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Beneficial effect of antibiotic treatment on alcohol-related liver pathology in mice is not due to reduction in the butyrate-producing gut microbial phyla
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Beneficial effect of antibiotic treatment on alcohol-related liver pathology in mice is not due to reduction in the butyrate-producing gut microbial phyla
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Content
Copyright 2021 Zeyu Zhang
BENEFICIAL EFFECT OF ANTIBIOTIC TREATMENT
ON ALCOHOL-RELATED LIVER PATHOLOGY
IN MICE IS NOT DUE TO REDUCTION IN THE
BUTYRATE-PRODUCING GUT MICROBIAL PHYLA
by
Zeyu Zhang
A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
MOLECULAR PHARMACOLOGY AND TOXICOLOGY
May 2021
ii
Acknowledgements
This work was funded by Rose Hills foundation Innovator Award (USC; to L. Asatyan),
NIAAA R01AA022448 (NIAAA, to D. Davies), USC School of Pharmacy and USC Good
Neighbors. I would like to thank my mentor Dr. Liana Asatyan for her support during my 2-year
master learning experience; Dr. Daryl L. Davies and Dr. Jing Liang for their help in our lab; Dr.
Junji Watanabe for his help with equipment training and histology evaluation. Thanks for my lab
member Rachel E.N. Reyes, Lei Gao, Alzahra J. Al Omran, Joshua Silva, Saki Watanabe, Isis
Janilkarn-Urena, Chen Xue, Jifeng Zhang, Luqing Qi, Catalina Vu, Rachel Paik, Carlos Delgado,
Greg Havton and Carson Folk for their help and support. Thanks for support from all my family
members and friends in China.
iii
Table of Contents
Acknowledgments………………………………………….……………………………………..ii
List of Tables………………………………………………………………………………………v
List of Figures……………………………………………………………………….…………….v
Abbreviations……………………………………………………………………………………..vi
Abstract……………………………………………………………………………………….….vii
Preface…………………………………………………………………………………………..viii
Chapter 1: Introduction……………………………………………………………..……………..1
1.1 Alcohol Consumption behavior, alcohol use disorder (AUD) and therapies.............1
1.2 Alteration of gut microbiota in AUD patients………………………………………2
1.3 Alcohol Liver Disease (ALD) and Early-stage liver steatosis……………………....3
1.4 Gut-liver axis……………………………………………………………………….4
1.5 Toll-like receptor (TLR) signaling and inflammatory cytokines…………………..5
1.6 Short-chain Fatty Acids (SCFA)…………………………………………………...6
1.7 Hypothesis and Aims……………………………………………………………….8
Chapter 2: Methods………………………………………………………………………………..9
2.1 Animal subjects and groups……………………………………….………………..9
2.2 Non-absorbable antibiotic cocktail (ABX) and short-chain fatty acids (SCFA)
treatment…………………………………………………………………….……11
2.3 “Drinking in the Dark” (DID) alcohol drinking behavior model………………….11
2.4 Two-bottle choice (TBC) alcohol preference drinking model…………………….12
2.5 Tissue collection…………………………………………………………………..13
2.6 Serum blood ethanol concentration levels……………………..…………………14
2.7 16S sequencing of mouse gut microbiome metagenomes…………………………15
2.8 Histology………………………………………………………………………….16
iv
2.9 RT-qPCR analysis for mRNA…………………………………………………….17
2.10 Data analysis……………………………………………………………..17
Chapter 3: Results………………………………………………………………………………..19
3.1 ABX/SB did not affect body parameters, but SB group consumed more liquid…..19
3.2 ABX-treated mice immediately had significant higher amounts of 20E intake
compared with H2O mice in the DID model……………………………………...21
3.3 SB treatment prevents ABX-induced increases in ethanol intake levels………….21
3.4 No significant differences in ethanol metabolism were observed in mouse serum
samples among treatment groups…………………………………………...……21
3.5 SB supplement altered ethanol preference in the TBC model…………………….23
3.6 ABX treatment groups had enlarged cecum and reductions in ceca microbiota
diversity and specific butyrate-producing bacteria populations…………………..24
3.7 Correlations between alcohol intake and intestinal bacteria populations………....27
3.8 Treatments did not appear to cause liver tissue morphology change while early-
stage steatosis appears after alcohol intake……………………………….………29
3.9 Antibiotic cocktail has protective effect against acute inflammation in liver……..34
Chapter 4: Discussion……………………………………………………………………………38
4.1 Higher alcohol consumption along with alterations in gut microbiota…………....38
4.2 Early-stage liver steatosis and acute liver inflammation………………………….39
4.3 Gut-brain axis…………………………………………………………………….40
Chapter 5: Conclusion………………………………………………………………………..…..41
References……………………………………………………………………………….……….43
v
List of Tables
Table 1 Group information
Table 2 16S sequencing and taxonomic analysis reaction conditions
Table 3 RT qPCR primers
List of Figures
Figure 1 Treatment and drinking behavior model experiment groups
Figure 2 Body parameters of mice in different treatment groups
Figure 3 Voluntary alcohol consumption and blood ethanol concentration
Figure 4 Preference of alcohol and SB in the TBC drinking model
Figure 5 Observed physiological changes between treatment groups
Figure 6 ABX reduced gut microbiota diversity while SB did not show observed effects
Figure 7 SB did not significantly change butyrate-producing bacteria populations
Figure 8 Total alcohol consumption is in correlation with alterations in intestinal flora phyla
Figure 9 H&E staining image sets of liver tissue sections
Figure 10 Oil Red O staining image sets of liver sections
Figure 11 Significant change in liver expression of inflammatory cytokines
Figure 12 Significant change in liver expression of innate immune response mediators
Figure 13 No significant change in Toll-like receptor 4 (TLR4)
vi
Abbreviations
20E 20% ethanol
ABX Antibiotic cocktail
ADH Alcohol dehydrogenase
ALD Alcohol Liver Disease
AP-1 Activator protein 1
AUD Alcohol Use Disorder
BEC Blood ethanol consumption
CB Clostridium butyricum
CNS Central nervous system
CYP Cytochrome P450
DID Drinking in the Dark
FMT Fecal microbiota transplantation
GPR G-protein receptors
H&E Hematoxylin & Eosin
HDAC Histone deacetylases
IHC Immunohistochemistry
IL Interleukin
IKK IκB kinase
IP Intraperitoneal
LPS Lipopolysaccharide
MAPK Mitogen-activated protein kinase
Mcp-1 Monocyte chemoattractant protein 1
MCT Monocarboxylate transporters
Mpo Myeloperoxidase
MyD88 Myeloid differentiation factor 88
NBF Neutral buffered formalin
PAMP Pathogen-associated molecular patterns
ROS Reactive oxygen species
SB Sodium butyrate
SCFA Short-chain fatty acids
SEM Standard error mean
SMCT Sodium-coupled monocarboxylate transporters
TBC Two-bottle choice
TLR Toll-like receptors
TNF Tumor necrosis factor
vii
Abstract
Patients diagnosed with continued, unhealthy use of alcohol usually compare with
alterations in gut microbiota diversity and compositions and these changes have been associated
with contributing to alcohol-related liver disease. It is therefore suggested that gut microbiota
edition can be considered as a potential therapy for alcohol liver diseases. In our previous study,
a designed low bioavailable antibiotic cocktail (ABX) treatment on C57BL/6J male mice using a
binge-like “Drinking in the Dark (DID)” mouse alcohol drinking model resulted in reduced gut
microbial diversity, with pronounced decreases in commensal Firmicutes phyla, which includes
members that produce short-chain fatty acids (SCFA). SCFA, for instance butyrate, fermented by
microorganisms, have protective benefits on human health. In this regard, ABX treatment caused
an increase in ethanol intake and sodium butyrate (SB) supplementation reversed this effect.
Based on all these findings, we hypothesized that ABX is also able to cause changes in the liver
due to reduced butyrate-producing microbial phyla. In our study, we tested the influences of
ABX and/or SB treatments on the liver in a 4-week DID paradigm. We found that ABX reduced
early-stage liver steatosis after DID exposure as assessed using H&E/Oil Red O tissue staining.
