), we next evaluated the downstream response of PGC-1α on mitochondrial
viability and ATP responses. Upon activation of PGC-1α and the subsequent activation of NRF
transcription factors, TFAM is produced and transported to the mitochondria to induce
mitochondrial transcription and biogenesis (Kunkel, Chaturvedi, & Tyagi, 2016; Picca & Lezza,
2015). Therefore, we expected to observe a reduction of mitochondrial TFAM expression in the
livers of ethanol-fed mice, due to our previous findings of reduced hepatic NRF2 (Silva et al.,
2020) and hyperacetylated PGC-1α (Fig. 2B), which is suggested to reduce the activation of
NRF1 required for TFAM transcription (Aquilano et al., 2013; Gureev, Shaforostova, & Popov,
2019; Piantadosi, Carraway, Babiker, & Suliman, 2008; Silva et al., 2020). Interestingly, hepatic
TFAM was found to be elevated by 30% in ethanol-fed mice relative to water-fed controls (†p <
0.05), suggesting a compensatory mechanism inherent in the ability of TFAM to bind or activate
mitochondrial genes. Administration of DHM alongside ethanol feeding resulted in a much
larger (86%) increase of TFAM expression compared to water-fed controls and a 54% increase
compared to ethanol-fed controls (**p < 0.01; Fig. 3A). Likewise, assessment of hepatic
mitochondrial complex III (UQCRC2) and complex V (ATP5A) were found to be significantly
reduced with chronic ethanol feeding. The administration of DHM significantly reversed this
79

effect in the liver (Fig. 3B). In correlation with our findings of ethanol decreasing expression of
ATP5A, we found that hepatic ATP concentrations were significantly reduced to 2.43 nmol/mg
(†p < 0.05 vs. water-fed; Figure 3C). This effect on ATP concentrations was significantly
reversed (3.898 nmol/mg) with daily DHM administration (*p < 0.05 vs. ethanol-fed; Fig. 3C)
and correlates with our findings of increased ATP5A in the livers of DHM-treated mice.
Furthermore, chronic ethanol feeding resulted in a 40% reduction of mtDNA/nDNA, suggesting
reduced mitochondrial content (†p < 0.05 vs. water-fed; Fig. 3D). Notably, DHM administration
significantly reversed this effect, resulting in a 20% increase compared to ethanol-fed controls
(*p < 0.05; Fig. 3D).

Fig 3. DHM administration significantly reversed EtOH-mediated stress on hepatic mitochondrial
content and ATP concentrations. A) Western blot image and ImageJ quantification of hepatic
TFAM protein expression normalized to untreated controls (n=3/group). B)Western blot and
Image J quantification of hepatic mitochondrial complex IV (UQCRC2) and complex V (ATP5A)
80

expression display elevations in mitochondrial complex proteins normalized to untreated controls
(n=3/group). C) Hepatic ATP concentrations (nmol/mg; n=6/group). D) mtDNA copy number in
the liver, measured as the ratio of mtDNA to nDNA (mtDNA/nDNA), and normalized to water-
fed mice (n=6/group). †p<0.05 vs. water-fed control (Ctl), ††p<0.01 vs. Ctl, *p<0.05 vs. EtOH-
fed control (EtOH), and **p<0.01 vs. EtOH.
Discussion
The outcomes from the present work represent the first demonstration that DHM
pharmacologically preserves mitochondrial viability following ethanol feeding that suppresses
biogenesis and induced injury. These data support the hypothesis that DHM pharmacological
effects on NAD bioenergetics result in the activation of the AMPK/Sirt-1/PGC-1α axis and
subsequent modification of the expression of mitochondrial respiratory complex proteins III and
V and ATP production. Beyond the hepatoprotective effects identified in our forced drinking ad
libitum model (Silva et al., 2020), we report herein that DHM also induces mitochondrial
adaptations via modification of hepatocellular bioenergetics and activation of signaling pathways
that are otherwise inhibited with chronic ethanol feeding. These mechanisms, combined with
those found to be engaged in anti-inflammatory responses, lipid oxidation, and mitochondrial
lipid transport, further illustrate the multifactorial benefits of DHM pharmacological responses in
the liver (Fig 4).
To investigate potential pharmacological mechanisms of DHM that benefit critical
metabolic signaling pathways in the liver, we assessed the expression of AMPK, Sirt-1, and Sirt-
3 in the liver following chronic ethanol feeding or ethanol feeding with daily DHM
administration. As expected with chronic ethanol metabolism and injury, a significant reduction
of AMPK activation and expression of both cytoplasmic Sirt-1 and mitochondrial Sirt-3 was
81

observed (Fig 1). The reduction of these key enzymes is likely contributed to by NAD depletion
resulting from NAD-dependent ethanol metabolism, production of toxic ACH, and additional
stressors in the liver (Fig 4). Consequently, the decrease of these Sirt enzymes in the liver
exacerbates ethanol-mediated damage by inhibiting pathways that favor oxidation and energy
expenditure, thereby contributing to mechanisms that promote hepatic lipogenesis and
inflammation (Choi et al., 2013; Fritz, Galligan, Hirschey, Verdin, & Petersen, 2012; Yin et al.,
2012, 2014). Notably, these effects are not limited to lipid metabolism, as the resulting NAD
+

depletion following ethanol metabolism can also contribute to several signaling dysregulations
involved in critical metabolic processes, such as glucose intolerance (Choi et al., 2013; Fritz et
al., 2012; Yin et al., 2014). Furthermore, hyperacetylation of mitochondrial ALDH2, and other
antioxidant enzymes, due to reduced Sirt-3 activity, likely reduces its detoxification of ACH and
ROS and contributes to mitochondrial instability (Ding, Bao, & Deng, 2017; Fritz et al., 2012).
Therefore, the multifactorial responses in the liver result in injury due to shifts in bioenergetics
responses and reduced enzymatic metabolism/clearance of ethanol, its toxic metabolite, ACH,
and associated increases in ROS. However, the DHM-mediated modification of NAD
+
in the
livers of ethanol-fed mice (Silva et al., 2020), likely increased by the activity of AMPK, partly
explains the preserved expression of Sirt-1/3 (Cantó et al., 2009) and supports our earlier
findings of DHM treatment inhibiting lipid synthesis, steatosis, and oxidative stress in the liver
(Silva et al., 2020). Further evaluation is necessary to better understand the DHM-mediated
effects on the transcriptional activity of Sirt-1 and Sirt-3 in the liver and other tissues affected by
shifts in NAD/NADH and reduced Sirt activity. Additionally, future investigations are critical to
address the mechanisms of DHM that promote increased NAD
+
concentrations, such as changes
in the expression and activity of nicotinamide phosphoribosyltransferase (NAMPT) and
82

nicotinate phosphoribosyltransferase (NAPRT) enzymes involved in NAD salvage (Audrito,
Messana, & Deaglio, 2020) and NAD
+
regeneration following mitochondrial respiratory chain
activity (Lieber, 2005).
As illustrated in Figure 4, PGC-1α is a downstream target of both AMPK and Sirt-1 that
requires phosphorylation by AMPK and the subsequent deacetylation by Sirt-1 for activation of
ROS, suppressing pathways, lipid oxidation, and mitochondrial biogenesis (Chaung et al., 2008;
Scarpulla, 2011). Consistent with previous findings, we identified that ad libitum chronic ethanol
feeding resulted in the hyperacetylation of PGC-1α that is due to the inhibition of Sirt
deacetylase activity and expression (Choi et al., 2013; Shepard & Tuma, 2009; You, Liang,
Ajmo, & Ness, 2008). DHM administration significantly reversed the hyperacetylation of PGC-
1α (Fig 2B), supporting earlier findings of increased lipid oxidation signaling and suppression of
oxidative stress. In total, this activity helps explain that DHM administration increases the
activity of cytoplasmic Sirt-1 on PGC-1α, and also appears to influence the deacetylase activity
of other Sirt family proteins (Fig 2C & D). This increase of PGC-1α deacetylation, in addition to
AMPK activation, is likely to result in the activation of PGC-1α, thereby contributing to multiple
benefits, including oxidative suppression, mitochondrial biogenesis, and hepatic glucose
homeostasis (Wu et al., 2016; Yoon et al., 2001). Moreover, these findings align with previous
investigations that report DHM administration improving nonalcoholic fatty liver disease
(NAFLD) outcomes (Chen et al., 2015), which is linked to preserved Sirt-3 deacetylation in the
mitochondria (Zeng et al., 2019). Results from the present study are in agreement with this
evidence, in that we found that DHM administration increased the expression of mitochondrial
Sirt-3 after ethanol feeding. We further expanded these results by illustrating the beneficial
83

effects of DHM to increase cytoplasmic deacetylation of PGC-1α linked to Sirt-1 activity while
maintaining the deacetylation activity of other Sirt members and hepatic NAD
+
levels.
Notably, we found that ethanol-fed mice using a forced drinking ad libitum model
resulted in the inhibition of mitochondrial content and reduced mitochondrial ATP output. The
copy number of mtDNA is reflective of mtDNA transcription and ATP production (Kunkel et al.,
2016), and was assessed to validate mitochondrial biogenesis and the restorative conditions of
the liver following chronic ethanol injury (Picca & Lezza, 2015). TFAM is a promoter-specific
enhancer of mtDNA and a major mitochondrial gene regulator that is produced in the nucleus
following NRF1 transcription and is transported to the mitochondria to increase mitochondrial
numbers (Picca & Lezza, 2015). Interestingly, TFAM was found to be elevated in the
mitochondria of ethanol-fed mice, similar to what has been reported with intragastric ethanol
feeding (Han et al., 2012), but the overall mtDNA content and ATP concentration were
significantly reduced. The elevation of TFAM expression and localization in the mitochondria
can likely be attributed to hepatic compensation and adaptations to chronic ethanol feeding.
However, the significant reduction of mtDNA number with increased TFAM localization
requires further elucidation of the potential dysregulation of TFAM binding to mtDNA or
changes in post-translational modifications that may contribute to decreased mtDNA
transcription. Similar to other investigations (Lamlé et al., 2008; Shin, Yang, & Ki, 2013), we
previously identified a decrease in hepatic nuclear NRF2 expression using a forced drinking ad
libitum model (Silva et al., 2020), which is involved in mitochondrial biogenesis through co-
activation with PGC-1α (Athale et al., 2012; MacGarvey et al., 2012). In the present study, we
measured an increase in TFAM expression in the livers of ethanol-fed mice. This outcome needs
further investigation to determine the activity of other potential factors and their involvement in
84

mitochondrial biogenesis and composition, such as NRF1 or hypoxia-inducible factor 1 (HIF-1)
signaling, following ethanol feeding. Likewise, changes in expression and signaling of TFAM,
PGC-1α, and NRF1 appear also to be regulated by zinc deprivation that can be observed with
ethanol feeding (Sun, Zhong, Zhang, & Zhou, 2016). Therefore, these findings of increased
mitochondrial TFAM and reduced biogenesis after ethanol feeding require further exploration
for mechanisms beyond NRF1/2 and PGC-1α. Nonetheless, we found that DHM administration
over the 8-week drinking period improved hepatic mitochondrial outcomes, as assessed by the
increases in TFAM expression, expression of mitochondrial complex proteins, and mtDNA
number, and this is potentially linked to the increased activity of PGC-1α (Fig 2B). Furthermore,
DHM preserved hepatic ATP concentrations with chronic ethanol feeding, thereby suggesting
improved mitochondrial viability. Overall, these findings illustrate the potential for DHM to
improve mitochondrial outcomes in response to injury and provide a novel pharmacological
mechanism of DHM that supports its utility for preventing/reducing ALD and other
mitochondrial-related disorders. Our findings of unaltered VDAC expression and enhanced ATP
production with DHM treatment require further investigation. It is well established that increases
of NADH can be utilized within the electron transport chain for ATP production. Although there
are elevations of NADH from ethanol feeding, it has been observed that ATP production is
diminished by about 50–60% (Bailey, Pietsch, & Cunningham, 1999; Zhong et al., 2014). The
reduction of hepatic ATP and the role of reduced mitochondrial lipid β-oxidation that results in
steatosis have been proposed to be linked to the closure of VDAC that alters mitochondrial ATP
output and uptake of fatty acids (Lemasters & Holmuhamedov, 2006; Zhong & Lemasters,
2018). Therefore, future investigation of the effects of DHM on mitochondrial VDAC activity
85

and conformation is necessary to understand its role and potential rate-limiting activity in these
hepatic responses.
As discussed above, the present work represents the first demonstration that DHM
reverses ethanol-mediated suppression of mitochondrial biogenesis and adds to the evidence that
DHM can be used to improve hepatic bioenergetics and mitochondrial outcomes that are
otherwise dysregulated with chronic ethanol feeding. Our findings suggest a therapeutic value
for DHM as a Sirt-activating compound that can modulate many aspects of oxidative stress and
mitochondrial function via preservation of hepatic bioenergetics. Importantly, building evidence
demonstrates that these factors contribute to many of the ethanol-induced pathologies and that
DHM supplementation can be used to benefit the management of various metabolic disorders
beyond ALD.
Acknowledgments
This work was supported by funding and grants from the NIH NIAA R01AA022448, USC
GoodNeighbors Campaign, USC School of Pharmacy, and American Foundation for
Pharmaceutical Education (AFPE).
Author Statement
Joshua Silva: Original draft preparation, Methodology, Data Analysis, Figure
Design. Maximilian H. Spatz:

