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The study of DHM effects on counteracting ethanol intoxications

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The study of DHM effects on counteracting ethanol intoxications

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The Study of DHM Effects on Counteracting Ethanol Intoxications

by

Xin Yu
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN
CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTERS OF SCIENCE
(PHARMACEUTICAL SCIENCES)
May 2019

2
ACKNOWLEDGEMENTS

Firstly, I would like to thank to my advisor Dr. Jing Liang who gave me the opportunity to work
in this fantastic lab and learn many new and practical techniques. Without their constant support
and encouragement in my research, I couldn’t finish this thesis.

I also like to thank Dr. Daryl Davies and Dr. Liana Asatryan for reviewing my thesis carefully
and providing me with useful suggestion in writing thesis.

I would also like to thank to all my lab members who serve as a great support throughout my
time in the lab. I would like to especially thank Joshua Silva for his guidance and technical
assistance.
Lastly, I want to express my greatest gratitude to my parents, without their support I couldn’t
have the ability to study in USC and achieve my degree.

3
Contents
List of Figures 5
Abbreviations 6
Abstract 7
Part I. Utilize the electrophysiological whole-cell voltage-patch-clamp recording to gain
knowledge of DHM activity on α5-GABAARs exposed to ethanol 9
Chapter 1 Introduction 9
1.1 Alcohol use disorders (AUD) 9
1.2 Alcohol’s actions on GABAARs 10
1.3 DHM effects on AUD 13
1.4 Studying α5-GABAARs and its agonist/antagonist- electrophysiological whole-cell
voltage-patch-clamp recording 14
1.5 Goals and specific aims 16
Chapter 2 Experimental Procedures 17
2.1 Materials and Equipment 17
2.2 α5-GABAARs expression in Oocytes 19
2.3 Electrophysiological whole-cell voltage-patch-clamp recording 20
2.4 Oocytes treatments 21
Chapter 3 Results and Conclusions 23
3.1 The response of α5-GABAARs to escalated concentration of GABA 23
3.2 Ethanol effects on α5-GABAARs 24
3.3 DHM like products reduce ethanol-induced potentiation effects on α5-GABAARs 25
Part II. In vitro study of hepatoprotective effects of DHM 27
Chapter 4 Introduction 27
4.1 Alcoholic liver disease (ALD) 27

4
4.2 Hepatoprotection effects of DHM 29
4.3 Oxidative metabolism of alcohol in liver 30
4.4 Goals and specific aims 34
Chapter 5 Experimental procedures 36
5.1 Cell culture 36
5.2 Trypan blue assay 36
5.3 MTT assay 36
5.4 Alamar blue assay 37
5.5 Acetaldehyde and Acetic acid measurements 37
5.6 Glutathione assay 38
5.7 Protein extraction and Western blot analysis 38
5.8 Confocal imaging 39
Chapter 6 Results and Conclusion 40
6.1 Higher amount of DHM can inhibit the proliferation of Hepatocellular carcinoma cells
40
6.2 DHM improves the mitochondrial respiratory capacity in VL-17A cells 41
6.3 DHM decreases the cytotoxicity caused by acetaldehyde 43
6.4 DHM accelerates the ethanol metabolism 44
44
6.5 DHM restores the GSH depletion in VL-17A cells 46
6.6 DHM reduces ethanol-induced intracellular lipid accumulation and SREBP-1 expression
48
6.7 DHM enhances the phosphorylation of AMPK and induces autophagy 50
Overall discussions 51
References 54

5

List of Figures

Figure 1.1 The basic structure of GABAARs................................................................................10
Figure 1.2 Pharmacological effect and brain distribution of α-GABAARs...................................11
Figure 1.3 The structure of DHM and pure DHM product...........................................................13
Figure 1.4 The electrophysiological whole-cell voltage-patch-clamp recording..........................15
Figure 2.1. Main procedures of Xenopus oocytes cDNA injection...............................................19
Figure 2.2 The timeline for agonist application to the Xenopus oocytes......................................21
Figure 2.3 The timeline for ethanol application to the Xenopus oocytes......................................22
Figure 2.4 The timeline for DHM-containing products application to the Xenopus oocytes........22
Figure 3.1 The response of α5-GABAARs to escalated concentrations of GABA........................23
Figure 3.2 The GABA current potentiation caused by ethanol treatments....................................24
Figure 3.3 The percentage of α5-GABAARs potentiation of pure DHM and DHM-containing
products..........................................................................................................................................25
Figure 3.4 DHM-containing compounds inhibit ethanol potentiated currents of α5-GABAARs..26
Figure 4.1 The lipid homeostasis regulated by SREBPs...............................................................28
Figure 4.2. The major enzymes involved in and the metabolites produced during the process of
ethanol microsomal oxidation.......................................................................................................30
Figure 6.1 The percentage of live HepG2 and VL-17A cells after 24 hours exposure to DHM..40
Figure 6.2 Mitochondrial activity detected by MTT assay...........................................................41
Figure 6.3. The Alamar blue luminescence of HepG2 and VL-17A cells....................................43
Figure 6.4. DHM increases the acetaldehyde metabolism in HepG2 and VL-17A cell models...44
Figure 6.5 Concentration of GSH produced in HepG2 cell and VL-17A cells.............................46
Figure 6.6 The synthesis and transportation of the GSH in liver cells..........................................47
Figure 6.7. Reduced expression of SREBP-1 and lipid accumulation..........................................48
Figure 6.8 DHM effects on expression of the AMPK-ULK1-mTOR in autophagy regulation....50

6
Abbreviations

AUD Alcohol use disorder
ALD Alcoholic liver disease
ADH Alcohol dehydrogenase
ACH Acetaldehyde
ALDH Aldehyde dehydrogenase
AWS Alcohol withdrawal syndrome
AMPK 5' AMP-activated protein kinase
AH Alcoholic hepatitis
DHM Dihydromyricetin
DAPI 4′,6-diamidino-2-phenylindole
DMSO Dimethyl sulfoxide
FAEE Fatty acid ethyl esters
GABA Gamma-Aminobutyric acid
GABAAR Gamma-aminobutyric acid type A receptor
HSCs Hepatic stellate cells
JNK1 c-Jun N-terminal kinase 1
mTOR Mammalian TORC
NADH Nicotinamide adenine dinucleotide
PFA Para- formaldehyde
PI3K Phosphoinositide 3-kinases
ROS Reactive oxygen species
SREBP Sterol regulatory element-binding protein
S1P Site-1 protease
SRE Sterol response element
TEVC Two-electrode voltage clamp
TNF-α Tumor necrosis factor alpha
TSC2 Tuberous Sclerosis Complex 2
ULK1 Unc-51 like autophagy activating kinase

7
Abstract

Alcohol use disorder (AUD) is a complex disease that is a major contributor to health problems
across the United States and the World. Building evidence links AUD with multiple system
pathologies, including liver disease, cardiac dysregulation, and gastrointestinal abnormalities.
There is a large body of investigations focusing on pathologies associated with chronic and
heavy alcohol intake, including the development of alcoholic liver disease (ALD) as it pertains to
alcohol-mediated increases in inflammatory processes and generation of reactive oxygen species
(ROS). However, the therapies against AUD and ALD are none or limited. These shortcomings
illustrate the important need for the identification and development of new therapies to treat the
onset of ALD induced by AUD. Dihydromyricetin (DHM), a bioactive flavanonol from the
Ampelopsis grossedentata species, is gaining interest by the scientific community due to its
broad range of pharmacological properties. This includes, for example, its ability to reduce
alcohol consumption, act as an antioxidant and potentially counteract ethanol’s detrimental
effects due to high levels of ethanol intake. To this end, building evidence from our laboratory
supports the use of DHM as a novel method to reduce alcohol consumption in rodents and to
improve liver health by providing protection against alcohol-induced liver injury and lipid
accumulation. The goal of my project (thesis) is to investigate DHM effects on certain GABAA
receptors (GABAARs). Furthermore, I investigate DHM effects on liver protection. The
technology of electrophysiological whole-cell voltage-patch-clamp recording was using to gain
knowledge of DHM activity on GABAARs, especially, α5-containing GABAARs in presenting
alcohol (ethanol) application. Ethanol (1mM to 300mM), and DHM (10nM to 1000nM),
concentration-dependently potentiated α5-GABAARs, while DHM could inhibit ethanol effects
on α5-GABAARs. This work confirmed that DHM protects the α5-GABAARs by counteracting