ABX also prevented the increase in mRNA for inflammatory cytokines (IL-6, TNF-α) and an
increase in myeloperoxidase (Mpo), a marker for neutrophils. SB supplementation did not
change the fat distribution caused by ABX. These findings demonstrate that in contrast to the
increase in ethanol intake, ABX treatment was more protective on the liver parameters and SB
did not have any additional effects over the ABX-induced changes. Manipulation of the gut
microbiome with ABX can be a therapeutic strategy to maintain liver health during excessive
alcohol consumption. Further study should focus on the specific microbial phyla and produced
protective metabolites for treating alcohol-related liver steatosis.
viii
Preface
My Master’s thesis project has been a collaboration between myself and my advisor and
mentor, Dr. Liana Asatryan. The basis of my project is from my passion to work on drug
treatment of Alcohol Use Disorder. Since my undergraduate study in pharmacy, I am dedicated
to devoting myself to patient care and drug development. My current study focuses on how
supplements are able to improve alcohol consumption and seeking behaviors.
My study and research experience are supported by many individuals. Most importantly,
my parents and all the other family members that have given me their love and understandings.
Secondly, all the lab members in Dr. Liana Asatryan’s research group and Dr. Daryl Davies’s lab
group who helped me in much of my daily work. Last but not least, thank for all my thesis
committee members who provided support by giving suggestions in ways to improve my
Master’s thesis.
1
1. Introduction
1.1 Alcohol consumption behavior, alcohol use disorder (AUD) and therapies
It is reported that global annual average alcohol consumption is 6.4 liter per person older
than age 15. The United States has a long history of on alcohol consumption since 1850
1
.
However, alcohol consumption is related to negative health impacts. Light-to-moderate
consumption of alcohol, which is considered safe, is defined as a maximum of 1 glass of wine a
day for females and a maximum of 2 glasses a day for males. Excessive drinking, in a relatively
short (i.e., 2 hours) also called binge drinking, and any heavily drinking behavior during
pregnancy or people under age 21, can cause either short-term or long-term health problems. The
CDC reports that in the United States, there are an average of 95,158 deaths caused by alcohol
each year and a potential loss of 2.8 million lives
1
. In the United States, 9.8% of all deaths are
caused by excessive drinking by working-age adults
2
. Alcohol Use Order (AUD), commonly
known as Alcoholism, is a chronic mental illness suffered in which patients cannot control their
drinking behavior by increasing their drinking and increasing their desire to drink. It is not
simply considered as a bad eating habit, but a mental illness
3
. Treatments for AUD trace back to
1700s, when Benjamin Rush suggested some ways to solve the loss of control in alcohol
drinking
4
. Acamprosate, disulfiram and naltrexone are currently three drugs approved by FDA,
and the mentioned varenicline, baclofen, nalmefene, gabapentin, topiramate, ondansetron, and
prazosin, are listed as current AUD potential drugs while not approved by FDA yet
5
. In the
beginning, common mechanism of AUD treatment is to inhibit the alcohol metabolism, which is
related to inhibition of enzymes like alcohol dehydrogenase (ADH) and aldehyde
dehydrogenase. Disulfiram was the first drug approved in 1951. It is an irreversible inhibitor of
acetaldehyde dehydrogenase, but it has some unexpected symptoms. For the drugs mentioned
2
that are not approved by the FDA, they are used as off-label drugs in European countries and
other regions. Baclofen, which have been reported safety issues, is considered with fabulous
effects overpassing side effects and able to be utilized if under a reasonable dosage. Gabapentin
has shown fabulous effects in better altering alcohol consumption and craving behaviors.
However, because of lack of supervision and low prices, it may be abused. The development of
AUD treatment comes with dozens of challenging issues. The major issue is the individual
situations of AUD patients, either their lack of understandings of AUD or the lack of effective
approaches for its prevention. High placebo response rates appear in trials, which is considered
as another big challenge
6
. The limited options with limited efficacy further confirm the key
needs of further research on pathophysiology and identification of new drugs.
1.2 Alteration of gut microbiota in AUD patients
The intestinal microbiome is defined as the gene codes of the intestinal flora in
composition of various microbes (including bacteria, archaea, fungi, and viruses), spreading
throughout the gastrointestinal environment of the intestine during body development. The
gastrointestinal tract contains trillions of microorganisms, most of which are Firmicutes and
Bacteroides
7
. Changes in the intestinal flora are related to expression factors and pathology
factors. The term describing changes of the composition of the intestinal microbiota is called
“dysbiosis”, which causes adverse effects on the host. Dysbiosis in intestinal flora has been
linked to several disease conditions, such as diabetes (type 2), inflammatory bowel disease (such
as colitis and Crohn’s disease), cardiovascular disease, central nervous system diseases (such as
Alzheimer’s disease and Parkinson’s disease), autoimmune diseases and cancer
8-13
. Alcohol
intake can also cause the alteration of gut microbiota composition, which is related to liver
3
disease development
14
. In rodent models, relative abundance of Firmicutes in gut microbiota of
alcohol-treated mice decreased, and the phyla of Bacteroides and Verrucomicrobia increased
15
.
The microbial community has also been tested for significant changes in the intestinal flora in
human studies, including overgrowth of the jejunum and alterations in the relative percentage of
abundance of lower Bacteroides and higher Proteobacteria
16
. Because of the important role
played by intestinal flora in disease models, fecal microbiota transplantation (FMT) is now a
possible method for AUD treatment. A Phase 1 trial of FMT showed that compared with the
placebo group, alcohol cravings and consumption behaviors in the AUD group were safe and
reduced in a short period of time, and the microorganisms had favorable changes
17
.
Psychobiotics, a new term defined as living organisms, probiotics and prebiotics which can bring
benefits to mental health, are also currently regarded as a new treatment for AUD
18
.
1.3 Alcohol Liver Disease (ALD) and Early-stage liver steatosis
Excessive drinking can lead to a spectrum of Alcohol Liver Disease (ALD). As for
chronic alcohol abuse, cytochrome P450, particularly cytochrome P450 2E1 (CYP2E1) is
upregulated and helps ADH in transferring alcohol into acetaldehyde. The reactive oxygen
species (ROS) produced by CYP2E1 work to have the pro-inflammatory effects of liver damage
caused by alcohol intake, including: (1) activation of NF-κB; (2) recruitment of innate immune
cells; (3) increased level of cytokines; (4) accelerating lipid peroxidation
19
.
Hydrogen peroxide, superoxide anions and hydroxyl radicals can be transferred into
stable form (such as peroxynitrites and nitric oxide) with strong toxicity in the liver tissue
20
.
Excessive alcohol intake for a long period of time usually leads to ALD, including hepatitis,
steatosis (acute and chronic), fibrosis and cirrhosis
21
. In some cases, increased ROS levels and
4
hepatotoxic exposure related endoplasm reticulum stress stimulate the accumulation of
cholesterol and inhibits the secretion of lipoprotein
22
. The increased enzyme activity of CYP2E1
is considered to play an important role in the development of alcoholic steatohepatitis because of
the high content of ROS during ethanol metabolism
23
.
In addition, alcohol dependence and liver pathology are related to intense changes of the
intestinal structure and metabolic potential of patients, which show different patterns and
virulence factors in relation to the metabolism procedures of ethanol and possible inflammatory
signaling pathways
24
.
1.4 Gut-liver axis
The “gut-brain axis” demonstrates the strong relationship between the intestinal flora and
the brain. Due to diverse liver diseases, the “gut-liver axis” is gaining increasing importance.