Methodology, data analysis, and review. Carson Folk:
Methodology, data analysis, and review. Arnold Chang: Methodology, data analysis, and
review. Enrique Cadenas: Scientific conceptualization, original draft preparation, editing,
and data interpretation. Jing Liang: Original draft preparation, editing, and data
interpretation. Daryl Davies: Scientific conceptualization, Writing- Original draft preparation,
Editing, Data Interpretation
86

CHAPTER 4
A novel pharmacotherapy approach using P-glycoprotein (PGP/ABCB1) efflux inhibitor
combined with ivermectin to reduce alcohol drinking and preference in mice
Joshua Silva
a
, Sheraz Khoja
a
, Liana Asatryan
a
, Eunjoo Pacifici
b
, Daryl L. Davies
a
*
a
Titus Family Department of Clinical Pharmacy, University of Southern California School of
Pharmacy, Los Angeles, California, United States
b
Department of Regulatory and Quality Sciences, University of Southern California School of
Pharmacy, Los Angeles, California, United States
*Address correspondence to:
Daryl L. Davies, Ph.D.
Titus Family Department of Clinical Pharmacy
University of Southern California School of Pharmacy
Los Angeles, California 90033
United States
Telephone: +1 323 442 1427 Email: ddavies@usc.edu
87

Abstract
Alcohol use disorder (AUD) has a major national impact, affecting over 18 million
people, causing approximately 88,000 deaths, and costing upward of $250 billion annually in the
United States. Unfortunately, FDA-approved AUD pharmaceuticals are few, and clinical benefits
are mostly ineffective in patients suffering from AUD. Therefore, the identification of novel
targets and/or innovative methods for the development of safe and effective medications
represents a critical public health need. Previously, we reported that avermectin compounds
(ivermectin [IVM] and moxidectin [MOX]) significantly reduced ethanol intake in male and
female mice. However, avermectin compounds are readily effluxed by P-glycoprotein
(Pgp/ABCB1) in the blood-brain barrier (BBB), resulting in reduced retention time by the drugs
in the central nervous system (CNS). As such, the doses of IVM or MOX and the time frame for
significant reductions of ethanol intake are not ideal. Here we evaluate a novel combinatorial
strategy involving IVM and tariquidar (TQ), a third-generation efflux inhibitor of Pgp, to reduce
the dosing necessary for improving alcohol (ethanol) consumption behavior. We tested male
C57BL/6J mice using a two-bottle choice study to evaluate ethanol consumption and preference.
We found that injecting 10 mg/kg of TQ 30 minutes prior to IVM resulted in a five-fold
improvement in the efficacy of IVM (dosed at 0.5 mg/kg), resulting in a significant reduction in
ethanol intake and preference. Notably, the reduction by IVM was well tolerated, and no adverse
effects were identified when tested at doses ranging from 0.50 mg/kg to 2.0 mg/kg. Collectively,
our findings indicate that IVM, in combination with TQ, increases its efficacy in the CNS for
reducing ethanol consumption. This work demonstrates a novel combinatorial drug strategy that
allows new opportunities for drugs with poor CNS retention, such as IVM, to demonstrate
improved potency and potentially improved safety.
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Highlights
• Tariquidar, a potent P-glycoprotein (Pgp) inhibitor, combined with ivermectin (IVM),
resulted in a five-fold improvement in the efficacy of IVM, resulting in a significant
reduction in ethanol intake and preference.
• We found that Pgp inhibition lowers the dose of IVM needed for reducing ethanol intake and
preference in male C57BL/6J mice.
• These findings set the stage for a novel combinatorial strategy to include other potential
AUD therapies and Pgp inhibitors.
• Our findings illustrate an innovative approach to address alcohol use disorders therapies and
other potential CNS disorders with limited therapeutic options, to improve clinical outcomes.
Keywords
alcohol use disorder
ethanol
ivermectin
p-glycoprotein/abcb1
tariquidar
Abbreviations
AUD, alcohol use disorder
BBB, blood-brain barrier
CNS, central nervous system
IVM, ivermectin
Pgp, p-glycoprotein
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P2X4R, P2X purinoreceptor 4
TQ, tariquidar