8
the excessive ethanol. Next, to obtain cellular insight of the DHM influences on ethanol
metabolism, I measured the effects of DHM plus/minus ethanol using human hepato-carcinoma
cells HepG2 and VL-17A, a transfected HepG2 cells. These cell lines express different levels of
the ethanol metabolizing enzyme alcohol dehydrogenase (ADH) and CYP2E1. The cells were
cultured at different concentrations of ethanol (50mM to 200mM) and treated with DHM to
evaluate ethanol metabolism, hepatoprotection, and changes in lipid metabolism. In addition,
colorimetric assays were performed to evaluate the benefits of DHM on ethanol-induced
mitochondrial stress and cytotoxicity. I found that DHM reduced the levels of ethanol-induced
metabolic activation when tested in high levels of ethanol. I also found that DHM treatment
decreased the production of the toxic metabolite acetaldehyde and increased the amount of the
acetic acid produced during ethanol metabolism. This latter finding suggests DHM is able to
accelerate the metabolism of ethanol and acetaldehyde, indicates DHM protective effects on
hepatocellular in vitro. Further, we hypothesized that DHM may play a positive role in
ameliorating ethanol-induced steatosis, we found that DHM markedly decreased the
accumulation of intracellular lipids and expression of the lipogenic transcription factor, SREBP1.
Collectively, our data elucidated DHM protective effects in hepatocellular, and suggesting that
DHM can be a novel method for liver protection.

9
Part I. Utilize the electrophysiological whole-cell voltage-patch-
clamp recording to gain knowledge of DHM activity on α5-
GABAARs exposed to ethanol

Chapter 1 Introduction

1.1 Alcohol use disorders (AUD)

Repeated alcohol use leads to the development of alcohol use disorders (AUD) characterized by
alcohol withdrawal syndrome (AWS), physical and psychological dependence and the loss of
ability to control excessive drinking (Shen et al., 2012).

Excessive alcohol use is the third leading cause of preventable death in the United States (Sayed
and French, 2016) and alcohol consumption is associated with 88 000 US deaths annually. The
NIH report demonstrates that in 2013, about 9% of adult men and 5% of adult women are
affected by AUD, however, only 14.6 % of people with alcohol abuse or dependence receive
effective treatments (Huebner and Kantor, 2011).

Although treatment techniques and tools to address AUD have multiplied over the last 30 years,
there is still no effective therapeutic agent for AUD without major side-effects because of the
multiple targets of alcohol in the body (Cohen et al.,2007). There are only three medicines
(Disulfiram, Naltrexone, Acamprosate) have been approved by the Food and Drug

10
Administration (FDA) to treat alcohol dependence. Therefore, it is crucial to discover and
develop other novel and effective medications for AUD with less side effects.

1.2 Alcohol’s actions on GABAARs

The GABA (γ-aminobutyric acid) receptors (GABAARs) are the major inhibitory
neurotransmitter receptors in the brain (Mizielinska et al., 2006), when the neurotransmitter
GABA binds to the GABAARs, the ion channel open and the Cl
-
flow into the cell which in turn
inhibit the neurons. GABAARs are heteropentamers consisted of 5 subunits (Figure1) made up
from 19 known subunits (α1–6, β1–3, γ1–3, δ, ε, θ, π and ρ1–3). Many GABAARs contain two α
subunits, two β subunits and one γ subunit. GABAARs have two GABA binding sites formed by
α and β subunits and one benzodiazepine binding site formed by one α subunits (α1, α2, α3 or
α5) and a γ subunit (Rudolph and Knoflach, 2011).

Figure 1.1 The basic structure of GABA ARs

11
The pie chart (Figure 1.2) lists the distribution of major types of α-GABAARs in the brain. When
the α-containing GABAARs are activated by GABA or benzodiazepines, different mental
inhibitory action will be mediated. For example, α2- GABAARs have been found to mediate the
anxiolytic-like activity (Low, 2000), whereas α1- GABAARs mediate the sedative action of
diazepam (Chuang and Reddy, 2018).

Figure 1.2 Pharmacological effect and brain distribution of α-GABA ARs

The study of alcohol’s actions on brain started from 1970, the first study found that ethanol
inhibited the release of the signaling molecule from the cortex (Phillis et al., 1971), the possible
mechanism is the inhibition of voltage-dependent ion channels by ethanol (Harris et al., 1980).
One prophetic study found that ethanol potentiated the neurotransmission by enhancing the
activity of the neurotransmitter GABA in the spinal cord (Davidoff et al., 1973). This discovery
was ignored until the mid-1980s, but since then, GABAARs have emerged as a major target for
ethanol’s actions and continued to be an area of intense research interest (Kumar et al., 2009).
The behavioral effects of acute ethanol administration mimic those of benzodiazepines and
barbiturates (Woo et al., 1979; Liang et al., 2014), drugs which are known to act on the

12
GABAergic system. This observation suggests the effects of ethanol on GABAARs, however, the
precise mechanisms underlying the physiological effects of ethanol exposure are still under
investigation (Fleming et al., 2009).

Several hypotheses have been put forward to explain ethanol’s effects on GABAergic
neurotransmission. First, ethanol can act directly on GABAARs to increase their response to
GABA (Roberto et al., 2003a; Boyle et al., 1993; Sanna et al., 2003; Weiner et al., 1994).
Second, ethanol acts at presynaptic part of neurons to increase the amount of GABA released
(Carta et al., 2003; Devlin et al., 1999). Third, ethanol acts indirectly to increase local synthesis
of GABAergic neuroactive steroids that potentiate GABAARs function (Sanna et al., 2003;
VanDoren et al., 2000).

13
1.3 DHM effects on AUD

Dihydromyricetin (DHM) (Figure 1.3) is a flavonoid derived from the natural plant named
Hovenia dulcis, which is traditionally use for treatment of alcohol hangovers (Shen et al., 2012;
Liang et al., 2014; Wang et al., 2016). DHM has been identified to exhibit the protective effects
against alcohol intoxication and alcohol tolerance by interacting with GABAARs (Liang et al.,
2014).

At the cellular level, DHM treatment antagonized potentiation of GABAARs induced by alcohol
consumption. Therefore, DHM could be used as a therapeutic candidate for alcohol use disorders
(Liang et al., 2014; Shen et al., 2012).

Figure 1.3 The structure of DHM and pure DHM product

14
1.4 Studying α5-GABAARs and its agonist/antagonist- electrophysiological whole-cell voltage-
patch-clamp recording

GABAARs are ligand-gated channel receptors, by binding to their specific neurotransmitter
GABA, the channels open and cause the influx of Cl
-
, therefore leading to the changes in
membrane voltage (Vm) (Naito et al., 2014). Since the Vm is variable, it is necessary to control
the Vm in order to study the chemical effect on GABAARs. This procedure, known as voltage
clamp, utilizes two intracellular electrodes, one to monitor Vm and the other to inject a current to
adjust Vm to desired values (Naito et al., 2014).