The gastrointestinal barrier can maintain the structure and function of the intestine in prevention
of harmful substances like toxins from entering other parts of the body. When the gastrointestinal
wall barrier is damaged, the compositions of intestinal flora and other toxic components are able
to get into the liver from the portal vein, affecting its functions as well as causing damage,
inflammation and different kinds of metabolic diseases
25
. Gastrointestinal malnutrition and
increased permeability will lead to the displacement of microorganisms and their products, and
these microbes and microbial products will enter the liver tissue and recognized by hepatic
stellate cells and Kupffer cells, which trigger and maintain inflammatory cascades
26
. Current
data show that in addition to venomous effects on hepatocytes caused by alcohol, the abnormal
alterations in intestinal flora initiate the activation of toll-like receptors (TLR) expressed on
5
Kupffer cells, which contributes to onset of liver diseases
27
. Meanwhile, it is important to
mention that the gut-liver axis is a bidirectional connection, so the liver can also inversely affect
the gut microbes, regulating microbe populations
28
. Bile acids are known as another
communicating bridge between the gastrointestinal tract and the liver. The interaction changes
bile acid components and affects its balance along with various metabolism procedures in the
body
29
.
1.5 Toll-like receptor (TLR) signaling and inflammatory cytokines
Increased bacterial translocation is related to chronic liver disease. Chronic liver disease
not merely results in characteristic infection diseases but produces chronic liver inflammation,
which are directed by invasion of antigens and through TLR activation, which are known as
pathogen-associated molecular patterns (PAMP). The TLR signaling works as the first defense
against pathogens by producing all different kinds of cytokines, for example interferons,
interleukin (IL) and tumor necrosis factor (TNF). IL-1β and TNF-α, known as pro-inflammatory
cytokines, are downstream targets of TLRs
30
. The ability of non-absorbable antibiotics and
TLR4 inactivation to inhibit liver fibrosis indicates the correlation between intestinal flora-TLR4
and liver fibrosis. Lipopolysaccharide (LPS), ligand of TLR4, is able to initiate the activation
state of inflammatory signals in a variety of cells in the liver and they express high levels of
TLR4 even in a resting state
31
. The resident macrophage Kupffer cell in the liver is one of the
first cells in the liver to encounter the intestinal bacterium and its products. Kupffer cells is a
good representative important population to be used to regulate the inflammatory response to
TLR agonists and their clearance. They express TLR4 and are highly sensitive to LPS.
6
Stimulated by LPS, Kupffer cells produce ILs, TNFα and several chemokines
32
. Myeloid
differentiation factor 88 (MyD88), is applicable to ordinary molecular adaptors of all TLRs
except TLR3. The MyD88-dependent cascade activates NF-κB signaling pathway as well as
activator protein 1 (AP-1) through the complex of IκB kinase (IKK) and mitogen-activated
protein kinase (MAPK), respectively. Followingly, NF-κB and AP-1 produce specific pro-
inflammatory cytokines
33
. NF-κB regulates inflammatory state and cell death as a key
transcription factor, and it exerts a main role in chronic liver diseases. NF-κB can be activated by
diverse stimulation and cell stress states, for instance TNF-α and TLRs. To effectively induce
cell death and following reactions (such as inflammatory reactions and fibrogenesis), NF-κB
activation in liver cells must be inhibited
31
.
1.6 Short-chain Fatty Acids (SCFA)
Short-chain fatty acids (SCFA) are fermented by intestinal flora. In addition to dietary
sources, they are also fermented from polysaccharides which are hard to digest (for instance
resistant starch and dietary fiber). There are diverse SCFAs in the intestine. Acetate is utilized in
lipids and cholesterol synthesis procedures in the host, while propionate is mainly used as a
substrate in the liver in the process of gluconeogenesis. SCFA is absorbed in the small and large
intestines by diffusion in a dissociated form or active transport by SCFA transporter
34
. They
are transferred by pH-dependent, H+-coupled sodium-coupled monocarboxylate transporters
(SMCTs) and monocarboxylate transporters (MCTs) across the membrane
35
.
Butyrate is the anionic portion of butyric acid and its salt form. The host’s energy uptake
and immune function rely on the regulations mediated by butyrate between the host and the
7
microorganism
36
. In the host, butyrate is usually absorbed and metabolized into very fast energy
(which is readily metabolized), even if the oral or intravenous dose is high, the plasma
concentration in the plasma will rise rapidly
37
. Butyrate is mainly used by host cells,
metabolized in the process of mitochondrial β-oxidation to generate NADH, H + and AcCoA,
and further generates ATP
35
.
Butyrate is the key supportive resource for colonic cells. It has been found that high-dose
systemic or local injection of butyrate can have neuroprotective effects, for instance enhancing
memory and restoring cognitive function
38, 39
. Butyrate activates G-protein receptors (GPRs),
such as GPR41 and GPR43. GPR41 expresses mainly on endothelial cells in blood vessels, while
GPR43 is predominantly on immune cells
40
. They are both activated with different specificities
and potency for ligands. The physiological levels of butyrate may affect neuronal inflammatory
states through different mechanisms. Histone deacetylases (HDACs) have also been considered
to play a part in neurodegenerative disorders
41, 42
. HDACs remove acetyl groups from histones,
so inhibition of HDACs can increase histone acetylation thus increasing gene transcription.
Butyrate is a known inhibitor of HDACs with anti-inflammatory effects in inflammatory diseases
43
. For instance, HDAC3 is related to inflammatory gene expressions and the monocyte
recruitments to the inflammation site through manipulating the NF-κB signaling pathway
44
.
Butyrate is also proved to be employed as an anti-inflammatory compound to improve
the intestinal barrier function and prevent colon cancer and neurodegenerative diseases. It
contributes to gut homeostasis; at the same time, it is involved in the functioning of other tissues
45
. Butyrate is specifically the fermentation product by Clostridium butyricum (CB) in the gut. It
is demonstrated that CB has the neuroprotective effects in multiple brain disorders and could
8
decrease pro-inflammatory cytokines, thus representing to be a promising candidate for treatment
of Alzheimer’s Disease
46
.
1.7 Hypothesis and Aims
My studies provided more support for alcohol consumption in relationship with changes in
the gut microbiota. This was accomplished by establishing a non-absorbable antibiotic cocktail
(ABX) model to simulate the destruction of the gut microbial environment in an adult male
C57BL/6J mouse model. In the binge drinking model, voluntary ethanol intake increased
significantly with ABX treatment. The ABX-treated mice also expressed alterations in the
intestinal flora. A significant reduction in diversity and a large-scale decrease in the Firmicutes
population were observed. This phylum contains major producers of major metabolites, which are
utilized to regulate the host system and functions, for instance butyrate. Based on this initial work,
we hypothesized that the lack of these SCFA-producing microorganisms appeared to be associated
with the significant increase in alcohol consumption after ABX treatment. Aiming to further
investigate the influence of microbial metabolites (i.e. SCFA) on alcohol consumption and seeking
behavior, in the following study, we expanded the research method of C57BL/6J adult male mice
and tested if the supplementation of sodium butyrate (SB) can inversely influence increased
ethanol intake caused by ABX. Mature mouse alcohol consumption and seeking behavior models,
known as Drinking in the dark (DID) and two-bottle choice (TBC), were designed as separate
cohorts. Consequently, SB supplements reversed the increased alcohol voluntary drinking caused
by ABX. Meanwhile, all mice preferred ethanol to their own treatments (total intake volume match
DID results). In order to investigate the potential mechanism of the increased drinking volume
9
caused by ABX treatment and the reversal of drinking behavior caused by SB, intestinal, brain and
liver tissues were collected for morphological evaluation and molecular analysis. My project
specifically focused on the effects of ABX and SB on the liver with the aim to explore the
mechanisms underlying ABX and SB mechanisms of action.
2. Methods
2.1 Animal subjects and groups
The C57BL/6J mouse strain is commonly used in many alcohol consumption behavior
studies. Especially, the male mice in this strain are more sensitive to alcohol
47-49
. 6-8 weeks
(wks) C57BL/6J adult male mice were purchased and shipped from Jackson Laboratory
(California, USA). The mice were placed in a single cage when it came in a special pathogen-
free vivarium. All the mice acclimate to the new experiment environment for at least 2 weeks
before the study. Unless additionally stated, mouse food and drinking water provided by the
facility were available at any time. The mice were housed in an automatic humidity, temperature
and light control room. Each light-dark day and night cycle was 12 hours (hours), and it was lit
from 12:00 am to 12:00 pm. Vivarium staff monitors all mice daily to ensure health. Mice body
weight (g), food consumption (g) and fluid intake (mL) were measured every other day of the
week (Mondays, Wednesdays, and Fridays). To minimize the impact on activities during the
dark phase at night and for the convenience of experiment, all measured values were recorded
during light phase. According to different treatment methods and rodent drinking models, mice
were randomly assigned into groups, and the average weight of each group was balanced from
the beginning of the study. What is more, the group of mice without ethanol treatment was used
10
as a control with 2-wks treatments. Groups and experiment design are presented in Figure 1 and
Table 1.