Introduction
Alcohol use disorder (AUD) has a significant health and social impact, affecting over 18
million people, causing approximately 88,000 deaths, and costing upward of $250 billion
annually in the United States (Sacks, Gonzales, Bouchery, Tomedi, & Brewer, 2015; Stahre,
Roeber, Kanny, Brewer, & Zhang, 2014). Additionally, alcohol-related disorders are estimated to
cause 5.9% of deaths worldwide, resulting in over 3 million deaths each year (World Health
Organization, 2011). Presently, there are three classes of drugs approved by the FDA for AUD,
but these drugs have resulted in only limited clinical success, as evidenced by the ongoing effects
of alcohol-related disorders. Furthermore, epidemiological research indicates that large portions
of individuals with AUD are not receiving any pharmacological or psychological therapy, nor are
they seeking treatment (Dawson et al., 2005). Therefore, the identification of novel targets or
innovative methods for the development of useful medications is necessary.
A novel target, the P2X receptor (P2XR), is a family of cation-permeable ligand-gated
ion channels activated by synaptically released extracellular adenosine 5′-triphosphate (ATP).
Recent work from our group and others suggests P2XR as a target for AUD pharmacotherapies
where alcohol has been shown to allosterically reduce ATP activation on several P2XR family
members (Asatryan et al., 2008; Franklin et al., 2014; Kidd et al., 1995; Li, Aguayo, Peoples, &
Weight, 1993). Ivermectin (IVM), a semi-synthetic macrocyclic lactone, is an FDA-approved
broad-spectrum antiparasitic avermectin that also acts as a selective positive allosteric modulator
of P2X4Rs (Asatryan et al., 2008; Geary, 2005; Jelínková et al., 2006; Popova et al., 2010;
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Silberberg, Li, & Swartz, 2007). We found that IVM significantly reduced or completely blocked
the inhibitory effects of alcohol on P2X4Rs when tested using a two-electrode voltage clamp
(Asatryan et al., 2010). Importantly, we discovered that IVM significantly reduced alcohol intake
and preference in male and female C57BL/6J mice with no significant effect on sucrose intake
(Wyatt et al., 2014; Yardley et al., 2012, 2014). Moreover, IVM was well tolerated, both acutely
and chronically, and no adverse effects were identified when tested at doses ranging from 0.65
mg/kg to 10 mg/kg (Yardley et al., 2012). In support of these findings, we also identified that the
knock-out (KO) of P2X4Rs in male mice resulted in higher alcohol consumption than their
littermate controls, and reduced the pharmacological effects of IVM on ethanol intake by 50%
(Wyatt et al., 2014). Together, these findings indicate that P2X4Rs represent a novel target for
drug development for AUD (Franklin et al., 2014; Wyatt et al., 2014). In addition, the findings
support the development of IVM as a novel therapy for AUD. However, the pharmacokinetic
properties of IVM are not ideal for a drug to treat AUD, as we found that it takes approximately
9 hours for IVM to accumulate in the CNS to exert its pharmacological activity (Yardley et al.,
2012). Therefore, modification of IVM pharmacokinetics via strategies that enhance IVM
penetration, retention time, and/or activity in the central nervous system (CNS) would
significantly improve the utility of IVM as an AUD therapy.
IVM is lipophilic and permeable to the CNS, but it is also a suitable substrate for the
efflux transporter P-glycoprotein (Pgp/ABCB1) (Amin, 2013; Kemper, Verheij, Boogerd,
Beijnen, & Van Tellingen, 2004; Osna et al., 2007; Potschka, 2010). Notably, KO of Pgp has
been shown to increase IVM retention time in the CNS (Geyer, Gavrilova, & Petzinger, 2009).
Therefore, Pgp is involved in reducing CNS retention of IVM, thereby influencing its
pharmacokinetic profile and delaying the onset of its pharmacological activity in the CNS.
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Collectively, this work indicates that co-administration of IVM with a Pgp inhibitor should
effectively reduce the dose of IVM necessary for reducing alcohol intake.
Tariquidar (TQ) is a well-tolerated, potent, and specific non-competitive inhibitor of Pgp
that has been investigated as a co-administered drug to enhance the absorption, retention time,
and efficacy of anti-cancer agents and HIV therapeutics (Fox & Bates, 2007; Müller, Pajeva,
Globisch, & Wiese, 2008; Su & Sinko, 2006). Moreover, one previous report found that IVM,
when administered in combination with TQ, resulted in the increased concentration of IVM in
the CNS as compared to being administered alone (Thoeringer, Wultsch, Shahbazian, Painsipp,
& Holzer, 2007). Therefore, similar to the KO of Pgp transporters, inhibition can increase the
concentrations of IVM in the CNS. With these findings, the current study tested the hypothesis
that co-administration of IVM with TQ can significantly reduce the dose of IVM necessary to
reduce ethanol intake and preference.
Methods
Animals and experimental design
Forty 6-week-old male C57BL/6J mice were purchased from Jackson Laboratories (Bar
Harbor, Maine, United States). The mice were housed in temperature-, light-, and 40 – 60%
humidity-controlled conditions with a 12-hour light/dark cycle (lights on at 12:00 AM). The
holding room was maintained at approximately 22 °C. All experimental procedures were
approved by the USC IACUC, and all methods were carried out in accordance with relevant
guidelines and regulations.
Ethanol drinking behavior
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The combinatorial effects of IVM and TQ on ethanol consumption were assessed using a
two-bottle choice drinking paradigm and within-subjects design. One tube contained tap water
and the other contained a 10% v/v ethanol (10E) solution in tap water. Mice were given free
access to 10E with bottle positions alternated every other day. Fresh fluids were provided three
times a week. Body weights were recorded daily. Every morning, daily fluid intake (to the
nearest 0.1 mL) was recorded from both bottles. Daily 10E intake was measured until it
stabilized (±10% variability from the mean dose of the last 3 days). After acclimating mice to
single housing and two bottles, mice received daily saline injections (intraperitoneal [i.p.]) until
10E intake values reached baseline and stabilized after 10 days. All mice were 8 weeks old at the
start of the ethanol-drinking and dose-escalation studies. In all cases, injections were
administered immediately prior to the period of 24-hour access to 10E versus tap water, so that
the change in drinking over 24 hours after IVM + TQ administration was measured. Mice (total
of 10 per dose and group) then received one injection (i.p.) with either TQ (10 mg/kg) and a
second injection of IVM (0.5–2.5 mg/kg) 30 minutes post-TQ, or an injection of TQ control (10
mg/kg), IVM control (0.5–2.5 mg/kg), or saline (control for injection effect, per se) followed by
a second i.p. injection of saline to control for volume and two injections. The dose of TQ (10
mg/kg) selected for this study was chosen due to a previous report of i.p. injections at this dose
improving exposure to imatinib in male BALB/c mice (Gardner, Smith, Figg, & Sparreboom,
2009). The doses of IVM tested within this study were 0.5, 0.75, 1.0, 1.5, 2.0, and 2.5 mg/kg,
either alone as control or co-administered with TQ (10 mg/kg). Using a within-subjects study
design, we designated 10 mice per group and randomized drug/vehicle treatments between all
groups after each study period, which ended once ethanol consumption values returned to
baseline. Mice groups were randomized so that all groups of mice received different treatments.
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The dose-escalation studies were conducted in ascending order, with 10 mice of one group
receiving IVM alone, IVM + TQ, saline (vehicle), or TQ only. Dose escalation studies of 10%
ethanol intake (g/kg/24 hours) and preference (calculated as [ethanol mL/total fluid mL] × 100)
continued until signs of toxicity were observed, with 2.5 mg/kg of IVM being the final dose
evaluated. The within-subjects design of the study allowed us to reduce the number of animals
required for the study and account for physiological differences among individuals. Animals
were evaluated until 10E drinking stabilized at baseline levels, usually 4–5 days after
administration, and then mice were injected with another dose. This pattern of IVM and TQ
administration continued until all doses of a particular study were complete. Consumption of 10E
returned to baseline levels prior to the administration of each subsequent dose of IVM tested.
Mice that showed signs of toxicity were excluded from the study and were under careful
observation until recovery.
Statistical analyses
Statistical analyses were performed with GraphPad Prism (GraphPad Software, Inc., La
Jolla, California, United States). ANOVA with repeated measures was used to examine the
interaction between drug treatment and days on all group ethanol intake (g/kg/24 hours) and 10%
ethanol (10E) preference baseline values (Figures 1 & 2). Bonferroni’s multiple comparisons
were used to identify drug treatment effects on ethanol intake (g/kg/24 hours) and 10E
preference in comparison to the baseline (day 0) values within all groups or a separate multiple
comparison analysis between all groups for comparison of treatment effects on matched days.
The baseline value for each group was calculated by averaging the daily ethanol consumption
(g/kg/24 hours) or 10E preference per each group and used as the day 0 baseline value for data
comparisons and presentation. All analyses included ethanol intake or 10E preference values
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after drug treatment (TQ, IVM, IVM + TQ, or saline vehicle) for comparison both within groups
(relative to baseline) and between groups (treatment effect on matched day comparisons). For
dose comparisons, all values of saline and TQ collected throughout the study (n = 60) were
averaged to provide n values and 5-day averages for comparison between all dose comparisons
by ANOVA with repeated measures followed by Bonferroni’s multiple comparisons. Figures 1
and 2 represent the daily averages of ethanol intake (g/kg/24 hours; Figure 1) and 10E preference
(Figure 2) measured by all mice, and dose comparisons of ethanol intake (g/kg/24 hours) and
10E preference are shown in Figure 3 as averages over the 5-day evaluation. For all studies,
significance was set at p ≤ 0.05.
Results
Doses of IVM controls (0.5–2.5 mg/kg), TQ controls (10 mg/kg), and IVM (0.5–2
mg/kg) + TQ were well tolerated throughout the study. No significant signs of toxicity were
observed as compared to that of controls in the five lowest doses of IVM tested (e.g., 0.5–2.0
mg/kg). However, IVM, when tested at 2.5 mg/kg plus TQ, resulted in behavioral signs of
toxicity in 2 out of 10 mice, including lethargy, tremors, and/or ataxia 1 hour post-injections.
One of the affected mice recovered from the treatment while the other did not.
Ethanol intake (g/kg/24 hours) values compared to baseline (day 0) within groups
Over the period of 10 days before drug administration and initiation of the study, mice
were acclimated with two bottles of 10% ethanol and water. As presented in Data S1A, the
average baseline values over the 10 days were found to be 10.55 g/kg/24 hours for group 1,
10.38 g/kg/24 hours for group 2, 10.78 g/kg/24 hours for group 3, and 10.86 g/kg/24 hours for
group 4 with no significant differences between groups or time (Data S1A). The average of the
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consumption values for each group and ethanol preference was used as the day 0 baseline value
presented in Figures 16 and 17. Additionally, no significant differences were observed in the
total fluid intake (mL/kg/24 hours) within or between each group treatment (Data S2A).
However, a repeated-measures 2-way ANOVA on baseline ethanol consumption values
identified a statistically significant interaction (days and treatments) on ethanol consumption
[F(90,840) = 6.437, p < 0.0001] and a significant main effect of time [F(30,840) = 6.599, p <
0.0001] (Fig 1).
Bonferroni’s correction for multiple testing between days of treatment within groups
found that the single administration of TQ + 0.5 mg/kg IVM in group 2 significantly reduced
ethanol intake in comparison to the day 0 baseline 24, 48, and 72 hours post-injection (days 2–4,
p ≤ 0.05). Additionally, the combination of TQ + 2.0 mg/kg IVM significantly reduced ethanol
intake relative to baseline 24 hours (day 22) and 48 hours (day 23) post-injection (p ≤ 0.05).
Multiple comparisons of treatment days against baseline in group 3 identified significant
reductions (p ≤ 0.05) of ethanol intake with administration of TQ + 0.75 mg/kg IVM 24 hours
(day 7), 48 hours (day 8), 72 hours (day 9), and 96 hours post-injection (day 10).
Multiple comparisons of treatment days against baseline in group 1 identified significant
reductions (p ≤ 0.05) of ethanol intake with the administration of TQ + 1.0 mg/kg IVM 24 hours
(day 12), 48 hours (day 13), and 72 hours post single injection (day 14). Additionally, the
administration of TQ + 2.5 mg/kg IVM in group 1 significantly reduced ethanol intake relative to
baseline 48 hours post-injection (day 28, p ≤ 0.05).
Multiple comparisons of group 4 showed significant reductions of ethanol intake in
comparison to the day 0 baseline with the administration of TQ + 1.5 mg/kg IVM 48 hours post-
injection (day 18, p ≤ 0.05). Additionally, administration of 2.5 mg/kg alone in group 4 showed a
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significant (p ≤ 0.05) reduction of ethanol intake compared to baseline 24 hours post-injection
(day 27). Treatments of TQ, IVM alone (0.5–2.0 mg/kg), and saline did not significantly differ
compared to baseline (day 0).
Ethanol intake (g/kg/24 hours) values compared between groups on matched days
A separate Bonferroni’s multiple comparison following a repeated-measures ANOVA of
daily ethanol intake values on days of treatment between groups found that a single
administration of TQ + 0.5 mg/kg IVM in group 2 significantly reduced ethanol intake on days
2, 3, and 4 in comparison to group 1 ethanol intake after treatment with TQ (p ≤ 0.05 for all
days), group 3 ethanol intake after treatment with 0.5 mg/kg IVM only (p ≤ 0.05 for all days),
and group 4 ethanol intake after treatment with saline (p ≤ 0.05 for all days).
The combinatorial administration of TQ + 0.75 mg/kg IVM in group 3 significantly
reduced daily ethanol intake on days 7, 8, and 9 in comparison to group 1 ethanol intake after
treatment with 0.75 mg/kg IVM alone (p ≤ 0.05 for all days), group 2 treatment with saline (p ≤
0.05 for all days), and group 4 treatment with TQ (p ≤ 0.05 for all days).
Combinatorial administration of TQ + 1.0 mg/kg IVM in group 1 significantly reduced
ethanol intake on days 12, 13, and 14 in comparison to group 2 ethanol intake values after
treatment with 1.0 mg/kg IVM alone (p ≤ 0.05 for all days), group 3 treatment with TQ (day 12,
n.s.; days 13–14; p ≤ 0.05), and group 4 treatment with saline (day 12, n.s.; days 13–14; p ≤
0.05).
Combinatorial administration of TQ + 1.5 mg/kg IVM in group 4 significantly reduced
ethanol intake on days 18, 19, and 20 in comparison to group 1 ethanol intake values after
treatment with 1.5 mg/kg IVM alone (days 18 and 19, p ≤ 0.05; day 20, n.s.), group 2 treatment
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with TQ (days 18–20; p ≤ 0.05), and a significant effect of the combination on day 18 (p <
0.0001) in comparison to group 3 ethanol intake values after treatment with saline.
Combination of TQ + 2.0 mg/kg IVM resulted in a significant reduction of ethanol intake
in group 2 from days 22, 23, and 24 in comparison to group 1 ethanol intake after treatment with
saline (day 22 and 23, p ≤ 0.05; day 24, n.s.), group 3 treated with 2.0 mg/kg IVM (day 22, p <
0.0001; day 23 –24, p ≤ 0.05), and group 4 after treatment with TQ (day 22, p < 0.0001; day 23–
24, p ≤ 0.05).
The combinatorial dose of TQ + 2.5 mg/kg IVM in group 1 significantly reduced ethanol
intake on days 27 and 28 in comparison to the ethanol intake values of group 3 after treatment
with TQ on matched days (day 27–28, p ≤ 0.05). The single combination dose also resulted in
significantly reduced ethanol intake values in comparison to group 2 after treatment with saline
on day 28 (p ≤ 0.05).
As expected, the single injection of 2.5 mg/kg IVM resulted in a significant reduction of
ethanol intake in comparison to the ethanol intake in group 3 after treatment with TQ on days 27,
28, and 29 (day 27–29, p < 0.0001), group 2 after treatment with saline (day 27, p ≤ 0.05; day 28,
p < 0.0001; day 29, p = 0.0001), and no difference in comparison to ethanol intake values of
group 1 treated with the single combination of TQ + 2.5 mg/kg IVM.
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Fig 1. IVM, combined with TQ, significantly reduced ethanol intake at lower doses. IVM (0.5 –
2.0 mg/kg) combined with TQ (10 mg/kg) significantly reduced ethanol intake in comparison to
baseline values and IVM dose controls. The four panels depict the average ± SEM of ethanol intake
(g/kg/24h) of four groups of mice randomized into treatment groups. All groups received either
IVM dosed at 0.5, 0.75, 1.0, 1.5, 2.0, and 2.5 mg/kg alone or with 10 mg/kg TQ, 10 mg/kg TQ
only, or saline i.p. injections. Day 1 is the average of the baseline value over 10 days for that group
prior to injections. Arrows indicate days of injection of the corresponding drug or controls.
*p<0.05 and **p<0.001 vs baseline values, and †p<0.05 vs. corresponding IVM dose control.
n=10/group; Repeated measure 2-way ANOVA followed by Bonferroni’s multiple comparisons.

Preference ratio for 10E values compared to baseline (day 0) within groups
Using a repeated-measures 2-way ANOVA that examined the day and effect of
treatments on baseline ethanol 10E preference values, a statistically significant interaction of
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days and treatments on ethanol consumption were identified [F(90,630) = 5.248, p < 0.0001] and
a significant main effect of time [F(30,210) = 2.640, p < 0.0001] (Fig 2).
After Bonferroni’s correction for multiple testing between days of treatment within
groups, the single administration of TQ + 0.5 mg/kg IVM significantly reduced 10E preference
of group 2 in comparison to the day 0 baseline 24, 48, and 72 hours post-injection (days 2–4, p ≤
0.05). All other single injections of either saline, 1.0 mg/kg IVM alone, 10 mg/kg TQ, and TQ +
2.0 mg/kg IVM resulted in no difference of 10E preference relative to baseline.
Multiple comparisons against baseline (day 0) within group 3 identified that the single
combinatorial administration of TQ + 0.75 mg/kg IVM significantly reduced 10E preference 24
and 48 hours post-injection (day 7 and 8, p < 0.0001). All other single injections (0.5 mg/kg
IVM, 10 mg/kg TQ, 2.0 mg/kg IVM, and saline) administered to group 3 showed no difference
when compared against baseline 10E preference values within the group.
Single administration of TQ + 1.0 mg/kg IVM significantly reduced 10E preference of
group 1 in comparison to the day 0 baseline 24, 48, and 72 hours post-injection (day 12–14, p ≤
0.05). All other single injections of 10 mg/kg TQ, 0.75 mg/kg IVM alone, saline, and the
combination of TQ + 2.5 mg/kg IVM resulted in no difference relative to baseline 10E
preference values within group 1.
Multiple comparisons against baseline (day 0) within group 4 identified that the single
combinatorial administration of TQ + 1.5 mg/kg IVM significantly reduced 10E preference 24
and 48 hours post-injection (day 17–18, p ≤ 0.05).
Administration (i.p.) of 2.5 mg/kg IVM alone significantly reduced 10E preference
relative to baseline 24 hours post-injection (day 27, p ≤ 0.05), with no difference observed the
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following days in comparison to baseline. All other single injections of saline and 10 mg/kg TQ
had no significant effect in comparison to the 10E preference baseline values within group 4.
Preference ratio for 10E values compared between groups on matched days
A separate Bonferroni’s multiple comparison following a repeated-measures ANOVA of
daily 10E preference values on days of treatment between groups identified that the single
administration of TQ + 0.5 mg/kg IVM in group 2 significantly reduced 10E preference on days
2, 3, and 4 in comparison to group 1 10E preference after treatment with TQ (day 2, p ≤ 0.05;
day 3–4, p < 0.0001) and group 4 treatment with saline (day 2–4, p ≤ 0.05). Furthermore, the
combination of TQ + 0.5 mg/kg IVM in group 2 significantly reduced 10E preference in
comparison to group 3 after treatment with 0.5 mg/kg IVM on day 2–3 (p ≤ 0.05).
The combination of TQ + 0.75 mg/kg IVM in group 3 significantly reduced 10E
preference in comparison to group 1 after treatment with 0.75 mg/kg IVM alone (day 7, p ≤ 0.05;
day 8–9, p < 0.0001), group 2 after treatment with saline (day 8, p ≤ 0.05; day 9, p < 0.0001),
and group 4 after treatment with TQ (day 7, p ≤ 0.05; day 8–9, p < 0.0001).
Combination of TQ + 1.0 mg/kg in group 1 significantly reduced 10E preference values
in comparison to group 2 after treatment with 1.0 mg/kg IVM alone (day 12 and 13, p ≤ 0.05;
day 14, p < 0.0001), group 3 after treatment with TQ (day 12, p ≤ 0.05; day 13, p < 0.0001; day
14, p ≤ 0.05), and group 4 after treatment with saline (day 12, p ≤ 0.05; day 13–14, p < 0.0001).
TQ + 1.5 mg/kg IVM significantly reduced 10E preference in group 4 in comparison to
group 3 after treatment with saline (day 17, p ≤ 0.05; day 18, p < 0.0001; day 19, p ≤ 0.05),
group 1 after treatment with 1.5 mg/kg IVM alone (day 17, p ≤ 0.05; day 18 and 19, p < 0.0001),
and group 2 after treatment with TQ (day 18 and 19, p < 0.0001).
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The administration of TQ + 2.0 mg/kg in group 2 significantly reduced 10E preference in
comparison to group 3 after treatment with 2.0 mg/kg IVM alone (day 22, p ≤ 0.05; day 23, p <
0.0001). There was no significant difference when compared against matched days after
treatment of either saline in group 1 or 10 mg/kg TQ in group 4.
The administration of TQ + 2.5 mg/kg IVM in group 1 significantly reduced 10E
preference in comparison to group 2 after treatment with saline (p ≤ 0.05) and group 3 after
treatment with TQ (p ≤ 0.05) on day 29. All other comparisons of treatments between groups
showed no significant difference.