The electrophysiological whole-cell voltage-patch-clamp recording is showing in Figure 1.4.
Through injection of the current into a cell with one microelectrode, the membrane voltage is
“clamped” as -70mV like the average neuron. When there is no GABA applied to activate the
GABAARs, there is no change of Vm. However, when the GABA is applied, the ion channels
open and let the Cl
-
ions influx, which causes the changes in the Vm. To keep the membrane
voltage as constant -70mV, the microelectrode needs to inject the current to counteract the
voltage change and this “injected” current is recorded, reflecting the activity of the GABAARs.

15

Figure 1.4 The electrophysiological whole-cell voltage-patch-clamp recording: A. When
GABA
A
Rs are not activated GABA, there is no change in Vm; B. When the GABA is applied,
GABA ARs are activated, Cl
-
influx into the cells and the change of Vm is recorded.

A B

16
1.5 Goals and specific aims

The goal of my project is to investigate DHM effects on certain GABAARs. The effects of
ethanol on GABAARs are different and the sensitivity of the GABAARs to ethanol varies with
subunit composition. It reported that ethanol potently (at low mM concentrations) facilitates α4-
or α6-GABAARs (Sundstrom-Poromaa et al., 2002; Wallner et al., 2003), but the results are hard
to be replicated and this inconsistency may be due to the volatile properties of ethanol,
differences in expression levels of GABAARs or unknown variables (Borghese et al., 2007). α5-
GABAARs are mainly located in extra-synaptic membrane of GABAARs synapses. They are
sensitive to ethanol (Wallner et al., 2003) and very easy to express in Xenopus oocytes as a
GABAARs study tool. Therefore, we carry up α5-GABAARs to study DHM effects on ethanol
and GABAARs. DHM has been proved to ameliorate the adverse effects caused by alcohol
consumption (Shen et al., 2012; Liang et al., 2014). Our preliminary data show that DHM can
potentiate α5-GABAAR current. To confirm the effects of DHM on ethanol potentiated α5-
GABAARs, I set up the experiments.

Specific aims:
(1) To test the hypothesis that ethanol is able to enhance the activation of α5-GABAARs in a
concentration dependent manner.
(2) To study the interactions among ethanol, DHM or DHM related compounds and α5-
GABAARs.
(3) To evaluate DHM containing products in the market (Morning recovery version1/version2,
Flyby and Thrive) have the different effects on reversing ethanol-induced toxics on α5-
GABAARs.

17
Chapter 2 Experimental Procedures

2.1 Materials and Equipment

Buffers: 5X concentrated Modified Barth’s solution (MBS) contained the following (in mM
concentration) NaCl 88, KCl 1.8, HEPES 5, Mg2SO4•7H2O 0.82, NaHCO3 2.4, CaCl2•2H2O
0.41, and Ca(NO3)2•4H2O 0.33, adjusted to pH 7.5 with 5M NaOH. 1X concentrated MBS was
diluted from 5X MBS with purified water immediately prior to using. Oocytes incubation
medium contains (in mM concentration) NaCl 96, KCl 2, MgCl2 1, CaCl2 1, HEPES 5, and
pyruvic acid 2.5 with 1% heat inactivated HyClone horse serum (VMR, San Dimas, CA), 1%
neomycin (Sigma or Molecular Probes) and 1% penicillin-streptomycin (Sigma or Molecular
Probes), adjusted to pH 7.4 with 5M NaOH, the oocytes incubation medium is stable for 1 month
store at 4 °C.

DHM-containing products in the market include Morning Recovery version 1 and 2 (Liquid, 82
labs), Flyby (Capsules, Flyby Ventures) and Thrive (Capsules, CHEERS alcohol health) were
used. They are advertised to be best products in relieving hangover with similar DHM
concentration(~4.7mM/dosage). The products were prepared as 1mM stock and different
concentrations of the products were applied to the oocytes. Morning recovery was diluted in 1X
MBS buffer directly, Thrive and Flyby were dissolved in dimethyl sulfoxide (DMSO) (Sigma-
Aldrich, St. Louis, MO), filtered and finally diluted in 1X MBS buffer.

Xenopus oocytes were purchased from Ecocyte Bioscience LLC, USA. Electrophysiological
experiments were conducted 48 hours after cDNA injection.

18
cDNA injection: Each oocyte was injected with 40 ng of DNA mixture (α5: β3: γ2=1:1:10) by
Nanoject II Nanoliter injection system (Drummond Scientific, USA). The oocytes were
incubated in incubation medium at 17 °C and used in electrophysiological recordings for 3-7
days after injections.

Electrophysiological whole-cell voltage-patch-clamp recording: Two-electrode voltage clamp
(TEVC) recordings were performed using the Warner instrument model OC-725C oocyte clamp
(USA). The Vm of oocytes were clamped as -70mV, and the currents were recorded on a chart
recorder (Barnstead/Thermolyne, USA).

19
2.2 α5-GABAARs expression in Oocytes

The micropipettes were pulled (P-30, Sutter Instruments, Novato, CA) from borosilicate glass
(1.2mm thick- walled filamented glass capillaries (WPI, Sarasota, FL)) which was filled with
mineral oil. The tips of micropipette were broken to expel the extra mineral oil to leave the space
for cDNA of α5-GABAARs. Arrange the oocytes in the injection dish, punch the oocytes at the
top middle site of nucleus part with micropipette and hit the nucleus of the oocytes which located
in the halfway of the oocytes. The brief procedures of the cDNA injection are listed in figure 2.1.

Figure 2.1 Main procedures of Xenopus oocytes cDNA injection: The major steps are (1) prepare
the injection dish and micropipette; (2) Set up the Nanoject II system; (3) Fill the micropipette
with the cDNA; (4) Punch the oocytes and inject cDNA

20
2.3 Electrophysiological whole-cell voltage-patch-clamp recording

Electrophysiological whole-cell voltage-patch-clamp recordings were performed according to the
techniques reported previously (Davies et al., 2004; Perkins et al., 2009). Briefly, micropipettes
that pulled from borosilicate glass were filled with 3 M KCl to yield resistances of 0.5-3 MΩ.
Electrophysiological recordings were conducted in an oocyte recording chamber (Type RC 3Z;
Warner Instruments, Hamden, CT) which is used to hold the impaled oocyte. The voltage and
current electrodes are fixed on a vibration resistant magnetic steel base, the Vm of the oocytes is
controlled to -70 mV by oocytes clamp OC-725C (Warner Instruments; Hamden, CT). The
oocyte recording chamber was continuously perfused with MBS ± drugs using a Dynamax
peristaltic pump (Rainin Inst Co., Emeryville, CA) at 3 mL/min using 18-gauge polyethylene
tubing (Becton Dickinson, Sparks, MD) and the clamped currents were recorded on a
Barnstead/Thermolyne chart recorder (Barnstead/Thermolyne, Dubuque, IA). All experiments
were performed at room temperature (20-23°C).

21
2.4 Oocytes treatments

1. Application of agonist
Oocytes expressed α5-GABAARs were exposed to 1 μM-1000 μM of GABA for 20 seconds at a
rate of 3 ml/min, with 5 minutes’ washout periods between applications of GABA to ensure
receptor is recovered completely.

Figure 2.2 The timeline of GABA application to Xenopus oocytes.

2. Application of ethanol
When using high GABA concentrations, like the concentration that produce 50% of the maximal
effect (EC50) of the α5-GABAARs, the potentiation of Cl
-
currents by ethanol is difficult to
measure because the sensitivity of the α5-GABAARs decreased at high GABA concentrations
(Mascia et al., 2000). Therefore, different concentrations of GABA were applied to the oocytes
and the concentration producing 20% of the maximal effect (EC20) was used. EC20 was used as
control pre-ethanol treatment, when the Cl
-
currents caused by EC20 were stable (within ± 10% of
each other), ethanol (1mM to 300mM) was applied. Oocytes were pre-incubated with ethanol for
20 seconds followed by co-application of ethanol and GABA for 20 seconds. Washout periods

22
(5-15 min, depending on ethanol concentration tested) were performed between GABA and
ethanol applications to ensure complete re-sensitization of α5-GABAARs.