Figure 1. Treatment and drinking behavior model experiment groups.
Mice are single housed upon arrival and randomly distributed to treatment groups after 2 wks acclimation. The treatment groups
are later abbreviated to non-alcohol control groups: 1) H2O only – H2O; 2) antibiotics cocktail only – ABX; 3) sodium butyrate
only – HSB; 4) antibiotics cocktail and sodium butyrate – ASB, and alcohol treated groups: 1) H2O and 20% Ethanol – H2O-
20E; 2) antibiotics cocktail and 20% Ethanol – ABX-20E; 3) sodium butyrate and 20% Ethanol – HSB-20E; 4) antibiotics
cocktail, sodium butyrate and 20% Ethanol – ASB-20E.
Table 1. Group information.
All mice were randomly distributed to different groups and make sure sample size is at least 5 (to better conduct data analysis
avoiding individual influences) in each group. DID and TBC were performed as two separate experiment cohorts.
DID: “Drinking in the dark” mouse ethanol consumption model; TBC: Two bottle choice mouse ethanol seeking model; 20E:
20% Ethanol.
11
2.2 Non-absorbable antibiotic cocktail (ABX) and short-chain fatty acids (SCFA) treatment
We utilized a non-absorbable antibiotic cocktail (ABX) treatment, which has pretty low
bioavailability. It has previously been proven that it can widely reduce intestinal flora
populations while not entering the circulatory system, and indirectly influence other parts of the
host after oral administration
50, 51
. We chose sodium butyrate (SB) as a representative of short-
chain fatty acids (SCFA) supplement, which was previously shown to crucially influence
behavior changes in hosts with deficiency in intestinal flora
51
. ABX includes vancomycin (0.2
mg/mL; Thermo Fisher, USA), neomycin (2.0 mg/mL; GoldBio, USA) and bacitracin (0.8
mg/mL; Sigma, USA). The concentration of SB (Sigma, USA) was adjusted from 4 mg/mL to 8
mg/mL after pilot study. To avoid potential fungi growth in mouse treatment liquid, anti-fungal
pimaricin (1.2 ug/mL) was employed. All treatments were added to drinking water as mice daily
liquid intake treatments and freshly made every 2 days. Each bottle contains around 15-20mL
treatment liquid to ensure mice have enough liquid intake during the whole experiment
procedure. SB was added to the fresh-prepared ABX (same concentration as the ABX treatment).
Hydrochloric acid was used to match the pH value in both SB-only group (HSB) and the
combined ABX-SB (ASB) group.
2.3 “Drinking in the Dark” (DID) alcohol drinking behavior model
The mice which are assigned to the 20% Ethanol (20E) intake groups were distributed to
four different treatment groups: H2O-20E, ABX-20E, HSB-20E and ASB-20E (n= 9 to 11)
randomly. From the beginning of the DID experiment and during the entire experiment period
(except for the 20E exposure hours), all mice were able to get access to their separate drinking
bottles within 24 hours (hrs) for a total of 2 weeks (wks). To further study the effects of ABX
12
and SB on ethanol consumption, we utilized an improved form of the “drinking in the dark
(DID)” model of mouse voluntary alcohol consumption, which has been proved to simulate a
large amount of ethanol consumption in this selected train of mice
52
. Each bottle was set to
contain around 10-12 ml of 20E, supplemented with fresh 20E every two days. During the
exposure to ethanol, each drinking bottle was replaced with a designated bottle containing 20E.
The appearance of the bottle was the same as the treatment bottle. During the preparation process
before the experiment, all bottles are checked every day to avoid leakage problems. During DID,
mice consumed 20E intermittently and limitedly after 3hrs from the start of the dark cycle for 2
hrs (15:00 – 17:00) from Monday through Friday, 5 days a week. After 2 hrs, drinking bottles
were replaced back to their treatment liquid and 20E intake were recorded in mLs. In order to
avoid errors between personnel readings, the reading standards of all personnel involved in the
experiment have been unified. All intake volumes are later converted into ml/g/2hrs for analysis
to reduce possible errors caused by mouse individuals.
2.4 Two-bottle choice (TBC) alcohol preference drinking model
To investigate the influences of ABX and SB on alcohol preference behavior in a “two-
bottle choice (TBC)” model, another study of C57BL/6J male mice aged 6-8 wks were divided
into five treatment groups: H2O/20E, ABX/20E, HSB/H2O, HSB/20E, and ASB/20E (n= 5 to 7)
randomly
52
. The mice housing in single cages provided with two bottles: a “test” and a
“control”. Control group bottles including H2O, ABX, HSB and ASB respectively were named
as bottle 1 of each group. In the test bottles (the second bottle), H2O was provided in the
HSB/H2O group, and 20E was provided in the ethanol groups. Before beginning the TBC
cohort, all mice can enter their drinking treatment bottles for 24 hrs after 2 wks of pre-treatment.
13
Then, mice were limited to get access to ethanol bottles and their own bottles for 2 consecutive
weeks, which the time matching the schedule used during DID to match these two drinking
models. Each bottle was set to contain around 10-12 ml of both test and control liquid,
supplemented with freshly made solutions every two days. In order to avoid any possible
position preference, positions of both bottles were altered daily. After 2 hrs, drinking bottles
were replaced back to their treatment liquid and 20E intake were recorded in mLs. Percentage of
test liquid intake to total two-bottle intake was calculated to demonstrate mice drinking
preference. To explain why HSB mice had higher daily liquid intake volume, the HSB/H2O
group mice (n=6) were later tested SB preference against sucrose in this sweet-sensitive strain of
mice
53
. The same experiment procedure was conducted with SB as the control bottle and 5%
sucrose in water as the test bottle.
2.5 Tissue collection
Mice weight were measured and then mice were euthanized by CO2. Blood was
immediately collected from the portal vein and the serum was isolated (sit at 4℃ overnight and
centrifuge under the condition of 4℃, 2000g, 10min in the following day). The anatomy picture
of the abdominal cavity was taken to illustrate the accumulation of abdominal fat and visual
changes in the appearance of the digestive tract. Liver samples for molecular analysis were flash
frozen and those for tissue sectioning were fixed in 10% neutral buffered formalin (NBF) at least
two days and transferred to 70% ethanol or 30% sucrose in a conical tube after. The conical
tubes were upside down and put in a 4℃ fridge. The livers were floating in the tube first, once
absorbing enough sucrose solution, they would sink. The livers were frozen in cold 2-methyl
isobutane with dry ice underneath the container. When livers became white, they were wrapped
14
with foil and stored at -80℃. GI tract tissues were collected, flash frozen and stored in -80℃ till
further analysis. Spleens were also collected and stored. The weight and length and were
measured, and photos of spleen were taken. They were fixed in 10% NBF at least 2 days and
transferred to 70% ethanol after. Cecum samples were collected and weighed. The length was
measured either before burying in dry ice or storing in -80℃ until analysis. Whole brains for
molecular analysis were flash frozen and those for tissue sectioning were fixed in 10% NBF for
least 2 days and transferred into 30% sucrose in conical tubes. The conical tubes were upside
down and put in a 4℃. The brains were floating in the tube first, but once they absorbed enough
sucrose solution, they would sink. The brains were frozen in cold 2-methyl isobutane with dry
ice underneath the container. When brains became white, they were wrapped with foil and store
in -80℃. All tissue blocks stored in 70% ethanol were processed after at least 2 days in the
ethanol (ThermoFisher Spin Tissue Processor Microm STP-120). Processing procedures are as
follows: 70% ethanol (90min) – 80% ethanol (90min) – 95% ethanol (90min) – 100% ethanol
(90min) – 100% ethanol (90min) – 100% ethanol (90min) – xylene (90min) – xylene (90min) –
paraffin (90min) – paraffin (90min). Then tissues were embedded into correct molds with
paraffin until sectioning (ThermoFisher Microm EC 350 Tissue Embedding Center). All tissue
blocks stored in 30% sucrose were frozen in 2-methyl butane and wrapped with foil. Then before
sectioning, frozen tissues were embedded into correct molds with OCT.