Fig 2. IVM, combined with TQ, significantly reduced 10% ethanol (10E) preference at lower
doses. IVM (0.5 – 2.0 mg/kg) combined with TQ (10 mg/kg) significantly reduced 10E preference
in comparison to baseline and IVM dose controls. The four panels depict the average ± SEM of
10E preference of four groups of mice randomized into treatment groups. All groups received
either IVM dosed at 0.5, 0.75, 1.0, 1.5, 2.0, and 2.5 mg/kg alone or with 10 mg/kg TQ, 10 mg/kg
TQ only, or saline i.p. injections. Day 0 is the average of the baseline value over 10 days for that
group prior to injections. Arrows indicate days of injection of the corresponding drug or controls.
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*p<0.05 and **p<0.001 vs baseline values, and †p<0.05 vs. corresponding IVM dose control.
n=10/group. Repeated measure 2-way ANOVA followed by Bonferroni’s multiple comparisons.
Weekly ethanol intake (g/kg) averages compared between treatment groups
A repeated-measures 2-way ANOVA that compared all ethanol intake values against
treatment groups over the 5-day period identified a statistically significant interaction of drug
treatment and days [F(52, 364) = 4.824, p < 0.0001], a significant main effect of time [F(4,28) =
18.13, p < 0.0001], and a significant main effect of treatment [F(13,91) = 12.85, p < 0.0001)]
(Fig 3). Bonferroni’s multiple comparison between the 5-day averages of ethanol intake and drug
treatment identified a significant reduction of ethanol intake in comparison to saline controls
when compared against a single i.p. administration of the combination of TQ + 0.5 mg/kg IVM
(p ≤ 0.05), TQ + 0.75 mg/kg IVM (p ≤ 0.05), TQ + 1.0 mg/kg IVM (p < 0.0001), TQ + 1.5
mg/kg IVM (p < 0.0001), TQ + 2.0 mg/kg IVM (p ≤ 0.05), and TQ + 2.5 mg/kg IVM (p ≤ 0.05).
Furthermore, in comparison to 10 mg/kg TQ, the multiple comparison analysis identified a
significant reduction in ethanol intake over the 5-day averages when compared to TQ + 0.5
mg/kg IVM (p ≤ 0.05), TQ + 0.75 mg/kg IVM (p ≤ 0.05), TQ + 1.0 mg/kg IVM (p < 0.0001),
TQ + 1.5 mg/kg IVM (p ≤ 0.05), and TQ + 2.0 mg/kg IVM (p ≤ 0.05). Multiple comparison of
TQ combined with IVM against its corresponding IVM-only dose control on ethanol intake over
the 5-day period identified a significant reduction in ethanol intake averages of TQ + 0.5 mg/kg
IVM vs. 0.5 mg/kg IVM (p ≤ 0.05), TQ + 0.75 mg/kg IVM vs. 0.75 mg/kg IVM (p ≤ 0.05), TQ +
1.0 mg/kg IVM vs. 1.0 mg/kg IVM (p ≤ 0.05), TQ + 1.5 mg/kg IVM vs. 1.5 mg/kg IVM (p ≤
0.05), and TQ + 2.0 mg/kg IVM vs. 2.0 mg/kg IVM (p ≤ 0.05). No significant difference was
identified when comparing the 5-day averages of ethanol intake between TQ + 2.5 mg/kg IVM
and 2.5 mg/kg IVM. Comparison of a single injection of 2.5 mg/kg IVM resulted in a significant
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reduction of ethanol intake over the 5-day averages in comparison to a single injection of 10
mg/kg TQ (p ≤ 0.05) and saline (p < 0.0001).
Weekly 10E preference averages compared between treatment groups
A separate Bonferroni’s multiple comparison between the 5-day averages of ethanol
intake and drug treatment identified a significant reduction of 10E preference in comparison to
saline controls when compared against single i.p. administration of the combination of TQ + 0.5
mg/kg IVM (p ≤ 0.05), TQ + 0.75 mg/kg IVM (p ≤ 0.05), TQ + 1.0 mg/kg IVM (p ≤ 0.05), and
TQ + 1.5 mg/kg IVM (p ≤ 0.05). Furthermore, in comparison to 10 mg/kg TQ, the multiple
comparison analysis identified a significant reduction in 10E preference over the 5-day averages
when compared to TQ + 0.5 mg/kg (p < 0.0001), TQ + 0.75 mg/kg IVM (p < 0.0001), TQ + 1.0
mg/kg IVM (p = 0.0001), TQ + 1.5 mg/kg IVM (p ≤ 0.05), and TQ + 2.0 mg/kg (p ≤ 0.05).
Multiple comparison of TQ combined with IVM against its corresponding IVM-only dose
control on ethanol intake over the 5-day period identified a significant reduction in ethanol intake
averages of TQ + 0.5 mg/kg IVM vs. 0.5 mg/kg IVM (p ≤ 0.05), TQ + 0.75 mg/kg IVM vs. 0.75
mg/kg IVM (p ≤ 0.05), TQ + 1.0 mg/kg IVM vs. 1.0 mg/kg IVM (p ≤ 0.05), TQ + 1.5 mg/kg
IVM vs. 1.5 mg/kg IVM (p ≤ 0.05), and TQ + 2.0 mg/kg IVM vs. 2.0 mg/kg IVM (p ≤ 0.05). All
other single injections showed no significant difference in effects.

A
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B

Fig 3. TQ (10 mg/kg) combined with IVM significantly reduces the dosing for ethanol
consumption and 10E preference over a period of five days. A) IVM (0.5 – 2.5 mg/kg) combined
with TQ (10 mg/kg) significantly reduces ethanol intake relative to baseline, with 0.5 – 2.0 mg/kg
IVM and TQ showing significant effects compared to IVM doses alone. B) IVM (0.5 – 1.5 mg/kg)
combined with TQ significantly reduces 10E preference in comparison to IVM controls. IVM (0.5
– 1.0 mg/kg) and TQ (10 mg/kg) significantly reduced 10E preference relative to saline baseline
values. Ctl = saline; TQ = tariquidar; IVM = ivermectin. *p<0.05 vs. saline control, and †p<0.05
vs. matched IVM dose control; n = 10/group. All values are shown as averages ± SEM. Repeated
measure 2-way ANOVA followed by Bonferroni’s multiple comparisons.

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Discussion
This is the first study to report on the effects of combining a Pgp inhibitor with IVM on
the self-administration of alcohol in rodents. Here we report that co-administering IVM (0.5,
0.75, 1.0, and 1.5 mg/kg) with TQ (10 mg/kg) in a single dose resulted in a significant reduction
in both ethanol consumption and preference as compared to IVM alone (Figs 1 - 3). Overall, this
finding represented a five-fold dose reduction for IVM to induce its effects on alcohol
consumption in male C57BL/6J mice. In total, this initial finding suggests that Pgp inhibition can
enhance IVM therapeutic action in the CNS, likely due, in part, to its increased retention and
activity on P2X4Rs. On the other hand, IVM alone did not show any significant effects on
ethanol intake until it was administered as a single dose at 2.5 mg/kg, supporting earlier findings
of IVM (2.5–10 mg/kg) on ethanol administration (Yardley et al., 2012).
TQ was used in our combinatorial approach due to its potent and specific inhibition of
Pgp and well-tolerated responses in combination with other therapies (Fox & Bates, 2007; Fox et
al., 2015; Müller et al., 2008; Su & Sinko, 2006). The pharmacological effects of inhibiting Pgp
with TQ have been reported to enhance bioavailability and CNS penetration for
chemotherapeutics (paclitaxel, docetaxel, and imatinib) via inhibition of Pgp (Breedveld et al.,
2005; Fox et al., 2015; Gardner et al., 2009). Likewise, the administration of TQ prior to
administration of radiolabeled IVM in male C57BL/6J mice resulted in an approximately three-
fold higher concentration of IVM in the brain (Thoeringer et al., 2007). Utilizing this strategy for
our proof of concept of improving IVM dosing for reducing alcohol consumption, we aimed to
inhibit the Pgp-mediated efflux of IVM to obtain therapeutic effects at lower doses. Previously,
we have shown that IVM is well-tolerated and effective at reducing ethanol self-administration
and preference in male and female C57BL/6J mice, with no difference in consumption of sucrose
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solutions, between the doses of 2.5 mg/kg and 10 mg/kg, with a maximum efficacy between 5
and 10 mg/kg (Yardley et al., 2012). This initial study in male C57BL/6J mice illustrates the
utility of this combination in male mice and most likely will produce similar effects in females,
as have been observed with IVM alone (Yardley et al., 2012). Nevertheless, the effects of this
combination in female mice remain to be tested before definite conclusions can be drawn.
We have previously reported that IVM administered at 30 mg is safe and well-tolerated in
alcohol-dependent patients administered an intoxicating dose of ethanol (0.8 g/dL) via
intravenous infusion (Roche et al., 2016). In that study, there was no clinical benefit observed.
This most likely was a result of several issues, including small sample size and/or low dose
and/or experimental design. In consideration of these findings, future clinical studies are
necessary to better determine the potential of IVM for patients suffering from AUD. This would
include, for example, dose-escalation studies, as well as appropriate parameters (i.e., motivation
to consume alcohol and/or alleviating withdrawal symptoms) to detect changes in alcohol
cravings. Doses up to 120 mg have been reported to be safe in humans (Guzzo et al., 2002).
Therefore, modification of IVM dosing and strategies in healthy human subjects should be
considered along with applicable self-administration studies for valid comparisons. To address
this, our current findings illustrate the utility of a combinatorial strategy that inhibits Pgp to elicit
reductions in alcohol consumption at lower doses of IVM. This strategy can thereby reduce the
dose of IVM, and other therapeutics targeting the CNS, as a means to induce medication effects
within safe and well-tolerated ranges.
To identify potential safety concerns of these initial findings, we dose-escalated IVM
until adverse events were observed. As reported, a dose of 2.5 mg/kg IVM with 10 mg/kg TQ
contributed to adverse effects in two mice, one of which did not recover. Similar findings were
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observed in Pgp/abcb1 KO mice, in which IVM doses of 0.4 mg/kg are considered to be lethal
(Ménez, Sutra, Prichard, & Lespine, 2012). However, the complete KO of Pgp/abcb1
significantly increased sensitivity to IVM toxicity. Our findings also suggest that inhibition of
Pgp with TQ increases sensitivity to IVM, and results in neurotoxicity when administered with
2.5 mg/kg IVM. These effects are partly explained by the increased brain to plasma
concentrations of IVM with Pgp KO and inhibition using TQ (Ménez et al., 2012; Thoeringer et
al., 2007). The increased sensitivity and neurotoxicity of IVM in the brain may be due to several
mechanisms involving IVM activity, such as its interactions with P2X4Rs, γ-aminobutyric acid
type-A receptors (GABAARs), glycine receptors (GlyRs), and neuronal α7-nicotinic receptors
(nAChRs) (Krause et al., 1998; Krůsek & Zemková, 1994; Shan, Haddrill, & Lynch, 2001).
Although TQ increases the CNS concentration of IVM and these potential interactions, the
increased sensitivity also permits the CNS effect of IVM on alcohol effects at significantly
lower, and potentially safer, doses. Furthermore, the activity of IVM on GABAARs that results in
anxiolytic effects may be of benefit in reducing anxiety-like behavior that corresponds with
increased alcohol consumption. Although these early data support the utility of the combinatorial
strategy, much work remains before definitive conclusions can be drawn.
Investigations evaluating the potential to inhibit Pgp efflux are not limited to the use of
TQ. Pgp inhibition can be achieved with several first-generation therapies, FDA-designated
dietary supplements, and bioflavonoids (Brand et al., 2006; Nabekura, Yamaki, Ueno, &
Kitagawa, 2008; Raghava & Lakshmi, 2012; Rodriguez-Proteau et al., 2006). Therefore, various
combinatorial strategies utilizing IVM and a Pgp inhibitor/competitor may be useful in reducing
the dose of IVM, and other potential AUD therapies, for mediating alcohol consumption
behaviors with long-term use. The use of differing Pgp inhibitors may also reduce the risk of
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IVM neurotoxicity, depending on its potency in inhibiting or competing for Pgp efflux.
Likewise, the inclusion of other avermectins that interact with P2X4Rs can be screened with Pgp
inhibitors to better evaluate potential combinatorial therapies for AUD that provide a better
safety profile, such as moxidectin (Huynh et al., 2017; Janko & Geyer, 2013; Khoja et al., 2018;
Ménez et al., 2012). Overall, this proof of concept study exemplifies the applicability of a novel
combinatorial strategy to pharmacologically reduce alcohol consumption and may be effective in
screening alternative combinations of either Pgp inhibitors or P2X4R modulating compounds.
In summary, we provide the first evidence demonstrating that Pgp inhibition significantly
lowers the dose of IVM needed for reducing ethanol intake and preference in male C57BL/6J
mice. Based on our initial findings, ongoing research will expand our analyses on this novel
combinatorial strategy to include other potential AUD therapies and Pgp inhibitors. Importantly,
our study encourages the investigation of innovative approaches to address AUD therapies and
other potential CNS disorders with limited therapeutic options and patient success.
Acknowledgments
This work was supported by funding and grants from the NIH NIAA R01AA022448
(DLD), USC GoodNeighbors Campaign (DLD), USC School of Pharmacy, and American
Foundation for Pharmaceutical Education (AFPE; JS). We would also like to thank C. Xue and J.
Zhang for their efforts and support during this project.
Authors’ statement
Josh Silva: Original draft preparation, Methodology, Data Analysis, Figure Design.
Sheraz Khoja: Review and Interpretations. Liana Asatryan: Review and Interpretations.
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Eunjoo Pacifici: Writing- Reviewing and Editing. Daryl Davies: Scientific conceptualization,
Writing- Original draft preparation, Editing, Data Interpretation.