Figure 2.3 The timeline for ethanol application to Xenopus oocytes. “Minus” refers to the
ethanol without GABA, “plus” refers to the mixture of ethanol and GABA. To get reliable
result, >30 oocytes were used

3. Application of DHM-contained compound
Based on the procedures of the ethanol application, when getting the stable Cl
-
currents
potentiated by ethanol, different DHM-containing compounds are applied in the presence of the
ethanol and GABA.

Figure 2.4 The timeline for DHM-containing products application to Xenopus oocytes

Another
Concentration
0 ’ ’ 5 ’ ’ 10 ’ ’11 ’ ’ 0 ’ ’ 5 ’ ’ 10 ’ ’ 11 ’ ’ 0 ’ ’ 5 ’ ’ 10 ’ ’11 ’ ’
0 ’ ’
GABA GABA - + GABA GABA - + GABA GABA - +

Add
Ethanol to
- , +
Group2 Group3 Group1

23
Chapter 3 Results and Conclusions

3.1 The response of α5-GABAARs to escalated concentration of GABA

Figure 3.1 The response of α5-GABA ARs to escalated concentrations of GABA (n>10)

The extent of GABA induced activation of α5-GABAARs is concentration-dependent, the higher
amount of GABA applied, the bigger response of α5-GABAARs observed. When the
concentration of GABA keeps increasing, the α5-GABAARs tend to be saturated and the
excessive amount of GABA will conversely suppress the activation of α5-GABAARs.

24
3.2 Ethanol effects on α5-GABAARs

Figure 3.2 The GABA current potentiation caused by ethanol treatments

Suppose the potentiated Cl
-
current is 100% when the α5-GABAARs are activated by 5 μM of
GABA, when different concentrations of ethanol are applied, the potentiated Cl
-
current
increased in a concentration-dependent manner.
This data verified that ethanol acts on α5-GABAARs and leads to depolarized inhibitory
postsynaptic potential.

25
3.3 DHM like products reduce ethanol-induced potentiation effects on α5-GABAARs

Figure 3.3 The percentage of α5-GABA ARs potentiation of pure DHM (control) and DHM-
containing products.
Figure 3.3 illustrated effects of different DHM-containing products in the markets on α5-
GABAARs. Pure DHM (control), Morning recovery, thrive and flyby have potentiation effects on
α5-GABAARs, however, other products like PS, LS, RES and NTOO inhibit the activation of the
α5-GABAARs. When the α5-GABAARs are inhibited, the structure of the GABAARs changed
and it hard to evaluate the product effects, therefore, the products that have similar effects of
pure DHM are evaluated in the following experiments.
DHM can cause the potentiated Cl
-
current of α5-GABAARs. The mechanism involved is still not
clear, but previous studies showed that DHM may have the ability to bind to the BZ site and
cause allosteric modulation of α5-GABAARs, that is, the binding of DHM to BZ sites will
potentiate the binding of GABA to GABA-binding sites.

26

Figure 3.4 DHM-contained products inhibit EtOH potentiated currents of α5-GABA ARs (n=9~12).

Figure 3.4 showed that DHM-containing products can reverse ethanol potentiated Cl
-
currents of
α5-GABAARs, morning recovery version 2 has stronger ability in inhibiting ethanol-induced
potentiation of α5-GABAARs in comparison with Flyby, Thrive and Morning recovery version 3.
This data could serve as evidence to evaluate the anti-hangover effects of those products.

Previous data showed that when only applying the ethanol or DHM (including DHM-containing
products) to the α5-GABAARs, the potentiated Cl
-
currents were observed. However, when both
of ethanol and DHM are applied to the α5-GABAARs, the ethanol-induced Cl
-
currents
potentiation is reduced by DHM and DHM-containing products. This result suggests that DHM
can inhibit the GABAergic activation caused by ethanol.

27
Part II. In vitro study of hepatoprotective effects of DHM

Chapter 4 Introduction

4.1 Alcoholic liver disease (ALD)

Alcoholic liver disease (ALD) is a result of overconsuming which comprises clinical symptoms
including fatty liver, alcoholic hepatitis (AH), and cirrhosis with its complications. ALD is a type
of chronic liver diseases, usually, by the time the liver damage is found, it is already irreversible.
It is one of the main causes of chronic liver disease worldwide and accounts for up to 48% of
cirrhosis-associated deaths in the United States (Yoon et al., 2016).

Steatosis is the earliest manifestation of ALD, it is a benign consequence of alcohol abuse and it
is reversible if the alcohol consumption is ceased. Steatosis is characterized as the deposition of
fat in liver, the main mechanism that contributes to the enhanced lipid synthesis in liver is the
higher expression of the lipogenic enzymes which are encoded by transcription factor sterol
regulatory element binding protein-1 (SREBP-1) (Eberle et al., 2004; Osna et al., 2017), the
homeostasis regulated by SREBP-1 is shown in Figure 4.1. Besides, alcohol consumption causes
the release of the fatty acid from the adipose tissue that have been taken up by the liver and
esterified into triglycerides, thereby exacerbating fat accumulation in the liver (Wei et al. 2013).

28
Figure 4.1 The lipid homeostasis regulated by SREBPs. Low level of sterol in cells will cause the
cleavage of the SREBP by site-1 protease (S1P), then the NH2-terminal bHLH domain is released
and transported to the nucleus in where it will bind to the sterol response element (SRE) in the
enhancer region of target genes and thereby increasing the sterol synthesis. Finally, the
increased sterol level will inhibit the cleavage of the SREBP (Horton et al., 2002).

Alcoholic hepatitis will be developed after continued alcohol, it is more severe than steatosis and
is associated with high short-term mortality. It is an inflammatory type of liver injury
characterized by swelling, dying hepatocytes and neutrophilic infiltration. Excessive exposure to
the ethanol causes the continuous activation of the resident macrophages in the liver and leads to
inflammatory (Osna et al., 2017). Hepatitis and chronic alcoholism will lead to the formation of
fibrosis and cirrhosis. The liver injury activates hepatic stellate cells (HSCs) and causes the
abnormal deposition of extracellular- matrix components that characterize fibrosis (Friedman
2008). Cirrhosis is a late stage of fibrosis (scarring) of the liver, the scar tissue forms to repair the

29
liver injury, as cirrhosis progresses, more and more scar tissue forms, making it difficult for the
liver to function and the advanced cirrhosis is life-threatening (Osna et al., 2017).

4.2 Hepatoprotection effects of DHM

Previous studies have suggested that DHM exhibits protective effects on the liver injury. DHM
has been found to exhibit anti-alcoholic, anti-lipid peroxidation and anti-inflammatory effects
(He et al., 2003; Shen et al., 2012) by improving the expression of the ethanol metabolic
enzymes and anti-oxidative enzymes or directly scavenging the free radicals.

Excessive consumption of alcohol leads to oxidative stress, the cellular homeostasis imbalance
characterized as reactive oxygen species (ROS) production overweighting the antioxidant
enzyme system (Li et al., 2017). DHM is able to restore the metabolic abnormality in
mesenchymal stem cells, improve the function of antioxidant system, and inhibit mitochondria-
dependent apoptosis (Li et al., 2016).

Accumulated evidences support that DHM can ameliorate non-alcoholic fatty liver diseases. In-
vivo study showed the effects of DHM on preventing fatty liver. It was observed that the
treatment with DHM decreased the body weight and level of triglycerides in a rat model of fatty
liver (Guo et al., 2019). Besides, in-vitro study also showed that DHM reduces oleic acid-
induced lipid accumulation in HepG2 cells (Xie et al., 2016).