2.6 Serum blood ethanol concentration levels
After TBC experiment, there was 1-week washout of all liquid intake. To see if ABX and
SB can affect ethanol metabolism, all mice were injected with ethanol (intraperitoneal (IP)
injection, 3.5 g/kg). After 45min, injected mice were euthanized (CO2 asphyxiation). The blood
15
was collected as mentioned above (from portal vein), and serum was prepared (sit at 4℃
overnight and centrifuge under the condition of 4℃, 2000g, 10min in the following day) and
stored in -80℃ until further analysis. The concentration of ethanol in mouse serum samples were
measured using ANALOX AM1 equipment according to manufacturer’s instructions (Analox
AM1, Analox Instruments Ltd., UK).
2.7 16S sequencing of mouse gut microbiome metagenomes
QuantiFlour dsDNA system (Promega, Madison, Wisconsin) was used to quantify DNA
isolated from the mouse cecum samples. Load DNA into Illumina’s library preparation
workflow. The V3-V4 region was amplified. In order to amplify from DNA samples, amplicon
PCR was performed. In each PCR reaction system, the volume of each component was shown in
Table 2. The PCR reaction step includes an initial denaturation step (95°C, 3 min), denaturation
(95°C, 30 s), annealing (55°C, 30 s) and extension (72°C, 30 s) cycles. In the end, stretching at
72°C for 5 min. Mag-Bind RxnPure Plus magnetic beads (Omega BioTek, Norcross, GA) were
utilized to extract PCR products from the final mixture. A second index PCR amplification was
performed in the same reaction condition as above in order to incorporate barcodes and
sequencing adapters into the final product. Mag-Bind®EquiPure library standardization kit
((Omega Biotek, Norcross, GA) was used for standardization, and then merged the library.
Agilent 2200 TapeStation was utilized to check the size of the merged library about 600 bases
and performing sequencing (2 x 300 bp Paired-end reading settings), amplification on MiSeq
(Illumina, San Diego, CA), and then used the BaseSpace application 16S Metagenomics
(Illumina, San Diego, CA) to analyze the 16S separated by sample/processing and observe as
16
aggregation Preliminary analysis and visualization of DNA sequence using RDP I Bayes
classification algorithm.
Table 2. 16S sequencing and taxonomic analysis reaction conditions.
16S sequencing of mouse gut microbiome metagenomes conditions are listed above.
2.8 Histology
Hematoxylin and eosin (H&E) staining is a common approach performed to do the tissue
staining used in histology. Formalin-fixed paraffin-embedded liver sections (3um and 5um) were
performed H&E staining to assess tissue morphology (BioTek Cytation5 Imaging Multi-mode
Reader, 4X; Zeiss Axio Scope. A1, 40X). Oil Red O is a fat-soluble dye and is usually
performed on frozen sections and some lipoproteins on paraffin sections. Because the processing
procedure will remove the lipid droplet in the liver tissue, Oil Red O staining (Lifeline Cell
Technology Kit) was utilized on fixed OCT-embedded frozen liver tissue sections (10um) to
assess hepatic steatosis stage (BioTek Cytation5 Imaging Multi-mode Reader, 4X).
17
2.9 RT-qPCR analysis for mRNA
RNA was extracted from mouse flash-frozen livers (miRNeasy Mini Kit, Qiagen)
following the manufacturer’s instructions (20-30mg liver tissue per sample). 1ug of RNA was
used to do cDNA transcription using Reverse Transcription System (Promega) and then diluted 1
to 5 using nuclease-free water. SYBR Green (Fisher Scientific) real-time qPCR (ABI
QuantStudio 12K Flex) was performed following the manufacturer’s instructions. The primers
used are listed below in Table 3 and GAPDH was selected as a housekeeping gene
(“housekeeper”) to do the 2
-ddCt
analysis method of mRNA expression. dCt was automatically
calculated by the software after each run. The H2O group is used as the control group with fold
change as 1 to show the baseline mRNA expression level of each gene.
Table 3. RT qPCR primers.
GAPDH is selected as the reference gene (“housekeeping” gene).
Il-1β: interleukin-1β; IL-6: interleukin-6; TNF-α: tumor necrosis factor-α; Mcp-1: monocyte chemoattractant protein1, Mpo:
myeloperoxidase; TLR4: Toll-like receptor 4.
2.10 Data analysis
Data analysis and graphs developed using GraphPad Prism (GraphPad Software Inc., San
Diego, CA). Group data are shown as mean ± standard error mean (SEM) for each treatment.
18
Significant changes were showed in form of * when p < 0.05 and ** when p < 0.01 after the
confirmation of FDR q < 0.05.
Mouse weight (g), food intake (g/g) and daily liquid intake (mL/g) were recorded every
other day to monitor the health situation of all groups of mice. All these data were analyzed
using two-way ANOVA with post hoc multiple comparison options between groups.
The effects of ABX and/or SB treatments on mouse alcohol drinking behavior was
performed in a DID drinking model. Mice voluntarily consumed 20% ethanol (20E) during the
dark cycle experiment procedure. Ethanol intake volume was recorded in the unit of mL, and
later transferred to g/kg/2hrs. Two-way ANOVA with post hoc multiple comparison options was
utilized to compare voluntary ethanol consumption between all treatment groups.
The effects of ABX and/or SB treatments on mouse alcohol seeking behavior was
performed in a TBC drinking model. All groups of mice were tested their ethanol preference to
daily liquid intake. HSB group mice were further tested SB supplement preference to H2O and
sucrose. In the TBC model, preference was calculated using 20E intake volume (mL) divided by
total consumption volume (mL) and body weight (g). Two-way ANOVA with post hoc multiple
comparison options was utilized to compare ethanol preference between all treatment groups.
Within treatment groups, unpaired non-parametric Mann-Whitney test and t-test were utilized to
compare liquid preference of the test bottle compared with the control bottle.
Cecum enlargement was observed in ABX-treated groups; thus cecum weights (g) were
measured. Mouse serum samples were prepared to investigate influence on ethanol metabolism
by measuring blood ethanol concentration (BEC) levels. Non-parametric Kruskal-Wallis tests
were utilized to analyze cecum weights and BEC levels as well.
19
To compare alterations in gut microbiome between treatment groups, qPCR analysis was
performed on cecum DNA extractions. Mann-Whitney tests were utilized to do data comparison
within treatment groups including groups with or without ethanol treatment. Non-parametric
Kruskal-Wallis tests were utilized aiming to compare microbiome diversity and bacterial
population changes. Non-parametric Spearman r correlations were performed to investigate
correlation relations between microbiota phyla and ethanol consumption.
In the study of inflammatory state in mouse liver tissues, RT-qPCR was done to analyze
related biomarkers. Non-parametric Kruskal-Wallis tests were utilized aiming to compare fold
changes between all treatment groups.
3. Result
3.1 ABX/SB did not affect body parameters, but SB group consumed more liquid
All treatment liquids were available 24hrs, 6wks (2wks pre-treatment and 4wks DID
experiment) except the time period performing DID or TBC (Figure 1 and Table 1). Mouse
weight, liquid intake and food consumption were measured to monitor mouse health. The food
consumption was not influenced by all treatments (Fig. 2B; two-way ANOVA, n=9-11 per
group, p = 0.78). All mice gained weight steadily throughout the whole experiment procedure,
but there were no significant changes in average weekly weights between treatment groups (Fig.