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CHAPTER 5
A Novel Dual Drug Approach that Combines Ivermectin and Dihydromyricetin (DHM) to Reduce
Alcohol Drinking and Preference in Mice
Joshua Silva1, Eileen Carry2, Chen Xue1, Jifeng Zhang1, Jing Liang1, Jacques Y. Roberge2, and
Daryl L. Davies1,*
1 Titus Family Department of Clinical Pharmacy, University of Southern California School of
Pharmacy, Los Angeles, California, United States
2 Molecular Design and Synthesis Group, Rutgers University Biomedical Research Innovation
Core, Piscataway, NJ, United States
* Correspondence: ddavies@usc.edu; Tel.: +1 323 442 1427
Abstract: Alcohol use disorder (AUD) affects over 18 million people in the US. Unfortunately,
pharmacotherapies available for AUD have limited clinical success and are under prescribed.
Previously, we established that avermectin compounds (ivermectin [IVM] and moxidectin) reduce
alcohol (ethanol/EtOH) consumption in mice, but these effects are limited by P-glycoprotein
(Pgp/ABCB1) efflux. The current study tested the hypothesis that dihydromyricetin (DHM), a
natural product suggested to inhibit Pgp, will enhance IVM potency as measured by changes in
EtOH consumption. Using a within-subjects study design and two-bottle choice study, we tested the
combination of DHM (10 mg/kg; i.p.) and IVM (0.5 – 2.5 mg/kg; i.p.) on EtOH intake and preference
in male and female C57BL/6J mice. We also conducted molecular modeling studies of DHM with
the nucleotide-binding domain of human Pgp that identified key binding residues associated with
Pgp inhibition. We found that DHM increased the potency of IVM in reducing EtOH consumption,
resulting in significant effects at the 1.0 mg/kg dose. This combination supports our hypothesis that
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inhibiting Pgp improves the potency of IVM in reducing EtOH consumption. Collectively, we
demonstrate the feasibility of this novel combinatorial approach in reducing EtOH consumption and
illustrate the utility of DHM in a novel combinatorial approach.

Keywords: dihydromyricetin; flavonoids; alcohol; alcohol use disorder; ivermectin
Citation: Lastname, F.; Lastname, F.; Lastname, F. Title. Molecules 2021, 26, x.
https://doi.org/10.3390/xxxxx
Academic Editor: Firstname Last-name
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Copyright: © 2021 by the authors. Submitted for possible open access publication under the terms
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(http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Alcohol use disorder (AUD), characterized as a problematic pattern of alcohol use, affects over 18
million people and costs upwards of $250 billion annually in the United States [1, 2]. Annual
alcohol-associated deaths in the United States were over 93,000 be-tween 2011 and 2015, with
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54.7% of those deaths caused by chronic conditions and 45.2% by acute conditions [3]. Currently,
there are three medications approved by the US Food and Drug Administration (FDA) to treat AUD:
disulfiram, naltrexone, and acamprosate. Notably, patients with AUD often seek counseling to
improve alcohol abstinence due to the lack of clinical success with pharmacological treatment [4].
The limited clinical success of available AUD therapeutics contributes to a low prescription rate of
9%, with medications only being prescribed to those who are likely to benefit from them, provided
that clinically meaningful effects are observed [5]. This lack of effective therapeutics illustrates the
necessity for identifying novel targets or innovative methods for the development of useful
medications for the treatment of alcohol dependence and the consequential damage associated with
alcohol abuse.
Beyond improving the patient’s likelihood of success of alcohol abstinence, therapeutics are
necessary to improve patient outcomes by reducing the risks of alcohol-related organ damage
(AROD). With a lack of effective therapies for AUD, the persistent alcohol (ethanol/EtOH) abuse
will exacerbate damage to the organs of the body, with substantial damage concentrated in the liver
[6]. Notably, patients suffering from AUD also have a significant risk of developing alcoholic liver
disease (ALD) in that the liver is the primary organ for metabolizing EtOH. Accordingly, the high
mortality rate of ALD is related to the high probability of a return to drinking, with only 30-40% of
newly diagnosed ALD patients remaining abstinent for one year [7]. This is not surprising when
considering the low success rate of AUD treatment, with relapse being a common occurrence on the
path to recovery [8, 9]. Thus, a therapeutic strategy that can reduce the severity of AUD symptoms,
meanwhile providing benefits to prevent AROD and ALD for the patient would be highly beneficial.
A novel target that our laboratory has been working on for AUD is the P2X4 receptor [10–15]. P2X
receptors are a family of cation-permeable ligand-gated ion channels activated by synaptically
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released extracellular adenosine 5’-triphosphate (ATP). Our lab has found that ivermectin (IVM),
an FDA-approved semi-synthetic macrocyclic lactone, acts as a positive allosteric modulator (PAM)
of P2X4 that reduces/eliminates the inhibitory effects of EtOH on P2X4 receptors [16–20].
Importantly, we demonstrated that IVM significantly reduced acute and chronic EtOH consumption
in female and male C57BL/6J mice. IVM was found to have no effect on sucrose intake when tested
at doses ranging from 0.65 mg/kg to 10 mg/kg and was well tolerated [12, 13, 15]. Adding to the
evidence for a role of P2X4 as a target for EtOH and IVM, we found that male P2X4 knock-out
(KO) mice resulted in higher EtOH intake than their respective controls and that there was a 50%
reduction in IVM efficacy on EtOH intake in the P2X4 KO mice [12]. Together, this evidence
suggests that P2X4 receptors represent a novel target for AUD drug development and support the
utility of IVM-mediated positive allosteric modulation of P2X4 receptors as a novel therapeutic for
AUD.
Although the utility of IVM for the treatment of AUD is supported, the pharmacokinetic (PK)
properties of this drug limit its bioavailability to the CNS relative to other avermectins. For example,
we found that moxidectin (MOX), a related avermectin, also modulated P2X4 receptors and
antagonized EtOH’s inhibitory effects on ATP activity in vitro. In addition, MOX significantly
reduced EtOH consumption in male and female C57BL/6J mice [11, 21]. The findings from this
work suggest that MOX has a superior PK profile in comparison to IVM, likely due to differences
in efflux from the blood-brain barrier (BBB) and higher lipophilicity [11, 13, 21–23]. Interestingly,
studies incorporating-glycoprotein (Pgp/ABCB1) deficient mice resulted in improved IVM PK
profile as compared to MOX, suggesting that Pgp efflux activity of IVM is more robust compared
to MOX in the CNS [22]. Furthermore, the high expression and activity of Pgp in the BBB is
understood to limit the retention of IVM in the brain [24–27]. In agreement with these reported
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outcomes, we recently found that a novel dual drug strategy that used tariquidar (TQ), a potent non-
substrate inhibitor of Pgp, in combination with IVM, significantly increased the potency of IVM as
demonstrated by a 5-fold dose reduction of IVM necessary to reduce EtOH consumption in male
C57BL/6J mice [28]. This work suggested that the regulation of Pgp efflux can enhance the positive
allosteric modulation of P2X4 receptors in the CNS and thus improve the potency of IVM for AUD
therapeutic benefits.
Building evidence suggests that the flavonol component of a traditional herbal medicine
[dihydromyricetin (DHM)] can be used to provide hepatoprotection by reducing the damage of
chronic EtOH consumption and other drug-induced liver injuries in rodent models [29–31]. In
addition, recent studies have shown that DHM can enhance the bioavailability of xenobiotic
substrates of Pgp, suggesting that DHM acts as a Pgp inhibitor alongside other mechanisms that
modify xenobiotic PK profiles [32–34]. Although the mechanism(s) associated with DHM’s ability
to inhibit Pgp are unclear, recent structure-activity relationship (SAR) studies of flavonols and Pgp
activity indicate that structural components of DHM are consistent with potent non-competitive Pgp
inhibition of ATP hydrolysis [35]. For example, the 3’-OH, 4’-OH, and 2,3-saturation of DHM are
associated with enhanced non-competitive Pgp inhibition [33, 36, 37]. Supporting these findings,
taxifolin, a flavonol with a similar structure to DHM, was found to inhibit Pgp ATPase at
concentrations as low as 100 nM through interactions with the Pgp nucleotide-binding domain
(NBD). These findings collectively suggest that DHM can act as a non-competitive Pgp ATPase
inhibitor, although further clarification on the mechanism is necessary. Therefore, we hypothesize
that DHM, a compound that we have found to have hepatoprotective benefits[31], can inhibit Pgp
and set the stage for a novel combination therapy that has the potential to reduce the severity of both
AUD and ALD. To begin to test this hypothesis, we utilized a dual drug delivery approach of IVM
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plus DHM to assess the outcome of this unique combinatorial strategy on EtOH consumption and
preference in C57BL/6J mice. Furthermore, we utilized an in silico approach to identify key ligand
residue interactions to help advance our understanding of the use of DHM as a novel Pgp inhibitor
in this combinatorial approach.
2. Results
2.1 DHM combined with IVM significantly increases IVM potency on EtOH intake and EtOH
preference
2.2 Male group baseline values
Male baseline values for EtOH consumption (g/kg) were found to be 11.89 g/kg/24-h for group 1,
11.76 g/kg/24-h for group 2, 12.64 g/kg/24-h for group 3, and 11.88 g/kg/24-h for group 4. No
differences were observed between male groups or time points for both EtOH consumption (Data
S1A) and 10E preference (Data S1B). Furthermore, Data S2 illustrates all daily measurements and
changes of EtOH consumption (Data S2A) and preference (Data S2B) in male mice post-drug
administration. Supplementary data S3A, S4A, and S5A illustrate the daily values of male water
intake (ml/kg/24-h), body weight (B.W.; %), and daily food intake (g), respectively. All doses of
IVM + DHM were well tolerated, and no adverse responses were observed in male mice throughout
the duration of this study.
EtOH intake (g/kg) averages compared between male treatment groups
Having identified significant differences in the daily EtOH consumption (Data S2) using a
randomized within-subjects design (Figure 1), we wanted to compare the changes in group averages
of EtOH consumption with respect to treatment. To do so, we conducted a 2-way ANOVA that
compared EtOH intake averages against the respective treatment groups over the 24-h period (Figure
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2A), in which we identified a significant effect of treatment [F(13, 91) = 4.787, p<0.0001].
Bonferroni’s multiple comparisons identified a significant reduction of EtOH intake post-
administration of DHM + IVM [1.0 – 2.5 mg/kg] in comparison to the average of all saline (Ctl)
post-treatment values collected over the 6-week study (*p<0.05 for all comparisons). Similarly, a
significant reduction of EtOH in-take was identified post-administration when comparing the
average of all post-treatment values of DHM controls compared to DHM + IVM [1.0 – 2.5 mg/kg]
and 2.5 mg/kg IVM only (#p<0.05 for all comparisons). The combinatorial drug strategy, when
administered as DHM + IVM (1.0 – 2.0 mg/kg), showed significant differences when compared to
their IVM controls (†p<0.05 for all comparisons). However, no differences were observed between
the combinatorial doses of IVM (0.5, 0.75, and 2.5 mg/kg) + DHM compared to the respective IVM
control doses.
10E preference averages compared between male treatment groups
We next wanted to identify differences in EtOH preferences after administration of the combinatorial
therapy. A 2-way ANOVA of 10E preference values post drug administration in male groups (Figure
2B) identified a significant main effect of treatment [F(13, 91) = 9.076, p<0.0001] on male EtOH
preference averages. Bonferroni’s multiple comparison identified a significant reduction of 10E
preference 24-h after administration of DHM + IVM [1.0 – 2.5 mg/kg] when compared to saline
(Ctl) averages (Ctl; *p<0.05 for all comparisons). Likewise, significant reductions of 10E preference
was observed when comparing DHM control averages to the DHM + IVM [1.0 – 2.0 mg/kg] post-
treatment averages (#p<0.05 for all comparisons). Multiple comparisons of EtOH preference
averages after administration of DHM + IVM compared to corresponding IVM controls identified a
significant reduction in EtOH preference for DHM + IVM [1.0 – 2.0 mg/kg] when compared to the
respective IVM control doses (†p<0.05 for all comparisons).
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Figure 1. Randomized within-subjects drug treatment layout for behavioral analysis. Male and
female C57BL/6J mice were separated into 4 cohorts and treated randomly each week with
incremental doses of IVM as either 1) IVM dose control (red), 2) IVM + DHM (green), 3) Saline
control (blue), and 4) DHM control (black).
A