30
In addition, DHM can also induce autophagy. DHM may promote AMPK and ULK1
phosphorylation and improve mitochondrial functions (Wu et al., 2017). mTOR, a master
regulator belonging to PI3K related kinase family, regulates the activation of autophagy. mTOR
can be phosphorylated and regulated by AMPK through regulating TSC2 and TSC1/2
phosphorylation. DHM has been reported to activate AMPK, leading to inhibition of mTOR and
activation of autophagy (Xia et al., 2014).

4.3 Oxidative metabolism of alcohol in liver

1. The main metabolism pathways of alcohol in liver

Figure 4.2. The major enzymes involved in and the metabolites produced during the process of
ethanol microsomal oxidation

After ethanol consumption, the ethanol could not be excreted directly through the kidney, it
needed to be metabolized first, mainly by the liver. Most of the ethanol is broken down by

31
alcohol dehydrogenase (ADH) which transform ethanol into an intermediate byproduct, a known
carcinogen called acetaldehyde (Lieber, 1997).

The acetaldehyde is relatively slowly metabolized into acetate by another enzyme called
aldehyde dehydrogenase (ALDH) (Lieber, 1997). Acetate is then broken down to carbon
dioxide and water mainly in tissues such as heart, skeletal muscle and brain cells other than liver
(Ginestaldacruz et al., 1975).

When ethanol concentration is low, the ethanol is mainly oxidized by ADH. However, as blood
alcohol concentration increases, so does the activity of CYP2E1 in metabolizing ethanol. The
excessive ethanol could be metabolized into acetaldehyde by cytochrome P450 enzyme, and then
the acetaldehyde could be oxidized by CYP2E1 or ALDH. Reactive oxygen species (ROS), such
as hydrogen peroxide and superoxide ions, generated by CYP2E1 are contributors to the pro-
inflammatory profile of alcohol-related liver damage (Karadayian et al., 2015; Seth et al., 2011).
Compounds called fatty acid ethyl esters (FAEEs) produced by the interacting between alcohol
and fatty acids also contribute to damage to the liver (Vonlaufen et al., 2007).

2. The consequences of the ethanol metabolism
As mention before, ADH is the major pathway for ethanol metabolism. During this process,
nicotinamide adenine dinucleotide (NAD
+
) is used as an electron acceptor and converted to
NADH. The shift of NAD
+
/NADH ratio can cause an increase in the rate of fatty acid synthesis
and a decrease in mitochondrial beta-oxidation of free fatty acids. Besides, the accumulated
acetate formed in the process of ethanol metabolism will cause the buildup of acetyl CoA, the

32
basic materials for lipid synthesis and finally, leading to lipid accumulation and the development
of steatosis.

Although the acetaldehyde produced by ADH is short lived, usually existing in the body only for
a brief time before it is further broken down into acetate, it has the potential to cause significant
damage because the reaction of metabolizing the acetaldehyde into acetate is relatively slow. The
formation of acetaldehyde during the process of ethanol metabolism will upregulate the lipid
synthesis pathway and cause lipid accumulation in liver. It also forms adducts with the protein in
hepatocytes and therefore causing severe damage to the liver by impairing protein secretion
(Freeman et al.,2005). Furthermore, acetaldehyde is highly toxic because it interferes with the
replication of DNA and inhibit the process of DNA repair (Seitz et.al, 2007). Studies have shown
that people who are exposed to large amounts of acetaldehyde are at greater risk for developing
certain cancers. Furthermore, acetaldehyde promotes enhanced GSH utilization/turnover and
significantly depletes GSH stores (Lieber, 1997), thus contributing to oxidative stress

The CYP2E1 pathway dominates following high ethanol consumption, the excessive amount of
ethanol promotes the synthesis of CYP2E1 and reduces its degradation in hepatocytes (Beier and
McClain, 2010). An animal study reported that for the mice who are lack of CYP2E1 enzyme,
they showed increased ratio of liver to body weights, serum endotoxin, and hepatic levels of
endobacteria (Valdes-Arzate et al., 2006). This study showed that the CYP2E1 is an important
pathway to protect the whole organism but causing damage to the liver. CYP2E1 has also been
shown to contribute to the formation of reactive oxygen species (ROS) in hepatocytes through
Kupffer cells (Cao et al., 2005). ROS are involved in liver injury through induction of tumor

33
necrosis factor alpha (TNF-α) and activation of c-Jun N-terminal kinase 1 (JNK1). These two
signaling pathways are involved in hepatocellular apoptosis (Colell et al., 1998). Ethanol itself
can also add to the production of ROS and oxidative stress by altering the levels of certain metals
(Tsukamoto et al. 1995) and reducing the synthesis of certain agents which can reduce the ROS
(Fridovich 1997).

34
4.4 Goals and specific aims

The goal in this part is to investigate the liver protective effects of DHM in vitro. Firstly, the
DHM influences on the cell viability of HepG2 and VL-17A cells had been studied. Previous
research reported that DHM has the ability to inhibit the proliferation of the hepatocellular
carcinoma cells, advocating the potential anti-cancer effect of DHM. In this thesis, different
fluorometric assays were performed to evaluate the uncertain effects of DHM on HepG2 and
VL-17A cells.

DHM is also described to reduce the ethanol-induced liver injury by increasing the expression of
the ADH and ALDH enzymes. As a result, the production of the acetaldehyde should decrease
while the generation of acetate is supposed to increase. To test this hypothesis and evaluate the
DHM effects on ethanol metabolism, the amount of acetaldehyde and acetic acids were
measured.

Another adverse effect of ethanol consumption is the production of oxidative stress, as an
antioxidant, DHM is supposed to decrease the ROS induced by alcohol. Specifically, the
decreased level of intercellular ROS and increased cytosolic catalase expression were discovered
with DHM treatment, suggesting that the antioxidant activity of DHM appears to be beneficial in
reducing ethanol-generated ROS. To further study and understand DHM’s antioxidant capability,
the levels of GSH, an important cellular component consumed for preventing oxidative stress,
were measured in this study.

35
Previous clinical trials indicated that DHM could improve hepatic steatosis in patients with non-
alcoholic fatty liver diseases, but the DHM effects on alcoholic steatosis remained elusive. To
verify DHM also has the beneficial effects on alcohol-induced lipid accumulation, steatosis assay
and western blot were performed.

Furthermore, DHM is said to be able to induce cell autophagy in human melanoma cells. The
mechanisms involved are the activation of the phosphorylation of AMPK and ULK1 and
inhibition of the mTOR which caused by DHM, western blot was utilized to test this hypothesis
and study the mechanism further.

Collectively, the main goal of this study is to assess the effects of dihydromyricetin (DHM) as a
hepatoprotective candidate in reducing hepatic injury caused by alcohol consumption.

Specific aims:
(1) To demonstrate the positive effects of DHM on HepG2 and VL-17A cells’ viability.
(2) To test the hypothesis that DHM is able to accelerate the ethanol metabolism by
decreasing the production of acetaldehyde and increasing the production of acetic acid.
(3) To test the hypothesis that DHM can increase the creation of GSH which is consumed a
lot in alcohol treated cells for preventing the damage caused by oxidative stress.
(4) To test the beneficial effects of DHM in reducing lipid accumulation.
(5) To study the effects of DHM in inducing autophagy.
These are addressed in the following Chapters.

36
Chapter 5 Experimental procedures

5.1 Cell culture
HepG2 cells (provided by the lab of Dr. Bangyan L. Stiles, University of Southern California.)
were cultured in MEM medium with 10% FBS, 5% penicillin-streptomycin, and grown in an
atmosphere containing 5% CO2 at 37°C (Faedmaleki et al., 2014). VL-17A cells (provided by
Dr. DL Clemens, University of Nebraska Medical Center and Veterans Affairs Medical Center,
Nebraska USA.) 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
(Faedmaleki et al., 2014; Osna et al., 2005).