2C; two-way ANOVA, n=9-11 per group, p = 0.47). For liquid intake, there were no significant
changes among H2O, ABX and ASB groups. However, HSB group of mice drank significantly
more SB compared to all the other 3 treatment groups (Fig. 2A; two-way ANOVA, n=9-11 per
group, ** p < 0.01). To test why the HSB group had significant higher daily liquid intake, TBC
20
was done to test mouse drinking preference of SB to H2O and 5% sucrose (described in section
4.5).
Figure 2. Body parameters of mice in different treatment groups.
A) HSB group mice had higher daily liquid intake throughout the experiment in comparison with the other 3 treatment groups.
(two-way ANOVA, n=9-11 per group, ** p<0.01) B) Food consumption (two-way ANOVA, n=9-11 per group, p=0.78) and C)
average weekly body weight (two-way ANOVA, n=9-11 per group, p=0.47) did not show significant differences among the 4
groups during the whole DID study. Results are presented in the form of mean ± standard error mean (SEM) for each treatment.
Significant changes were presented as * (p < 0.05) and ** (p < 0.01).
21
3.2 ABX-treated mice immediately had significant higher amounts of 20E intake compared
with H2O mice in the DID model
The treatments were given to mice 2 wks prior to the 4wks DID experiment; H2O and
ABX were available to mice throughout the DID period. The significant higher ethanol intake in
ABX mice group in comparison with H2O mice group was observed immediately at week 1 of
DID; this increase was stable throughout the DID period (Fig. 3A; two-way ANOVA, n=9-11
per group, ** p < 0.01).
3.3 SB treatment prevents ABX-induced increases in ethanol intake levels
SB is used as a representative of SCFA treatment and HSB group mice were observed to have no
impact on ethanol intake. However, ASB mice had significant lower levels of ethanol in
comparison with ABX group mice (Fig. 3A; two-way ANOVA, n=9-11 per group, ** p < 0.01).
3.4 No significant differences in ethanol metabolism were observed in mouse serum
samples among treatment groups
Blood ethanol concentration (BEC) was tested using mouse serum samples and there were no
significant differences observed between treatment groups (Fig. 3B; Kruskal-Wallis test, n=3-4
per group, p = 0.22).
22
Figure 3. Voluntary alcohol consumption and blood ethanol concentration.
A) ABX-treated mice had significant higher 20% ethanol (20E) intake, while SB supplement lowered ABX-induced higher
ethanol intake level in the DID mouse voluntary alcohol consumption model (two-way ANOVA, n=9-11 per group, ** p<0.01).
B) ABX and/or SB treatments did not have significant impact on blood ethanol consumption (BEC) at 45 min time point after
ethanol (3.5g/kg) IP injection in mouse serum samples (Kruskal-Wallis test, n=3-4 per group, p=0.22). Results are presented in
the form of mean ± SEM for each treatment. Significant changes were presented as * (p < 0.05) and ** (p < 0.01).
23
3.5 SB supplement altered ethanol preference in the TBC model
To investigate mouse alcohol seeking behavior, the TBC model was utilized in another
cohort of experiment. The preference of 20% ethanol (20E) to the treatment liquid was tested in
all groups of mice for 2wks. The time period was designed to match with the DID experiment to
avoid any possible influences. It is important to mention that SB supplement groups (HSB, ASB)
mice had lower preference of 20E compared with H2O and ABX groups mice (Fig. 4A; two-way
ANOVA, n=5-6 per group, ** p < 0.01). It is interesting that there was no significant preference
observed in alcohol preference between H2O and ABX group mice (Fig. 4A; two-way ANOVA,
n=5-6 per group, p = 0.56), although significant differences in voluntary alcohol intake were
observed in the DID drinking model. As predicted, all mice treated in different groups had
significantly higher percentage of ethanol preference compared to their daily treatment liquid
(Fig. 4B-E; Mann-Whitney test, n=5-6 per group, ** p < 0.01). Because of the higher daily liquid
intake in HSB group mice, they were investigated for alcohol drinking preference of SB against
H2O and sucrose (5% sucrose dissolved in drinking water) under same experiment conditions as
ethanol preference tests. Mice preferred to drink more HSB against H2O (Fig. 4F – Left; Mann-
Whitney test, n=6, p = 0.02). Then a 2-week washout procedure was performed, and those mice
were tested for the preference of SB against sucrose, thus same group of mice were used in HSB
preference tests against H2O and sucrose accordingly. Mice had higher preference for HSB,
however this was substantially lower than the preference observed for sucrose intake (Fig. 4F –
Right; Mann-Whitney test, n=6, ** p < 0.01).
24
Figure 4. Preference of alcohol and SB in the TBC drinking model.
A) SB supplement groups (HSB, ASB) mice had lower preference of 20E compared with H2O and ABX groups mice (two-way
ANOVA, n=5-6 per group, p = 0.56). B) – E) All groups of mice had higher 20E preference against their daily treatment liquid
(Mann-Whitney test, n=5-6 per group, ** p < 0.01). F) HSB group mice had preference of SB to H2O (left) (Mann-Whitney test,
n=6, p =0.02) and, while preference of sucrose to SB (right) (Mann-Whitney test, n=6, ** p < 0.01). Results are presented in the
form of mean ± SEM for each treatment. Significant changes were presented as * (p < 0.05) and ** (p < 0.01).
3.6 ABX treatment groups had enlarged cecum and reductions in ceca microbiota diversity
and specific butyrate-producing bacteria populations
Enlarged cecum was observed in ABX-treated groups after 4wks of DID experiment.
Also, these groups of mice had less abdominal fat accumulation (Fig. 5 – Top panel). Cecum
weights were measured, and it was shown that those weights of ABX-treated groups were
significantly higher than non-treated groups (Fig. 5 – Bottom panel; Kruskal-Wallis test, n=9-11
per group, **p < 0.01).
25
Gut microbiome was analyzed using ceca samples. ABX treatment groups which include
ABX and ASB, had significant gut microbiome alterations, meanwhile SB supplement was not
observed to significantly change gut microbiota composition. Unique number of species was
compared between ABX treated and non-treated groups, those treated mice had significantly
smaller numbers of species (Fig. 6A; Kruskal-Wallis test, n = 6 per group). Α-diversity Shannon
Index was utilized to show that ABX treatment led to obvious reductions in diversity, which
indicates that taxa richness and evenness showed significant differences (Fig. 6B; Kruskal-Wallis
test, n = 6 per group).
There were significant differences in Firmicutes genus populations between ABX treated
and non-treated groups. Bacteria populations fermenting SB, such as Clostridium IV, Clostridium
XIVa and Lachnospiraceae were shown significant genus-level reduction in ABX-treated group
mice in comparison with non-treated mice (Fig. 7; Kruskal-Wallis test, n = 6 per group).
However, SB supplement was not able to affect butyrate-producing microbiota populations
within cecum (Fig. 7; Kruskal-Wallis test, n = 6 per group).
26
Figure 5. Observed physiological changes between treatment groups.
A) Mouse necropsy images showing enlarged cecum (red arrows) and reduced abdominal adipose tissue (blue stars) in ABX-
treated groups. B) ABX-treated groups (ABX and ASB) demonstrated significant higher cecum weights in comparison with non-
ABX treated groups (H2O and HSB) (Kruskal-Wallis test, n=9-11 per group, **p < 0.01). Results are presented in the form of
mean ± SEM for each treatment. Significant changes were presented as * (p < 0.05) and ** (p < 0.01).
27
Figure 6. ABX reduced gut microbiota diversity while SB did not show observed effects.
A) ABX-treated groups (ABX and ASB) of mice had reduced gut microbiota diversity while SB-treated groups (HSB and ASB)
were not observed to have altered number of specific species analyzed within cecum samples (Kruskal-Wallis test, n = 6 per
group). B) Similar to Fig. 6A, SB supplement did not influence ⍺-diversity (Shannon Index Measure) in comparison with non-SB
treated mice (Kruskal-Wallis test, n = 6 per group). Results are presented in the form of mean ± SEM for each treatment.
Figure 7. SB did not significantly change butyrate-producing bacteria populations.