B
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Figure 2. DHM (10 mg/kg) combined with IVM reduces the dosing necessary to significantly
decrease EtOH consumption and 10E preference in male C57BL/6J mice over a period of 24-h. A)
IVM (1.0 – 2.5 mg/kg) combined with DHM (10 mg/kg) significantly reduced EtOH intake relative
to saline treatment (Ctl), with 1.0 – 2.0 mg/kg IVM and DHM showing significant effects compared
to IVM doses alone. B) IVM (1.0 – 2.0 mg/kg) combined with DHM significantly reduces 10E
preference in comparison to IVM controls. IVM (1.0 – 2.0 mg/kg) and DHM (10 mg/kg)
significantly reduced 10E preference relative to saline values. Ctl=saline; DHM=dihydromyricetin;
IVM=ivermectin. *p<0.05 vs. Ctl values, †p<0.05 vs. corresponding IVM dose control, and #p<0.05
vs. DHM control; n=48/group for DHM and saline groups; n=8/group for IVM and IVM + DHM
groups. All values are shown as averages ± SEM. 2-way ANOVA followed by Bonferroni’s multiple
compari-sons.
2.3 Female group baseline values
Female baseline values were 13.4 g/kg/24-h for group 1, 15.2 g/kg/24-h for group 2, 14.4 g/kg/24-h
for group 3, and 13.8 g/kg/24-h for group 4. No differences were observed between time or groups
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for both EtOH consumption (Data S1C) and 10E preference (Data S1D). Furthermore, Data S3
illustrates all daily measurements of EtOH consumption (Da-ta S3A) and preference (Data S3B) in
female mice post-drug administration. Data S3B, S4B, and S5B illustrate no difference in female
responses with treatment in water intake (ml/kg/24-h), body weight (B.W.) (%), and daily food
intake (g), respectively. All doses of IVM + DHM were well tolerated, and no adverse responses
were observed in female mice throughout the duration of this study.
EtOH intake (g/kg) averages compared between female treatment groups
We next wanted to compare the changes in group averages of EtOH consumption after treatment in
female C57BL/6J mice. Using a 2-way ANOVA, we found a significant effect of treatment [F(13,
91) = 4.927, p<0.0001] (Figure 3A) on EtOH intake values in female mice. Bonferroni’s multiple
comparison tests determined a significant reduction of EtOH intake after administration of DHM +
IVM [1.0 – 2.5 mg/kg] and 2.5 mg/kg IVM in comparison to all saline (Ctl) controls (Ctl; *p<0.05
for all comparisons). Similarly, a significant reduction of EtOH intake was observed when
comparing the post-treatment averages of DHM controls against DHM + IVM [1.0 – 2.5 mg/kg] and
2.5 mg/kg IVM (#p<0.05 for all comparisons). Bonferroni’s multiple comparisons of DHM + IVM
compared to the corresponding IVM controls identified significant reductions of EtOH consumption
with DHM + IVM [1.0 – 2.0 mg/kg] in comparison to the respective IVM controls (†p<0.05 for all
comparisons). When comparing the average EtOH intake values, no significant differences were
observed between 2.5 mg/kg IVM alone vs. DHM + 2.5 mg/kg IVM.
10E preference averages (24-h post-administration) compared between female treatment
groups
10E preference averages compared between female treatment groups
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We next conducted a 2-way ANOVA to compare all 10E preference measurements against DHM
and saline controls, as well as the respective IVM dose. We identified a significant main effect of
treatment on female EtOH intake averages [F(13, 91) = 5.469, p<0.0001] (Figure 3B). Bonferroni’s
multiple comparison identified a significant reduction of 10E preference 24-h after administration
of DHM + IVM [1.0 – 2.5 mg/kg] and 2.5 mg/kg IVM compared to saline (Ctl) controls (*p<0.05).
Similarly, a significant reduction of 10E preference in comparison to treatment with DHM was
observed when compared against DHM + IVM [1.5 – 2.5 mg/kg] and 2.5 mg/kg IVM only
(#p<0.05). Multiple comparisons of DHM + IVM compared to corresponding IVM controls on 10E
preference identified a significant reduction in EtOH consumption averages for DHM + IVM [1.0 –
2.0 mg/kg] in female mice (†p<0.05 for all comparisons).
A

B
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Figure 3. DHM (10 mg/kg) combined with IVM significantly reduces the dosing for EtOH
consumption and 10E preference in female C57BL/6J mice over a period of 24-h. A) IVM (1.0 – 2.5
mg/kg) combined with DHM (10 mg/kg) significantly reduced EtOH intake relative to saline
treatment (Ctl), with 1.0 – 2.0 mg/kg IVM and DHM showing significant effects compared to IVM
doses alone. B) IVM (1.0 – 2.0 mg/kg) combined with DHM significantly reduces 10E preference
in comparison to IVM controls. IVM (1.0 – 2.0 mg/kg) and DHM (10 mg/kg) significantly reduced
10E preference relative to saline values. Ctl=saline; DHM=dihydromyricetin; IVM=ivermectin.
*p<0.05 vs. Ctl values, †p<0.05 vs. correspond-ing IVM dose control, and #p<0.05 vs. DHM
control; n=48/group for DHM and saline groups; n=8/group for IVM and IVM + DHM groups. All
values are shown as averages ± SEM. 2-way ANOVA followed by Bonferroni’s multiple
comparisons.
2.4 EtOH consumption and preference averages compared between treatment groups and sex
EtOH consumption averages (24-h post-injection) compared between treatment groups and sex
To identify sex-specific differences in the combinatorial potency, we utilized a repeat-ed-measures
2-way ANOVA that examined sex and the effect of treatments on EtOH (g/kg/24-h) values followed
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by Bonferonni’s multiple comparisons (Figure 4A). We found a statistically significant main effect
of treatment between EtOH consumption values [F(6, 42) = 9.419, p<0.0001]. EtOH intake values
(g/kg/24-h) were found to be significantly reduced after administration of DHM + IVM [1.0 – 2.5
mg/kg] in both male and female C57BL/6J mice when compared to saline (Ctl) controls (*p<0.05
for all comparisons). Comparing male to female values of EtOH intake (g/kg/24-h) showed no
difference between sexes, suggesting no sex-specific differences in EtOH consumption responses.
10E preference averages (24-h post-injection) compared between treatment groups and sex
To identify potential sex-specific differences in the therapeutic potency on 10E preference, we
utilized a repeated-measures 2-way ANOVA that examined sex and the effect of treatments on 10E
preference values followed by a Bonferonni’s multiple comparisons test (Figure 4B). Using a RM
2-way ANOVA, we found a statistically significant main effect of treatment on 10E preference [F(6,
42) = 12.95, p<0.0001]. EtOH preference averages were found to be significantly reduced after
administration of DHM + IVM [1.0 – 2.5 mg/kg] in both male and female mice when compared to
saline (Ctl) controls (*p<0.05 for all com-parisons) with no differences between sexes.

Figure 4. DHM (10 mg/kg) combined with IVM significantly reduces the dosing for EtOH
consumption and 10E preference over 24-h in male and female C57BL/6J mice with no sex-specific
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differences. A) DHM combined with IVM (1.0 – 2.5 mg/kg) significantly reduced EtOH
consumption 24-h post-treatment with no sex-specific differences between normalized consumption
values. Similarly, B) DHM combined with IVM (1.0 – 2.5 mg/kg) significantly reduced 10E
preference 24-h post-treatment with no sex-specific differences between normalized consumption
values. IVM=ivermectin; DHM = dihydromyricetin; Baseline = day 0 values presented in Figs 1 &
3 for males and females, respectively. *p<0.05 vs. sex-matched baseline values (n=48/group for
DHM and saline controls; n=8/group for IVM and IVM + DHM groups). All values are shown as
averages ± SEM. 2-way ANOVA followed by Bonferroni’s multiple comparisons.
2.5 In silico modeling studies
SAR studies of similar flavonoids support DHM to be a non-competitive Pgp inhibitor through
interactions with the NBD. However, studies investigating non-competitive Pgp inhibition by DHM
are lacking. Thus, to simulate non-competitive Pgp inhibition, in silico modeling studies were
performed into the NBD1 of the Cryo-EM structure of human Pgp (PDB:6C0V) [38]. ATP, taxifolin,
and DHM were successfully docked into the NBD1 bind-ing site, with binding enthalpies of -16.1,
-6.2, and -6.3, respectively (Fig 6), with lower binding enthalpies representing more favorable
binding interactions. The comparable binding enthalpies between DHM and taxifolin suggest that
DHM also acts as a non-competitive Pgp inhibitor. Further, docking results illustrate a near-perfect
overlap between the lowest energy conformations of taxifolin and DHM (Figure 5), indicating a
maintained binding conformation among DHM and taxifolin. Key residue-DHM inter-actions are
displayed in Figure 6. Specifically, hydrogen bond interactions occur between the 4-carbonyl and 2-
hydrogen of both DHM and taxifolin with Arg905 and Gln1175, respectively. These findings
suggest that DHM acts as a non-competitive Pgp inhibitor through interactions at the NBD,
maintaining key ligand-residue interactions of taxifolin.
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Figure 5. A) Chemical structures of taxifolin (a potent Pgp ATPase inhibitor) and DHM, depicting
variation only at the 5’ position of ring B. B) Overlay of lowest binding energy conformations of
taxifolin (green) and DHM (yellow) in NBD1 of human Pgp (PDB: 6C0V), depicting near-perfect
overlap.