5.2 Trypan blue assay
Coated the six-well plate with poly-D-lysine (Invitrogen), the cells were detached by trypsin and
seeded in six-well plates (1000,000 cells/well). Treat the cells with 5μM, 50μM and 100μM of
DHM and incubate for 24 hours. Recollect the cells, mix with 0.4% of trypan blue as 10:1 ratio
and read the live cells percentage by Countess II FL Automated Cell Counter (Thermo Fisher
Scientific). The percentages of viable cells were compared with the control. The experiments
were performed independently at least in triplicate.

5.3 MTT assay
Both of HepG2 cells and VL-17A cells were plated in 96-well plates (100,000 cells/well), the
cells were treated with ethanol only (50mM and 100mM) and ethanol (50mM and 100mM) with
the presence of 5μM of DHM. Following 24 h, culture medium was removed and the same

37
volume of medium, containing same amount of MTT solution, was added into each well and
incubated at 37 °C for 4 h. To avoid the influence caused by the medium, remove all but 25μL of
medium from the cells and 0.01 M DMSO was added to dissolve the formazan created. Incubate
the plate at 37 °C for another 10 minutes, the absorbance was read by Perkin Elmer 2103
EnVision Multilabel plate reader (Perkin-Elmer, Rodgau, Germany) at 540 nm. The experiments
were performed independently at least in triplicate.

5.4 Alamar blue assay
HepG2 and VL-17A cells were seeded at the density of 100,000 cells per well in 96-well plates.
The cells were treated with acetaldehyde and DHM, the plates were incubated under standard
conditions of 37 °C and 5% CO2 in a humidified atmosphere and the absorbance was read by
Perkin Elmer 2103 EnVision Multilabel plate reader (Perkin-Elmer, Rodgau, Germany) at 570
nm after 24 h. The experiments were performed independently at least three times.

5.5 Acetaldehyde and Acetic acid measurements
Acetaldehyde and acetic acid production were measured using the Acetaldehyde and Acetic acid
Assay Kit (Megazyme, Bray, Ireland) in 96-well plates. Acetaldehyde ammonia trimer and acetic
acid were used as the standard according to the manufacturer’s instructions for the acetaldehyde
assay and acetic acid assay, respectively. VL-17A cells and HepG2 cells were incubated with 50
mM ethanol and either 100 nM - 50μM DHM, or untreated for two hours before measurements
using the Perkin Elmer 2103 EnVision Multilabel plate reader (Perkin-Elmer, Rodgau,
Germany). The experiments were performed independently at least three times.

38
5.6 Glutathione assay
The measurement of the production of glutathione were performed using the Glutathione Assay
Kit (Cayman, MI, USA) in 96-well plates. Disulfide dimer GSSG was used as the standard. VL-
17A cells and HepG2 cells were incubated with 50mM and 100mM ethanol with or without the
existence of 5μM DHM, or untreated for 24 hours before cell lysate. The cells were collected and
deproteinated by MPA reagent (Sigma-Aldrich 239275) and triethanolamine (Sigma-Aldrich,
Item No. T58300), the absorbance was read by Perkin Elmer 2103 EnVision Multilabel plate
reader (Perkin-Elmer, Rodgau, Germany) at 412 nm, the GSH assays were performed
independently for three times.

5.7 Protein extraction and Western blot analysis
HepG2 and VL-17A cells (9x10
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, p-ULK1, ULK1, p-mTOR, mTOR and SREBP1) 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

39
images were visualized with enhanced chemiluminescence detection reagent and Chemi-Doc
(Bio-Rad) imaging device. Anti-rabbit 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.

5.8 Confocal imaging
Grow the cells on coverslips in 6-well plates (40% confluency). Treat the cells with 50 mM –
100 mM ethanol, DHM, and a 2:1 ratio of oleic acid to palmitic acid (4 mM fatty acids), incubate
for 24 hours at 37 degrees. Wash the cells with PBS for three times, use 4% para- formaldehyde
(PFA) solution for cell fixing. Stain the cells with DAPI and Nile red at room temperature for 15
minutes in the dark. Take out the coverslips and sealed on the glass slides by nail polish. PBS
washouts were conducted before and after chemical treatments, the staining results were imaged
using Confocal Microcrope (Zeiss LSM 780 AxioObserver.Z1).

40
Chapter 6 Results and Conclusion

6.1 Higher amount of DHM can inhibit the proliferation of Hepatocellular carcinoma cells

Figure 6.1 The percentage of live cells after 24 hours exposure to DHM. A) HepG2 and B) VL-17A
cells were cultured in 5 μM–100 μM DHM for 24 hours before trypan blue assay. Data
represented as mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01 were
considered statistically significant compared with corresponding ethanol controls; n=3.

As demonstrated in Figure 6.1, when the cells were treated with 5 μM of DHM, the percentage
of cells alive is similar to that of the untreated group, this result suggests that the proliferation of
HepG2 and VL-17A cells is not affected by the lower concentration of DHM.
However, the increased concentration of the DHM caused the increased number of dead cells.
Especially for the cells treated with 100 μM of DHM, the percentage of live cells are much lower
compared with the untreated group, for HepG2 cells, the percentage of live cells decreased about
23% but for VL-17A cells, only 9%. The trypan blue data illustrates that the excessive amount of
*
*

A B

41
the DHM inhibits the proliferation of both cell lines, however, the extent of inhibition varies
according to the cell lines.

6.2 DHM improves the mitochondrial respiratory capacity in VL-17A cells

Figure 6.2 Mitochondrial activity detected by MTT assay. A) HepG2 and B) VL-17A cells were
exposed to 50 and 100 mM of ethanol and treated with 5 μM DHM or untreated for 24 hours.
Data represented as mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01
were considered statistically significant compared with corresponding ethanol controls; n=3.

DHM has been shown to promote mitochondrial biogenesis and improve mitochondrial
functions. However, MTT data showed the contradictory result. When the HepG2 and VL-17A
cells were treated with 5 μM of DHM, the absorbance is lower compared with untreated group,
indicating the reduced mitochondrial activity and function. Therefore, DHM may exhibit its anti-
proliferation effects on HepG2 and VL-17A cells by interfering the biogenesis of mitochondria.
For HepG2 cells, because they lack the critical enzymes (ADH/CYP2E1) for ethanol
metabolism, only small amounts of toxic byproducts or ROS will be produced, the treatment of
* *

A B

42
ethanol does not influence the mitochondria function of HepG2 cells significantly. However, the
existence of DHM leads to lower mitochondria activity, indicating that DHM may activate other
pathways in HepG2 cells to metabolize the ethanol and as a result, other toxic metabolites
created and caused the mitochondrial damage.
The data for VL-17A cells confirmed our hypothesis that DHM can improve the mitochondrial
function and activity reduced by ethanol. Figure 6.2 showed that for VL-17A cells treated with
50 mM and 100 mM ethanol in combination with DHM, the cell viability increased 26% and
19%, respectively, suggesting the positive role DHM played in reducing the ROS and oxidative
stress.

43
6.3 DHM decreases the cytotoxicity caused by acetaldehyde

Figure 6.3. The Alamar blue luminescence of (A) HepG2 cells and (B) VL-17A cells treated with
ACH and DHM. Data represented as mean ± SD of three independent experiments.
*p < 0.05 and p** < 0.01 were considered statistically significant compared with corresponding
ethanol controls; n=3.