As shown before, ABX-treated groups, were related to decrease in populations of Firmicutes. SB treatment group (HSB and
ASB) mice were not observed to have alterations in the population of A) g_Lachnospiraceae, B) g_Clostridium IV, and C)
g_Clostridium XIVa in cecum in comparison with H2O and ABX group mice. Results are presented in the form of mean ± SEM,
n = 6 per group.
3.7 Correlations between alcohol intake and intestinal bacteria population
4 wks DID data were analyzed per mouse from each group and a correlation test was performed
with Firmicutes, Actinobacteria, Bacteroidetes and Verrucomicrobia phyla population counts
(Fig. 8A-D; Spearman r correlation, n=6 per group *p < 0.05, **p < 0.01). Data were shown as
28
counts per phyla in comparison with total 20E consumption (g/kg/40hrs). Total ethanol intake
was shown to have a significant inverse correlation with Firmicutes and Actinobacteria
population counts. (Fig. 8A-B; Spearman r correlation, r = -0.6, **p < 0.01 in both population).
Total alcohol consumption was also shown to have significant positive correlation with
Bacteroidetes and Verrucomicrobia numbers in population (Fig. 8C-D; Spearman r correlation, r
= 0.5 *p < 0.05 and r = 0.7 **p<0.01, respectively)
Figure 8. Total alcohol consumption is in correlation with alterations in intestinal flora phyla.
4 wks DID data was calculated per mouse and averaged (H2O – blue circles, ABX – red circles, HSB – dark blue triangles, ASB
– dark red triangles). A) and B) Total ethanol intake was shown to have significant inverse correlation with Firmicutes and
Actinobacteria population counts. C) and D) Total ethanol intake was also shown to have significant positive correlation with
Bacteroidetes and Verrucomicrobia population counts. Results are presented in the form of mean ± SEM for each treatment.
Significant changes were presented as * (p < 0.05) and ** (p < 0.01).
29
3.8 Treatments did not appear to cause liver tissue morphology change while early-stage
steatosis appears after alcohol intake
H&E staining was performed to observe morphology changes between groups with or
without 20E treatment. Under 4X field, there is no significant morphology changes between
different treatment groups. However, using 40X field, there were significant differences between
ABX treated and untreated groups. In the H2O-20E group, like all the other alcohol treated
control mice, lipid accumulation appeared in liver tissues. However, in the HSB-20E group,
more empty space was observed compared with all the other groups. Compared with non-ABX
groups, ABX and ASB groups significantly had more continuous tissue morphology with less
space within and between cells, showing protective effect by ABX treatment. To test the lipid
accumulation state in the liver, Oil Red O staining was utilized. Matching with what was shown
in the H&E stain, within non-20E groups, there was no obvious lipid accumulation differences
between different treatments. Within 20E group, the HSB-20E group has more intense red space
compared with all the other groups. The H2O-20E group had more intense red space compared
with ABX treated groups, especially those areas surrounding the blood vessels.
30
Figure 9a. H&E staining of liver sections between non-20E groups (4X).
H&E staining is performed to observe morphology changes between groups without 20E treatment. There is no significant
morphology difference (under comparison of liver tissue damage) among all treatment groups without 20E intake. (BioTek
Cytation5 Imaging Multi-mode Reader)
31
Figure 9b. H&E staining of liver tissue sections between 20E groups (4X).
H&E staining is performed to observe morphology changes caused by group treatments combining with 20E effect. There is no
obvious morphology (tissue damage, lipid droplet, inflammation positions etc.) changes under 4X field. (BioTek Cytation5
Imaging Multi-mode Reader)
32
Figure 9c. H&E staining of liver tissue sections between 20E groups (40X).
H&E staining is performed to observe morphology changes caused by group treatments combining with 20E effect. The groups
with ABX treatments either with SB or without show less space within or between cells. In H2O-Et group, there is more space in
each cell unit compared with all the other groups. In HSB-Et group, there is more space surrounding the cells compared with the
ABX and ASB groups. (Zeiss Axio Scope. A1)
33
Figure 10a. Oil Red O staining of liver tissue sections between non-20E groups (4X).
Oil Red O staining was utilized to observe lipid accumulation in the liver between groups without 20E treatment. There were no
obvious lipid accumulation differences between the groups without 20E treatment. (BioTek Cytation5 Imaging Multi-mode
Reader)
34
Figure 10b. Oil Red O staining of liver tissue sections between 20E groups (4X).
Oil Red O staining was utilized to observe lipid accumulation in the liver tissue caused by group treatments combining with 20E
effect. The HSB-20E group, has more intense red space compared with all the other groups. The H2O-20E group has more red
stained tissue compared with ABX treated groups, especially those areas surrounding the blood vessels. (BioTek Cytation5
Imaging Multi-mode Reader)
3.9 Antibiotic cocktail has protective effect against acute inflammation in liver
Adipocytes secrete interlekukin-6 (IL-6) and Tumor Necrosis Factor -α (TNF-α), the
concentrations of which correlate with the distribution of fat tissue
54
. Both of them are the main
causes which induce production of acute phase proteins production and the inflammatory state.
Interlekukin-1β (IL-1β) signaling contributes to tumor growth and progression through activation
of numerous transcription factors, including NF-κB. All these mentioned cytokines are tested
with fold changes of mRNA expression in liver tissues. ABX treatment significantly reduce
mRNA expression of IL-6 and TNF-α with 20E treatment compared with H2O-20E group.
35
(Figure 11A and B; Kruskal-Wallis Test, n=3 or 4 per group, * p = 0.0291 and * p = 0.0192
respectively). There is no significant change shown in IL-1β (Figure 11C; Kruskal-Wallis Test,
n=3 or 4 per group). Monocyte chemoattractant protein-1 (MCP-1/CCL2) regulates
monocytes/macrophages movement working as an important chemokine
55
. Neutrophil
granulocytes abundantly express myeloperoxidase (Mpo), which carries out their antimicrobial
activity by producing hypohalous acids. Both factors are measured to evaluate inflammatory
state in the liver tissues. ABX and SB treatment increased MCP-1 mRNA expression after 20E
treatment compared with H2O-20E group (Fig. 12A; Kruskal-Wallis Test, n=3 or 4 per group, p
= 0.0571 and p = 0.0625 respectively). ABX treatment significantly reduce MPO mRNA
expression with 20E treatment compared with H2O-20E group (Fig. 12B; Kruskal-Wallis Test,
n=3 or 4 per group, * p = 0.0401). Activation of TLR4 plays a vital role in innate immune
system activation by initiating the NF-κB signaling pathway and inflammatory cytokine
production. No significant differences were observed in TLR4 expression between all the
treatment groups. The IL-6, TNF- α and MPO results have same trend, indicating liver acute
inflammation state, while IL-1β and MCP-1 in the HSB-20E group has the highest expression
level.
36
37
Figure 11. Significant change in liver expression of inflammatory cytokines. A) – C) are IL-1β, IL-6 and TNF-α mRNA
expression in liver tissues respectively.
A) and B) ABX treatment significantly reduce IL-6 and TNF-α mRNA expression with 20E treatment compared with H2O-20E
group (Kruskal-Wallis Test, n=3 or 4 per group, * p = 0.0291 and * p = 0.0192 respectively). C) There is no significant change
shown in IL-1β (Kruskal-Wallis Test, n=3 or 4 per group). Results are presented in the form of mean ± SEM for each treatment.
Significant changes were presented as * (p < 0.05) and ** (p < 0.01).
Il-1β: interleukin-1β; IL-6: interleukin-6; TNF-α: tumor necrosis factor-α.
Figure 12. Significant change in liver expression of innate immune response mediators.
A) ABX and SB treatment increased MCP-1 mRNA expression after 20E treatment compared with H2O-20E group (Kruskal-
Wallis Test, n=3 or 4 per group, p = 0.0571 and p = 0.0625 respectively). B) ABX treatment significantly reduce MPO mRNA
expression with 20E treatment compared with H2O-20E group (Kruskal-Wallis Test, n=3 or 4 per group, * p = 0.0401). Results
are presented in the form of mean ± SEM for each treatment. Significant changes were presented as * (p < 0.05) and ** (p <
0.01).