Figure 6. Lowest energy binding conformation of DHM (yellow) in NBD1 of human Pgp
(PDB:6C0V). Atoms are displayed in the following colors: oxygen (red), nitrogen (blue), hydrogen
(white), and carbon (grey, except DHM). Hydrogen bonds and distances are displayed in green. As
depicted, in the lowest binding energy conformation, the 2-hydrogen and 3-carbonyl of DHM form
hydrogen bond interactions with Gln1175 and Arg905 residues, respectively.
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3. Discussion
This is the first investigation demonstrating the utility of DHM as a regulator of Pgp activity that
improves IVM potency on EtOH consumption in both male and female C57BL/6J mice. We found
that the combination of DHM with IVM significantly increased IVM potency effects in reducing
EtOH consumption and preference consistently in both sexes. These effects are likely due in part to
DHM’s Pgp inhibiting activity resulting in increased CNS bioavailability of IVM. This finding is in
agreement with our recent work, demonstrating that TQ improved IVM potency in male C57BL/6J
mice [28]. In contrast, IVM alone at these effective doses did not show any significant effects on
EtOH intake until it was administered at 2.5 mg/kg, further supporting our earlier findings of IVM
(2.5 – 10 mg/kg) on EtOH consumption [13, 28]. In support of DHM acting as a non-competitive
Pgp inhibitor, molecular docking studies into NBD1 of Pgp revealed consistent ligand-residue
interactions among DHM and taxifolin, a similar flavonoid and potent Pgp ATPases inhibitor.
Combined, our findings support DHM as a promising Pgp inhibitor that can significantly enhance
IVM potency in AUD models.
Molecular modeling studies have played a key role in interpreting and predicting non-competitive
Pgp inhibition [39, 40]. Notably, a direct correlation between Pgp NBD docking results of flavonoids
and in vitro Pgp inhibition has been established [41], strongly supporting the validity of flavonoid
docking into NBD of Pgp. With this in mind, we chose to utilize in silico modeling studies to
illustrate critical DHM-Pgp interactions and mechanisms of Pgp inhibition. Upon docking into
NBD1 of human Pgp, nearly identical binding conformations and enthalpies were observed between
DHM and taxifolin, a potent Pgp ATPase inhibitor (Fig 6). Our results demonstrate that 2,3-
saturation and 4-carbonyl of both DHM and taxifolin are involved in key ligand-residue interactions
and support DHM as a Pgp ATPase inhibitor. The involvement of 2,3-saturation in binding pocket
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interactions correlates with SAR studies associating 2,3-saturation with enhanced potency of Pgp
inhibition [35]. As SAR studies are reported for flavonoids similar to DHM, these data, coupled with
modeling studies, could be utilized to guide the design of DHM analogs aimed at enhancing Pgp
inhibition. For example, others have shown that O-methylation, prenylation of ring A at the 6 or 8
carbon, and addition of a 4’-n- octyl group results in enhanced potency Pgp inhibition. However,
DHM modifications, which design towards Pgp inhibition/binding should be evaluated for
differences in hepatoprotective effects observed with natural DHM [31, 42–45]. Also, enhanced
potency of Pgp inhibition could increase the risk of IVM neurotoxicity in the brain, in which IVM
has been found to involve several mechanisms such as interactions with P2X4Rs, γ-aminobutyric
acid type-A receptors (GABAARs), glycine receptors (GlyRs), and neuronal α7-nicotinic receptors
(nAChRs) [46–48]. As such, future studies should also consider investigating the potential of
enhancing the hepatoprotective effects of DHM while maintaining Pgp activity. These questions will
be addressed in future investigations. Nonetheless, molecular modeling studies can provide a
powerful tool to investigate the potential of DHM analogs for enhanced or maintained Pgp
inhibition.
In support of our predictions of DHM enhancing the potency and safety of this combinatorial
approach, we found that 10 mg/kg DHM in combination with IVM was well-tolerated up to the
maximum dose that we tested (2.5 mg/kg). These observations of IVM + DHM being well tolerated
at the maximum dose (2.5 mg/kg IVM) suggest an improvement in the safety of this combination of
Pgp inhibition compared to TQ administration's neurotoxic effects (10 mg/kg) combined with IVM
at 2.5 mg/kg [28]. This improvement in drug tolerability is likely related to the hydrophilic properties
of DHM that result in its rapid clearance, compared to potent longer-lasting inhibitors that block the
conformation change of Pgp, such as TQ [28, 49]. Importantly, we found that the combination of
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DHM with IVM between the doses of 1.0 – 2.0 mg/kg enhanced IVM's potency to reduce EtOH
intake in both male and female C57BL/6J mice, with no sex-specific differences. Interestingly, the
combined dosing of IVM (above 1.0 mg/kg) with DHM did not provide any added effects in reducing
EtOH intake. This lack of additional improvement by DHM + IVM may be due to a “ceiling effect.”
Therefore, potential modifications of the DHM structure that benefit its PK profile may likely
influence this “ceiling effect” and would be interesting to investigate for differences in combinatorial
approaches. Of additional consideration is the potential impact that DHM may have on EtOH
consumption. Previously, it has been reported that DHM can reduce EtOH voluntary intake in male
SD rats when provided orally in tap water at 0.05 mg/mL and evaluated using a two-bottle choice
paradigm [50]. In contrast, in the present study, we did not see a reduction in EtOH voluntary intake
by DHM when administered at 10 mg/kg (i.p.). This could be due to the different types of animal
models (SD rats vs. C57BL/6J mice) and metabolic differences resulting from differential metabolic
enzyme expression and activity (e.g., cytochrome P450 [CYP] enzymes)[51], methodological
differences in the delivery of DHM (i.p. vs. voluntary consumption) and/or frequency of DHM
administration (once per week vs. daily administration). It has also been reported that the activity of
DHM on GABAAR potentiation is critical to the anti-intoxication effects of DHM [50, 52].
Therefore, it is of interest to explore the effects of DHM on saccharin and/or sucrose using the
drinking model alongside investigations of changes when combined with IVM. Additionally, the
enhanced metabolism of EtOH in the liver at the doses of 5 and 10 mg/kg DHM [31, 42] is of interest
when utilizing this combination and should be evaluated for changes in EtOH metabolism when
administered as a combinatorial therapy. Continuing investigations utilizing other animal mod-els
and alcohol drinking assessments (e.g., drinking in the dark for evaluations of changes in “binge
drinking”) will lead to a greater understanding of the benefits of DHM, either alone or in combination
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with IVM, as well as the effects of DHM on commonly tested tastants. Although these factors were
not investigated or observed in this present study, our findings highlight the utility of DHM as a
natural hepatoprotective molecule [31, 42, 43, 53–56] that improves the potency of IVM in reducing
voluntary consumption of EtOH. Furthermore, the development of this therapeutic combination
supports our hypothesis that utilizing different types of Pgp inhibitors can improve the safety of this
approach. However, future studies are necessary to better evaluate the observed ceiling effect of the
combined therapy for potential differences relating to both alcohol intake and potential liver effects.
This novel combinatorial approach is not limited to the use of the investigated compounds (e.g.,
IVM as a P2X4 PAM). As mentioned earlier, MOX acts as a P2X4 PAM with a superior PK profile
to IVM and significantly reduces EtOH drinking behavior [11, 21]. Therefore, various combinatorial
strategies utilizing different P2X4 PAMs and Pgp inhibitors need to be tested to identify
combinations with the greatest therapeutic potential. In addition to identifying multiple P2X4 PAMs
and/or Pgp inhibitors, future studies are necessary to expand the utility of these combinations in
other animal models of AUD. For example, investigations focusing on the utility of this
combinatorial approach in models of binge-like drinking (e.g., Drinking in the Dark [DID]
Paradigm) and alcohol withdrawal would add therapeutic value to the current findings by expanding
the therapeutic potential of this therapy beyond EtOH voluntary intake. Furthermore, the approach
of using the DHM flavonoid in combination with IVM suggests that novel combinatorial strategies
utilizing flavonoids, and other potential Pgp inhibitors, with known pharmacological responses can
likely provide added systemic benefits against EtOH- or drug-mediated organ injury.
Previously, human studies found that IVM administered at a dose of 30 mg is well-tolerated in
alcohol-dependent patients that were intravenously delivered an intoxicating dose of EtOH (0.8
g/dL; n=11) [57]. Unfortunately, no clinical benefit in cue-induced cravings was observed from this
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small human subject investigation. However, it is important to note that this study was not designed
nor powered to investigate IVM's potential to reduce the patients’ level of alcohol consumption or
change consumption habits. In addition, due to the time constraints of the clinical support staff, the
effects of IVM were measured approximately 6 hours after IVM administration. Our preclinical
work suggests that IVM activity is not observed until approximately 9 hours after administration
[11, 21]. Nonetheless, the lack of clinical benefit leads us to ask the question, is there a way to
improve IVM potency for IVM activity in the CNS to support the use of IVM for AUD? As presented
in our recent work [28] and the present study, the answer is yes. With this in mind, we predict that
this dual drug strategy could be employed to improve the therapeutic potential of IVM in patients
seeking pharmacotherapy for AUD. Notably, many patients undergoing AUD pharmacotherapy
often revert to alcohol abuse, and thus patients need to be aware of potential risks of alcohol abuse
with ongoing treatments [58]. The initial human safety study, as described above, lessens this
concern as no significant adverse effects by IVM were noted compared to placebo [57].
Furthermore, consideration of the subsequent effects of alcohol abuse must be considered when
developing therapies for patients suffering from AUD. Individuals that suffer from chronic alcohol
abuse often develop systemic injuries, particularly the liver (e.g., ALD), where much of the damage
is concentrated due to its primary role in EtOH metabolism [59–65]. To address this, our approach
of combining a hepatoprotective agent with Pgp inhibiting activity (i.e., DHM) and IVM elicits
reductions in EtOH consumption at lower and safer doses of IVM and can potentially provide
benefits to the liver following EtOH metabolism [31, 42, 56]. Based on the aforementioned
combinatorial findings, we are currently investigating the potential benefit of targeting both AUD
and ALD via modifications of DHM hepatoprotection when combined with IVM. In support of this
hypothesis, therapeutic benefits have been observed with IVM on nonalcoholic fatty liver disease,
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hyperglycemia, and lipidemia using in vitro and in vivo models, and these preclinical benefits are
likely related to the activity of IVM on hepatic farsenoid X receptors (FXR) [66–68]. Based on these
findings, ongoing research in our laboratory is focusing on the combination of DHM and IVM on
the liver for potential changes in DHM mediated activation of AMP-activated protein kinase
[AMPK] [31, 42, 69, 70] and/or IVM activity on FXR when combined as a combinatorial therapy.
Furthermore, this combinatorial therapy's combined effects should also be investigated in other
tissues, as Pgp inhibition or competition is likely to affect the PK profile in other Pgp expressing
organs.
In summary, we provide strong evidence for the utility of a combinatorial approach that implements
a Pgp inhibitor and P2X4 PAM, IVM, to enhance the potency of IVM on EtOH intake behavior.
Additionally, we are the first to report that using DHM in combination with IVM enhances the dosing
effects and improved the safety of this approach when compared to the neurotoxic responses
observed with the administration of TQ (10 mg/kg) and IVM (2.5 mg/kg) [28]. Supporting the
mechanism of this promising synergistic effect, we report the first molecular modeling study to
identify key DHM-NBD interactions that strongly support DHM to be a non-competitive Pgp
inhibitor. Based on these initial findings, ongoing research will expand our analyses of this novel
combinatorial strategy to elucidate the potential hepatoprotection of administering DHM in
combination with IVM as an innovative therapy to target both AUD and ALD. Importantly, this
study encourages the investigation of novel multi-targeted therapy combinations to benefit the
treatment of AUD and other CNS disorders lacking therapeutic options, meanwhile providing
pharmacological mechanisms that protect the patient from alcohol- or drug-induced organ damage.
4. Materials and Methods
Animals and experimental design
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Thirty-two male and thirty-two female C57BL/6J mice (6-weeks of age) were purchased from
Jackson Laboratories (Bar Harbor, Maine, United States). C57BL/6J mice have been studied over
many decades and are often utilized in alcohol studies due to their high alcohol intake and preference
compared to other strains [71–73]. All mice were individually housed in light-, temperature-, and 40
– 60% humidity-controlled conditions with a 12-hour light/dark cycle, and the vivarium was
maintained at 22 °C. Experimental procedures were approved by the USC IACUC (protocol # 10977
approved 11/08/2020), and all studies were carried following the relevant regulations and guidelines.
Two-bottle choice EtOH drinking behavior
The combination of IVM with DHM on EtOH consumption was evaluated using a two-bottle choice
drinking paradigm providing free access to chow and two bottles (tap water and 10% ethanol [10E])
over the course of the 6-week study, as previously described [28]. Baseline EtOH consumption
values were collected over a 10-day acclimation period in which all mice were administered two
saline injections (i.p.) to control for the number of injections and volumes utilized within the study.
These daily values (Data S1) were then averaged and used as the day 0 (baseline value) in
Supplementary Data S2 & S3. Using a within-subjects design, randomized groups of mice (8
mice/group) received an i.p. injection with either DHM (10 mg/kg) followed by a second i.p.
injection of a single dose of IVM (0.5–2.5 mg/kg) 15 minutes post-DHM. The remaining three
groups were randomized into control groups receiving either saline, DHM (10 mg/kg), or the
respective IVM dose control followed by a second saline i.p. injection (as a control for injections
and volume). All treatments (saline, DHM, IVM alone, and IVM + DHM) were administered once
per week to the designated randomized group. Following the treatment period, all mice were then
evaluated for changes in EtOH consumption and preference, followed by a 1-week wash-out period
to ensure elimination of the treatment before the transition to the next dose of IVM randomized to
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another group. The dose of DHM utilized in this study was selected due to our previous work
demonstrating reduced ALD in male C57BL/6J mice [31, 42]. The IVM doses tested in this study
were 0.5, 0.75, 1.0, 1.5, 2.0, and 2.5 mg/kg. IVM doses were administered ± DHM in ascending
order in randomized groups after a 1-week wash-out period for all treatments, established as a return
to baseline EtOH consumption, to ensure that the next administration of control(s) or combinatorial
therapy would not confound the previous intervention. Dose escalation studies of EtOH intake
(g/kg/24 hours) and 10E preference continued until the final dose of IVM (2.5 mg/kg) that we have
found to have significant effects on EtOH consumption when administered alone [13, 14, 28].
Statistical analyses
GraphPad Prism (GraphPad Software, Inc., La Jolla, California, United States) was used to conduct
all statistical analyses. EtOH consumption and 10E preference values for all 4 randomized groups
and treatments were analyzed using a repeated-measures (RM) 2-way ANOVA either as daily
changes (Figs 1 [males] & 3 [female]) or compiled as aver-ages for comparisons of combinatorial
administration vs. IVM alone between groups of the same sex (Figs 2 & 4) using Bonferonni’s
multiple comparisons test. Likewise, all data collected (n=48 total/sex for saline and DHM; n=8/sex
for IVM controls and IVM + DHM) were compiled as averages of post-treatment values collected
throughout the studies and compared to the post-treatment values of the opposite sex (Fig 3) using a
Bonferonni’s multiple comparisons test. For data presented in Figs 2 - 4, all values obtained from
saline and DHM treatment groups collected throughout the study (n = 48) were averaged from all
daily values over the 6 separate dose trials. These values were then compared against the averages
of IVM alone or IVM + DHM values (n=8/dose) to compare all dose treatments to the total average
values using a RM 2- way ANOVA followed by Bonferroni's multiple comparisons tests. Likewise,
for data presented in Fig 4, all values obtained from saline and DHM controls (n=48/sex) were
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averaged and compared against the respective IVM dose control (n=8/sex) and IVM + DHM dose-
matched treatments (n=8/sex) for assessment of differences against control values within the same
sex and treatment effects in the opposite sex. Statistical significance was set at p ≤ 0.05 for all
studies.
In silico modeling studies
To simulate non-competitive Pgp inhibition, in silico modeling studies were performed into the
nucleotide-binding domain (NBD)1 (ATP binding site) of the Cryo-EM structure of human Pgp with
bound ATP (PDB:6C0V) [38]. Molecular Operating Environment (MOE®) software was utilized
for modeling studies and energy minimization of all compounds. First, to ensure all ligands were in
their lowest energy structural conformations, energy minimization was conducted on ATP, DHM,
and taxifolin. Next, all ligands were docked with 1000 poses, using a triangular matcher method,
and induced fit refinement. Of the docking poses, docking conformation of the lowest binding
energy, signifying more favorable binding interactions, was selected for each ligand. To assess the
accuracy of the docking simulation, we compared the lowest energy docking conformation of ATP
with that of the Cryo-EM bound ATP. Complete structural overlap between docked and Cryo-EM
ATP was observed, supporting the validity of our ligand docking results into NBD1 of human Pgp
(PDB:6C0V).
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1:
title, Table S1: title, Video S1: title.
Author Contributions: JS, EC, JYR, and DD conceived of the investigation and developed the
analysis plan. JS, EC, CX, and JZ conducted the studies and collected and analyzed the data. JL
contributed to the manuscript edits, concepts, and discussions regarding DHM activity. All authors
contributed to data interpretation, figure development, manuscript drafting, and editing.
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Funding: This work was supported by NIH NIAA R01AA022448 (DLD), the American Foundation
for Pharmaceutical Education (AFPE; JS), USC GoodNeighbors Campaign, USC School of
Pharmacy, and Rutgers Office for Research. All authors declare no potential conflict of interest.
Institutional Review Board Statement: “The study was conducted according to the guidelines of the
USC IACIC, and all studies were carried following the relevant regulations and guidelines (protocol
code 10977 approved 12/5/2018).
Conflicts of Interest: The authors declare no conflict of interest.