For both cell lines, when treating with 100 μM of acetaldehyde (ACH), the alamar blue
luminescence decreased significantly which indicates that 100 μM of ACH causes severe cellular
cytotoxicity.
The cell viability of HepG2 and VL-17A increased significantly with the presence of the DHM.
For HepG2 cells, the middle concentration of DHM (5 μM) exhibited better effect in reducing
cellular toxicity. While for VL-17A cells, the higher concentration of DHM, the better anti-
toxicity effect was observed. In general, DHM showed hepatocellular protective ability by
promoting the metabolism of ACH.

A B

HepG2 + ACH Viability
Alamar Blue Luminescence (AU)
Untreated
100 uM ACH
1 uM DHM + 100 uM ACH
5 uM DHM + 100 uM ACH
10 uM DHM + 100 uM ACH
0.0
0.2
0.4
0.6
0.8

VL-17A + ACH
Alamar Blue Luminscescence (AU)
Untreated
100 uM ACH
1 uM DHM + 100 uM ACH
5 uM DHM + 100 uM ACH
10 uM DHM + 100 uM ACH
0.0
0.2
0.4
0.6
0.8
1.0
* * * *

44
6.4 DHM accelerates the ethanol metabolism

Figure 6.4. DHM increases the acetaldehyde metabolism in HepG2 and VL-17A cell models.
HepG2 cells and VL-17A cells were exposed to 50 mM ethanol and treated with 100 nM – 50
μM DHM, the production of ACH (A) and acetic acid (B) were measured by fluorometric
methods. **p < 0.05 and **p < 0.01 were considered statistically significant compared with
corresponding ethanol controls; n=3, ACH = acetaldehyde.

The amount of acetaldehyde and acetic acid produced in both cell lines are measured by
colorimetric assays.

A
B

H e p G 2
[A c e tic A c id ] (u M )
U n tre a te d
E 5 0 + D M S O
E 5 0
1 0 µ M D H M
2 0 µ M D H M
3 0 µ M D H M
4 0 µ M D H M
5 0 µ M D H M
0
1 0
2 0
3 0

[A c e tic A c id ] (u M )
U n tre a te d
E 5 0 + D M S O
E 5 0
1 0 µ M D H M
2 0 µ M D H M
3 0 µ M D H M
4 0 µ M D H M
5 0 µ M D H M
0
1 0
2 0
3 0
V L -1 7 A
HepG2
HepG2
VL-17A
VL-17A
*
*

45
Compared with HepG2 cells treated with 50 mM of ethanol, the acetaldehyde produced is lower
while the acetic acid generated is higher in VL-17A cells treated with 50 mM of ethanol, this
result suggests the greater metabolic capacity of VL-17A cells.
Although HepG2 cells only express a low level of ADH, the amount of ACH produced is not as
low as we expected, meaning that other ethanol metabolism pathway might be activated by an
excessive amount of ethanol.
Concentrations of DHM ranging from 1 μM to 30 μM significantly enhanced the metabolism of
ethanol, meanwhile, 10 μM to 20 μM seemed to be effective in enhancing ALDH metabolism of
acetaldehyde in both cell lines, as observed by the reduction in acetaldehyde and increased
production of acetic acid.
However, DHM concentrations above 30 μM did not reduce the concentration of acetaldehyde or
increasing the concentration of acetate in both cell lines, thereby eliciting no response in regard
to the ethanol metabolic pathway. Therefore, it appears that concentrations of DHM between 1
μM and 20 μM may be helpful in enhancing ethanol metabolism.

46
6.5 DHM restores the GSH depletion in VL-17A cells

Figure 6.5 Concentration of GSH produced in (A) HepG2 cells and (B) VL-17A cells treated with
50mM, 100mM ethanol with and without 5uM of DHM. *p < 0.05 and **p < 0.01 were
considered statistically significant compared with corresponding ethanol controls; n=3.

In ethanol metabolism process, the GSH is used for ethanol detoxication. Ethanol metabolism is
associated with a reduction in the availability of reduced glutathione (GSH), thereby reducing the
overall protection of the cell against oxidative stress caused by ethanol and related factors. The
GSH depletion is observed in VL-17A cells treated with 50 mM and 100 mM ethanol, but for
HepG2 cells treated with low concentration of the ethanol, there is no GSH depletion as the
toxicity caused by ethanol is lower. When the HepG2 cells are administered with 100 mM
ethanol, the toxicity increased which resulted in decreased GSH level.
However, the addition of DHM does not increase the GSH concentration in HepG2 cells, which
is contrary to our hypothesis. The possible illustration is that DHM may promote other pathways
to metabolize the ethanol which in turn causes the creation of “bad” metabolites and therefore,
leading to higher consumption of GSH.

A B
*
*
*

47
For VL-17A cells, as the concentration of ethanol increases, the higher amount of GSH
consumed. With the presence of DHM, the GSH consumption decreased. The data for VL-17A
cells proves the beneficial effect of DHM in restoring GSH depletion. Although the mechanism
involved is under investigation, one possible explanation is that DHM can active the nuclear
factor (erythroid-derived 2)-like 2 (NERF2) pathway (Qiu et al., 2017). While in response to
ROS, NERF2 dissociates from kelch like ECH associated protein 1 (KEAP1), enabling the
NERF2 to translocate to the nucleus where it binds to the antioxidant response element (ARE) at
the promotor part and activates the transcription. The increased expression of the xCT
transporters will increase the production of GSH (Inoue et al., 1984; Qiu et al., 2017).
Figure 6.6 The synthesis and transportation of the GSH in liver cells

48
6.6 DHM reduces ethanol-induced intracellular lipid accumulation and SREBP-1 expression

Figure 6.7. Reduced expression of SREBP-1 and lipid accumulation. (A) Representative western
blot image of HepG2 and VL-17A cells cultured in ethanol and treated with 5μM DHM or
untreated for 24 hours and immunoblotted with anti-SREBP1 mAb. (B) Representative confocal
images of Nile red staining of HepG2 and VL-17A cells cultured in ethanol and treated with 5μM
DHM.
Figure 6.7 (A) showed that DHM reduced the expression of the SREBP1 which is upregulated
when the level of sterol in cells are relatively low. Usually, the higher amount of SREBP1, the
higher amount of lipid produced. Therefore, by reducing the generation of the transcription

A
B

49
factor involved in sterol synthesis, DHM inhibits the lipid accumulation caused by ethanol
consumption.
Figure 6.7 (B) illustrated that in comparison with the untreated group, the fatty acid
accumulation was observed in HepG2 and VL-17A cells treated with ethanol. With the presence
the DHM, less fatty acid groups detected, implying that DHM can reduce the lipid accumulation
in HepG2 and VL-17A cells when they are exposed to ethanol.

50
6.7 DHM enhances the phosphorylation of AMPK and induces autophagy
Figure 6.8 DHM effects on expression of the AMPK-ULK1-mTOR in autophagy regulation

After treating with 5μM DHM, the expression of p-AMPK of HepG2 is improved but the
expression of the total AMPK is not influenced, suggesting that DHM can upregulated the
activation of phosphorylated AMPK.
Although the image is not very clear, the levels of p-ULK1 and ULK1 are increased when
HepG2 cells are treated with lower concentration of ethanol (50mM and 100mM) in combination
with DHM, this may indicate that DHM is able to induce the cell autophagy by upregulating the
expression of p- ULK1 and ULK1.
When the cells are incubated with a higher amount of ethanol (200mM), the expression of the p-
mTOR is lower, meaning that the excessive amount of the ethanol will inhibit the activation of
mTOR and thus inducing autophagy. However, the addition of the DHM to the cells exposed to
200 mM ethanol, the expression of p-mTOR increases while the expression of the total mTOR
decreases, this contradicts with our hypothesis that DHM can induce the autophagy by inhibiting
the activation of the mTOR.