Mcp-1: monocyte chemoattractant protein -1; Mpo: myeloperoxidase.
38
Figure 13. No significant change in Toll-like receptor 4 (TLR4).
There were no significant fold changes in mRNA expression of TLR4 shown either within groups or among different treatment
groups. Results are presented in the form of mean ± SEM for each treatment.
4. Discussion
4.1 Higher alcohol consumption along with alterations in gut microbiota
Dysbiosis in the intestinal flora can influence behaviors. In this study, we tried to connect
alterations in gut microbiota with mouse alcohol consumption behaviors. Mice treated with ABX
had higher alcohol consumption, and at the same time, the butyric acid producing bacteria in
Firmicutes phyla at the genus level were significantly reduced, including Lachnospiraceae,
Clostridium IV and Clostridium XIVa. Based on a previous report and our preliminary study, we
selected the SB concentration of 8 mg/mL drinking water. In the pilot study, we used 4mg/mL as
SB treatment. There is a trend to decrease the alcohol intake, but there was no significant
difference within the first 2 wks DID. Thus, in this experiment, we increased the SB
concentration to 8mg/mL. Different from the ABX group mice, the 20E consumption volume of
ASB mice was significantly lower and was close to the intake levels of H2O and HSB group
39
mice. These different volumes of alcohol consumption are not due to different treatments in
alcohol absorption and metabolism since we observed same level of blood ethanol concentrations
after a bolus IP injection of ethanol in all treatment groups of mice. These findings demonstrate
that alterations in gut microbiota are able to alter the drinking behavior in different mouse
alcohol drinking models.
4.2 Early-stage liver steatosis and acute liver inflammation
Alcohol drinking is usually connected with inflammation state in the body. ABX group
(ABX-20E) mice had highest alcohol intake, however, they did not develope liver steatosis as
what control groups did (H2O-20E). Matched by decreased IL-6, TNF-α and MPO mRNA
expression in ABX-20E group compared with H2O-20E group demonstrated the protective
effect caused by ABX, indicating that the innate immune system played a role. TLR4 is related
to development of the inflammation state by endotoxins and activation of its downstream
signaling pathway. However, the mRNA expression of TLR was stable. The drinking model we
used in this study, i.e. DID paradigm, resembles a binge-like voluntary drinking, wherein the
blood ethanol concentrations reach high levels. We could expect higher levels of steatosis,
however we did not see that. Only early-stage liver steatosis was observed. It could be due
overall low amounts and time to ethanol exposure, as well mouse sex and age. We expected that
supplementation with SB will be protecting from ethanol-induced inflammation based on its
known anti-inflammatory nature. As expected, SB supplementation did reduce inflammatory
response in the liver; however, this effect was not as potent as the one induced by ABX and the
differences did not get to be significant. Moreover, SB by itself caused some level of
inflammation and steatosis in the liver. ABX helped reduce these effects when applied together
40
with SB. This could be related to the dose concentration of SB. Further study needs to be
performed to see how SB supplement along with alcohol drinking can influence the metabolism
in the liver.
4.3 Gut-brain axis
There are links between the intestinal flora and different tissues. The gut-liver axis, as
mentioned in section 2.4, is one of them. The gut-brain axis is another bidirectional pathway,
through the vagus nerve, the endocrine system and the immune system that mediates the
communication between the intestine and brain activities. The imbalance of intestinal flora is
related to abnormal neurodevelopment, neuroinflammation and abnormal brain function. Recent
studies link the gut microbiome and neurological diseases through the gut-brain axis. It is also
demonstrated that the destruction of the gut microbiome ecosystem and its functions will be able
to directly or indirectly affect the disease state of central nervous system (CNS), which involves
neuroinflammation and neurodegeneration caused by microglia. It is vital to analyze the
neuroinflammatory state in the brain to see if ABX also has a protective effect on the brain. Glial
cells are vital in CNS development and maintenance. Microglia act as macrophages in the central
nervous system and play a vital role in phagocytosis. Astrocytes are supporting cells with many
functions in the central nervous system. These functions include providing structural support,
insulating the receptor surface, and buffering the extracellular compartment.
Immunohistochemistry (IHC) staining is a good way to compare the morphological changes of
these cells. Based on the morphology and quantitative results, the effect of ABX can be better
understood.
41
All in all, this study showed that SB produced by “good” intestinal flora species is able to
reversely influence ABX-induced increase in voluntary alcohol drinking in a binge-like mouse
drinking model. This is because the fact that although a higher taste preference of SB is observed
in mice, SB reduces both the ethanol intake and preference. Ongoing tests which compare the
microbiota composition between ABX and ASB group mice will enhance the value of stability in
intestinal flora in drinking behavior.
The gut microbiome has a vital impact on host development, immune homeostasis, and
participation in the development of central nervous system diseases, making it an ideal candidate
for new possible treatments. These approaches include the usage of prebiotics and probiotics and
improvement in lifestyle regarding diet and exercise.
5. Conclusion
DID experiment found that ABX treatment significantly increased the mouse alcohol
consumption behavior immediately after 2wks pre-treatment. Importantly, SB, as a supplement,
is able to reverse this increased alcohol intake. In the TBC study, all groups of mice preferred to
alcohol drinking compared to their daily liquid treatments. Moreover, HSB group of mice prefer
SB in the drinking water to the H2O treatment. At the same time, increased alcohol consumption
along with different treatments did not influence the alcohol metabolism in serum. The ABX
treatment altered the gut microbiota composition in mice, and specifically reduced the microbe
diversity in the gut. However, no significant alterations were observed in the SB treatment group.
Early-stage liver steatosis is observed in non-ABX groups, more severe in HSB group. ABX was
also shown to have protective effects on acute liver inflammation, while less fat accumulation
was observed. All these findings show that antibiotic treatment can increase mouse alcohol
42
consumption behavior, and, at the same time, has the protective effect on liver steatosis
development and liver acute inflammation. While SB treatment is able to reverse the increased
alcohol intake, it was not able to alter the gut microbiota and improve steatosis and inflammation
development in the liver.
43
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Abstract (if available)
Abstract
Patients diagnosed with continued, unhealthy use of alcohol usually compare with alterations in gut microbiota diversity and compositions and these changes have been associated with contributing to alcohol-related liver disease. It is therefore suggested that gut microbiota edition can be considered as a potential therapy for alcohol liver diseases. In our previous study, a designed low bioavailable antibiotic cocktail (ABX) treatment on C57BL/6J male mice using a binge-like “Drinking in the Dark (DID)” mouse alcohol drinking model resulted in reduced gut microbial diversity, with pronounced decreases in commensal Firmicutes phyla, which includes members that produce short-chain fatty acids (SCFA). SCFA, for instance butyrate, fermented by microorganisms, have protective benefits on human health. In this regard, ABX treatment caused an increase in ethanol intake and sodium butyrate (SB) supplementation reversed this effect. Based on all these findings, we hypothesized that ABX is also able to cause changes in the liver due to reduced butyrate-producing microbial phyla. In our study, we tested the influences of ABX and/or SB treatments on the liver in a 4-week DID paradigm. We found that ABX reduced early-stage liver steatosis after DID exposure as assessed using H&E/Oil Red O tissue staining. ABX also prevented the increase in mRNA for inflammatory cytokines (IL-6, TNF-α) and an increase in myeloperoxidase (Mpo), a marker for neutrophils. SB supplementation did not change the fat distribution caused by ABX. These findings demonstrate that in contrast to the increase in ethanol intake, ABX treatment was more protective on the liver parameters and SB did not have any additional effects over the ABX-induced changes. Manipulation of the gut microbiome with ABX can be a therapeutic strategy to maintain liver health during excessive alcohol consumption. Further study should focus on the specific microbial phyla and produced protective metabolites for treating alcohol-related liver steatosis.
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Beneficial effect of antibiotic treatment on alcohol-related liver pathology in mice is not due to reduction in the butyrate-producing gut microbial phyla
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