135

CHAPTER 6
CONCLUSIONS
Excessive ethanol consumption continues to be a global healthcare problem with enormous
social, economic, and clinical consequences and accounting for approximately 3.3 million deaths
in 2012 (World Health Organisation, 2014). Unfortunately, as unhealthy levels of ethanol intake
remain unchecked, there are significantly increased risks for AROD, ALD, and other pathologies
associated with chronic ethanol consumption. Due to the significant metabolism of ethanol in the
liver, resulting in the production of acetaldehyde, ROS, and induction of inflammatory
responses, alongside contributing risk factors, this organ is often the primary site of concentrated
damage. Currently, there are no FDA-approved therapies for the treatment of ALD. Therefore,
these investigations aimed to develop DHM as a novel therapy for the prevention/reduction of
ALD. Collectively, these investigations support the hypothesis of my Dissertation that DHM is a
bioactive flavonoid with hepatoprotective function against liver injury that occurs with chronic
ethanol consumption. In particular, my work found that DHM reverses many of the effects of
ethanol in the liver by activating hepatic AMPK signaling, mitochondrial viability, and
increasing hepatic NAD
+
pools to enhance ethanol metabolism and metabolic signaling
pathways, thereby reversing the damage of ethanol in the liver. Additionally, I found that DHM
has novel Pgp binding properties within the NBD of human Pgp and can be utilized as a Pgp
inhibitor in a combinatorial approach to benefit therapeutic potency for repurposed P2X4 PAM
compounds (e.g., IVM) to target AUD in animal models.
To investigate the potential mechanisms of DHM hepatoprotection against ethanol, I
investigated the role of DHM treatment on ROS-producing enzymes, Nrf2 induction of antioxidant
enzymes in response to ethanol metabolism (Gong and Cederbaum, 2006; Osna and Donohue,
136

2007; Leung and Nieto, 2013), and the hepatic signaling activity of AMPK. I found that DHM
significantly reduced the expression of CYP2E1 in the livers of ethanol-fed mice and increased
the expression of Nrf2 and a downstream antioxidant product, HO-1, supporting previous
investigations of orally administered DHM (75 and 150 mg/kg) (Fig 6; Qiu et al., 2017).
Furthermore, these effects were found to reduce ROS stress in the liver after chronic ethanol
feeding. I also identified that DHM significantly increased AMPK signaling, resulting in the
inhibition of lipid synthesizing pathways and increasing markers of lipid transport to the
mitochondria for the oxidation of fatty acids. Therefore, the combination of DHM’s activity on
antioxidant responses and the induction of lipid oxidation pathways reversed ethanol effects in the
liver, thereby promoting liver protection and reducing/preventing fatty liver. These collective
liver-protective effects are likely due to the dual activation of AMPK, Nrf2 antioxidant inducing
activity, and increases in hepatic bioenergetics that explain the observed anti-inflammatory
responses and reduced lipid accumulation in the liver following treatment with DHM.
In association with the resulting changes in hepatic AMPK activity, I also found that
ethanol-feeding resulted in the inhibition of mitochondrial content and reduced mitochondrial
ATP output. Notably, daily administration of DHM reversed these mitochondrial outcomes in the
liver of ethanol-fed mice. In particular, I found that DHM administration improved hepatic
mitochondrial outcomes, as assessed by the increases in TFAM expression, expression of
mitochondrial complex proteins, and mtDNA number, and this is potentially linked to the
increased activity of PGC-1α (Silva et al., 2020b). Furthermore, DHM was found to preserve
hepatic ATP concentrations with chronic ethanol feeding, thereby suggesting improved
mitochondrial viability and energy demands. The modification of mitochondrial viability was
found to be partly enhanced by changes in hepatic levels of NAD
+
and the subsequent
137

improvements in NAD-dependent enzymes, such as the Sirtuin deacetylase activity on PGC-1α
that is critical for metabolic regulation and mitochondrial biogenesis. Our findings of DHM
activity on AMPK/Sirt-1/PGC-1α and mitochondrial biogenesis illustrate the potential for DHM
to improve mitochondrial outcomes in response to drug/alcohol-induced injury and provide a
novel pharmacological mechanism of DHM that supports its utility for preventing ALD and
other mitochondrial-related disorders.
With the lack of FDA-approved therapies for ALD, novel therapies are critically needed
for clinical improvement. Notably, the treatment of excessive ethanol intake, the underlying
cause of ALD and AROD, is necessary to improve the patient’s success of abstaining from
ethanol use and limiting the progression and development of ALD and AROD. To further
develop the utility of DHM for ALD and AUD, I sought to identify key interactions of DHM that
might benefit the BA and PK profile of therapeutics targeting AUD, specifically P2X4 PAMs
that have been identified to play a role in ethanol consumption. To address this, I investigated the
utility of combining DHM with identified P2X4R PAMs (e.g., IVM). In screening DHM’s
chemical structure and its ability to bind to Pgp similar to other Pgp-inhibiting flavonoids, I
identified key interactions of DHM with the NBD of human Pgp that matches that of a related
flavonoid, taxifolin. In doing so, I became interested in investigating the potential benefit of
targeting both AUD and ALD via modifications of DHM hepatoprotection when combined with
IVM. Previously, I found that using a potent Pgp inhibitor, TQ, was effective in enhancing the
potency of IVM, resulting in a 5X dose reduction on ethanol consumption in male C57BL/6J
mice (Silva et al., 2020a). Therefore, by combining DHM with IVM, I was interested in
developing and screening unique drug combinations capable of targeting both ethanol use and
the subsequent damage that results from excessive consumption. From my investigations, I found
138

that DHM partly acts as a Pgp inhibitor to improve the potency of IVM, resulting in a 2.5X dose
reduction in both female and male animal models. Differences in IVM potency between TQ and
DHM were expected due to DHM’s reduced potency for Pgp and its hydrophilic properties,
resulting in rapid elimination and clearance. Importantly, this combination can likely benefit the
multi-targeted treatment of both AUD and ALD. Overall, these latter findings suggest that the
utility of DHM can be expanded to target Pgp efflux, which could improve the therapeutic
potency of IVM and other Pgp substrates in the CNS, meanwhile providing added potential
therapeutic benefits.
In the presented studies, I delivered DHM via i.p. injections to increase its BA rather than
using gavage administration or other oral delivery methods. I recognize that i.p. delivery of DHM
is not ideal for the clinic, but it allows us to draw our first conclusions without the confound of
bioflavonoid BA issues. Ongoing studies will enhance methods to deliver DHM orally with the
goal of maintaining beneficial responses in the liver. Investigations from other laboratories have
started focusing on improving the BA of DHM, but issues remain (Wang et al., 2016; Xiang et al.,
2017; Zhao et al., 2019). As such, my studies using i.p. delivery set the foundation and benchmarks
that can be used in upcoming studies as we continue to advance DHM to the clinic. To address
these limitations, ongoing investigations in our laboratory are underway to formulate and/or
enhance the chemical structure of DHM to provide added benefits both as a Pgp inhibitor and as a
hepatoprotective agent. As part of this endeavor, we intend to improve the stability and absorption
of DHM via formulating it for oral administration and identifying hepatic responses similar to
those observed with i.p. injections. Furthermore, my investigations of combining DHM with IVM
for the multi-targeted treatment of AUD and ALD require further studies that identify hepatic
responses beneficial for protection against ethanol injury. To address these limitations of the
139

combinatorial approach utilizing DHM, we are continuing our efforts to specify the applicability
of DHM as a novel Pgp inhibitor. Therefore, DHM is currently being optimized for Pgp inhibition
and is undergoing evaluation for liver protective benefits when combined with IVM to enhancing
IVM potency on alcohol consumption. Importantly, these collective studies encourage the
investigation of unique multi-targeted combination therapies to benefit the treatment of AUD and
other CNS disorders, meanwhile providing added pharmacological mechanisms that protect the
patient from alcohol- or drug-induced organ damage.

140

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Abstract (if available)

Abstract Alcohol use disorder (AUD) affects over 18 million people in the US. Unfortunately, pharmacotherapies available for AUD have limited clinical success and are under prescribed. Furthermore, excess alcohol (ethanol) consumption is a significant cause of chronic liver diseases, accounting for nearly half of the United States' cirrhosis-associated deaths. Ethanol-induced liver toxicity, resulting in the development of alcoholic liver disease (ALD), is linked to ethanol metabolism and its associated increase in proinflammatory cytokines, oxidative stress, and the subsequent activation of Kupffer cells. Dihydromyricetin (DHM), a bioflavonoid isolated from Hovenia dulcis, can reduce intoxication and potentially protect against chemical-induced liver injuries. But there remains a lack of information regarding the mechanisms of DHM on ethanol metabolism and hepatoprotection. As such, the described investigations herein, tested the hypothesis that DHM supplementation enhances ethanol metabolism and reduces ethanol-mediated fatty liver, thus promoting hepatocellular health. Through investigations utilizing a forced drinking model that induces ALD in C57BL/6J mice, I found that DHM supplementation (both 5 and 10 mg/kg intraperitoneal [i.p.]) ameliorated the development of fatty liver and hepatic inflammation. Ethanol-mediated lipid accumulation and DHM effects against lipid deposits were determined using H&E stains, triglyceride measurements, and intracellular lipid assays. Likewise, I identified that DHM reversed ethanol-mediated inhibition of key metabolic signaling pathways (AMP activated protein Kinase [AMPK]) in the liver that is associated with ALD pathology, suggesting metabolic and anti-inflammatory benefits of DHM. To expand on these metabolic benefits, I also evaluated the effects of DHM on hepatic energy levels and mitochondrial responses to understand the diverse mechanisms of DHM and its potential to ameliorate mitochondrial-related damage following chronic ethanol injury. Through these investigations, I found that DHM maintained hepatic mitochondrial density and ATP and that these effects are partly due to the DHM-mediated activation of AMPK and the Sirtuin metabolic regulators critical for antioxidant and mitochondrial pathways. Furthermore, using a two-bottle choice study, I found that the combination of a potent P-glycoprotein (Pgp) inhibitor, tariquidar (TQ

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Asset Metadata

Creator Silva, Joshua (author)

Core Title Development of dihydromyricetin (DHM) as a novel therapy for alcoholic liver disease (ALD) and alcohol use disorder (AUD)

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Clinical and Experimental Therapeutics

Publication Date 03/22/2021

Defense Date 03/11/2021

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag alcohol use disorder (AUD),alcoholic liver disease (ALD),dihydromyricetin,ethanol,fibrosis,OAI-PMH Harvest,steatosis

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Davies, Daryl L. (committee chair), Cadenas, Enrique (committee member), Liang, Jing (committee member)

Creator Email jaysilva90@gmail.com,silvajos@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-430022

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Legacy Identifier etd-SilvaJoshu-9343.pdf

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Document Type Dissertation

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Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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