51
Overall discussions

The first study which focuses on the DHM beneficial effects on alcohol use disorder provides a
preliminary demonstration of the interactions between ethanol and α5-GABAARs receptors,
ethanol is able to potentiate the α5-GABAARs at low concentration (3 mM), the potentiated Cl
-

current is proportional to the ethanol concentration. The mechanism involved could be that like
benzodiazepines, ethanol can cause allosteric modulation of α5-GABAARs and lead to the
enhanced ability of α5-GABAARs to bind with GABA.
DHM is the traditional treatment for the hangover, but the communications among DHM,
ethanol and α5-GABAARs are unclear. This study revealed the effect of DHM or its related
products on α5-GABAARs and ethanol potentiated α5-GABAARs. Besides, I also set up a
screening system to compare the hangover cure effects of DHM-containing products in the
markets, this system could be utilized in the fields of drug discovery and drug quality control.
However, my data illustrated what happened among ethanol, DHM and α5-GABAARs, the
principle and mechanism are still unclear. My hypothesis is that DHM may influence the
scaffolding protein named gephyrin which anchors the α5-GABAARs on the membrane, thus
causing the changes of α5-GABAARs’ activity and plasticity. Besides, there might be some
interactions between DHM and alcohol. DHM is a flavonoid with six hydroxyl groups, the
hydrogen bond could form easily between DHM and ethanol, this connection may lead to the
weaker interactions between ethanol and α5-GABAARs.
Except for interactions between ethanol and brain receptors, liver is the main organ that
metabolizes the ethanol, therefore, the ethanol effects on liver can’t be ignored. Excessive and
continuous alcohol consumption cause the damage to the liver and lead to the development of

52
ALD, the beneficial effects of DHM on counteracting ethanol-induced injury were investigated
by setting up in vitro experiments.
Firstly, DHM demonstrated different performances in HepG2 and VL-17A cells. The protective
effect is more significant in VL-17A cells, because the restore of the damaged mitochondria and
GSH depletion had only been observed in VL-17A cells rather than HepG2 cells. This could be
illustrated by the difference between two cell lines, the levels of ethanol metabolic enzymes
ADH and CYP2E1. For HepG2 cells, due to the lack of those enzymes, the other ethanol
metabolism pathways (e.g., nonoxidative alcohol metabolism) might be activated by DHM and
cause the production of toxic metabolites that can’t be “digested” by DHM. On the other side, for
VL-17A cells, ADH and CYP2E1 dominate the ethanol metabolism, the expression of ADH and
ALDH could be enhanced by DHM thereby decreasing the toxicity caused by acetaldehyde.
Secondly, DHM is able to ameliorate liver injury induced by alcohol consumption. The previous
data in our lab showed that the ROS generated during ethanol metabolic process is reduced in
both cell lines and the expression of the catalase, an essential enzyme to promote the degradation
of oxidative stress, is enhanced by DHM. Besides, the restore of the GSH in VL-17A cells
provides another evidence to substantiate the ability of DHM to suppress the ethanol-induced
ROS. Additionally, my data validates that DHM significantly reduces the ethanol-mediated lipid
accumulation and expression of SREBP-1, a transcription factor that plays a role in lipid
biosynthesis, in both HepG2 and VL-17A cells.
Finally, to further study the signaling pathway involved and to test other beneficial effects of
DHM, I did western blots to measure the changes of the signaling proteins related to the
autophagy pathway. For HepG2 cells treated with ethanol, DHM activates the phosphorylation of
energy sensor AMPK, activated AMPK stimulates the lipid oxidation and cell autophagy to

53
restore the energy balance. In addition to AMPK, previous studies suggest that DHM can also
provoke the autophagy by inhibiting the mTOR, this is contradictory to our data that DHM
increased the expression of phosphorylated mTOR. Therefore, the concrete and solid conclusion
cannot be made here that DHM does or doesn’t play a positive role in inducing autophagy before
we get more data and much clearer images.
Overall, this thesis demonstrated the beneficial effects of DHM on ethanol modulation of α5-
GABAARs and ethanol-induced liver toxicity and steatosis and provided the preliminary data for
future animal work to further study the potential of DHM in treating alcohol use disorders and
alcoholic liver diseases.

54
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Abstract (if available)

Abstract Alcohol use disorder (AUD) is a complex disease that is a major contributor to health problems across the United States and the World. Building evidence links AUD with multiple system pathologies, including liver disease, cardiac dysregulation, and gastrointestinal abnormalities. There is a large body of investigations focusing on pathologies associated with chronic and heavy alcohol intake, including the development of alcoholic liver disease (ALD) as it pertains to alcohol-mediated increases in inflammatory processes and generation of reactive oxygen species (ROS). However, the therapies against AUD and ALD are none or limited. These shortcomings illustrate the important need for the identification and development of new therapies to treat the onset of ALD induced by AUD. Dihydromyricetin (DHM), a bioactive flavanonol from the Ampelopsis grossedentata species, is gaining interest by the scientific community due to its broad range of pharmacological properties. This includes, for example, its ability to reduce alcohol consumption, acts as an antioxidant and potentially counteract ethanol’s detrimental effects due to high levels of ethanol intake. To this end, building evidence from our laboratory supports the use of DHM as a novel method to reduce alcohol consumption in rodents and to improve liver health by providing protection against alcohol-induced liver injury and lipid accumulation. The goal of my project (thesis) is to investigate DHM effects on certain GABAA receptors (GABAARs). Furthermore, I investigate DHM effects on liver protection. The technology of electrophysiological whole-cell voltage-patch-clamp recording was used to gain knowledge of DHM activity on GABAARs, especially, α5-containing GABAARs in presenting alcohol (ethanol) application. Ethanol (1mM to 300mM), and DHM (10nM to 1000nM), concentration-dependently potentiated α5-GABAARs, while DHM could inhibit ethanol effects on α5-GABAARs. This work confirmed that DHM protects the α5-GABAARs by counteracting excessive ethanol. Next, to obtain cellular insight of the DHM influences on ethanol metabolism, I measured the effects of DHM plus/minus ethanol using human hepato-carcinoma cells HepG2 and VL-17A, transfected HepG2 cells. These cell lines express different levels of the ethanol metabolizing enzyme alcohol dehydrogenase (ADH) and CYP2E1. The cells were cultured at different concentrations of ethanol (50mM to 200mM) and treated with DHM to evaluate ethanol metabolism, hepatoprotection, and changes in lipid metabolism. In addition, colourimetric assays were performed to evaluate the benefits of DHM on ethanol-induced mitochondrial stress and cytotoxicity. I found that DHM reduced the levels of ethanol-induced metabolic activation when tested in high levels of ethanol. I also found that DHM treatment decreased the production of the toxic metabolite acetaldehyde and increased the amount of acetic acid produced during ethanol metabolism. This latter finding suggests DHM is able to accelerate the metabolism of ethanol and acetaldehyde, indicates DHM protective effects on hepatocellular in vitro. Further, we hypothesized that DHM may play a positive role in ameliorating ethanol-induced steatosis, we found that DHM markedly decreased the accumulation of intracellular lipids and expression of the lipogenic transcription factor, SREBP1. Collectively, our data elucidated DHM protective effects in hepatocellular and suggesting that DHM can be a novel method for liver protection.

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Creator Yu, Xin (author)

Core Title The study of DHM effects on counteracting ethanol intoxications

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/28/2019

Defense Date 05/10/2019

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

Tag alcohol dehydrogenase,alcohol use disorder,alcoholic liver disease,aldehyde dehydrogenase,CYP2E1,dihydromyricetin,electrophysiological whole-cell voltage-patch-clamp recording,ethanol metabolism,GABAA receptors,hepto-carcinoma cells,OAI-PMH Harvest,steatosis

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Language English

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Advisor Liang, Jing (committee chair), Asatryan, Liana (committee member), Davies, Daryl (committee member)

Creator Email smile33330@qq.com,xyu852@usc.edu

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