MISS XIUXIAN LIN (Orcid ID : 0000-0003-2134-6396)

Article type : Critical Review

Accepted Article
Reversing epigenetic alterations caused by alcohol: a promising

therapeutic direction for alcoholic liver disease

Xiu-Xian Lin1,2, Guang-Hui Lian3, Shi-Fang Peng4, Qing Zhao1,2, Ying Xu1,2, Dong-Sheng

Ou-Yang1,2, Wei Zhang1,2, and Yao Chen1,2*

1Department of Clinical Pharmacology, Xiangya Hospital, Central South University,

Changsha, Hunan, China, 2Institute of Clinical Pharmacology, Central South University,

Changsha, Hunan, China, 3. Department of gastroenterology, Xiangya Hospital, Central

South University, Changsha, Hunan, China, 4Department of Hepatology and Infectious

Diseases, Xiangya Hospital, Central South University, Changsha, Hunan, China.

*Correspondence: Associate Prof. Yao Chen, Department of Clinical Pharmacology,

Xiangya Hospital, Central South University, Changsha, Hunan 410078, China. Tel:

+86-731-84805380; Fax: 86-731-82354476; E-mail: [email protected]

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/acer.13863

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 Acknowledgments

This work was supported by the national science and technology plan (Grants
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2017ZX09304014), the National Scientific Foundation of China (Grants 81302850),

the Changsha Science and Technology Plan (Kq1602014) and the Natural Science

Foundation of Hunan Province (S2018JJ2629). The funders had no role in the study

design, data collection and analysis, decision to publish or preparation of the

manuscript.

Other authors’ contact emails:

Xiu-Xian Lin：[email protected]

Guang-Hui Lian: [email protected]

Shi-Fang Peng：[email protected]

Qing Zhao：[email protected]

Ying Xu：[email protected]

Dong-Sheng Ou-Yang：[email protected]

Wei Zhang：[email protected]

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 Reversing epigenetic alterations caused by alcohol: a promising

therapeutic direction for alcoholic liver disease

Accepted Article
Abstract Alcoholic liver disease (ALD), a liver function disorder caused by excessive

alcohol intake, is a serious threat to global public health and social development. Toxic

metabolites and reactive oxygen species (ROS) produced during the metabolism of

alcohol can alter the epigenetic state including DNA methylation, histone modifications,

and expression of microRNAs. Epigenetic alterations can conversely involve various

signaling pathways, which could contribute to the initiation and progression of ALD. To

elucidate the relationship between epigenetic alterations and alcohol damage not only

reinforces our understanding on pathogenesis of ALD, but also provides novel targets

for clinical diagnosis, treatment, and drug research of ALD. In the present review, we

have summarized the research progress of epigenetic alterations and related

mechanisms caused by alcohol in the pathogenesis of ALD. Considering the invertibility

of epigenetic alterations, treatment of ALD through epigenetic modification with

common less harmful compounds is also related.

Key words： Alcoholic liver disease, Epigenetic alteration, DNA methylation,

Histone modification, MicroRNA.

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 Introduction

Prolonged and excessive alcohol use usually causes alcoholic liver disease (ALD).
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Depending on presenting clinical symptoms and histopathology, ALD can be further

categorized as steatosis, alcoholic hepatitis (AH), fibrosis, cirrhosis. Alcohol

abuse-induced mobility impairment accounted for 9.6% (7.7%–11.18%) of

disability-adjusted life years (DALYS) which was reported by 58 studies from 17 Global

Burden of Disease (GBD) study regions in 2010 (Whiteford et al., 2013). ALD-induced

liver cirrhosis has become the 12th largest global threat to mortality, and now accounts

for 0.9% of total mortality, endangering public health and social development (Rehm et

al., 2013).

The pathogenesis of ALD is complicated, with many environmental factors

involved, including fat metabolism (Naveau et al., 2010), other liver disease (Nahon et

al., 2016), intestinal microbiota (Llopis et al., 2016) and nutrient status (Stickel and

Hampe, 2012). Alcohol is the most important original risk factor for ALD, which can lead

to ALD in various ways (Askgaard et al., 2015). On the one hand, the metabolism of

alcohol can produce abundant ROS, resulting in endoplasmic reticulum (ER) stress in

hepatocytes. Moreover, the metabolite acetaldehyde can cause harmful adduct

formation and induce genetic material transformation(Anstee et al., 2015). On the other

hand, alcohol can also increase intestinal permeability and alter gut microbial

homeostasis, which facilitate the Gram-negative bacteria endotoxin LPS to access portal

circulation and result in the activation of Kupffer cells, thus aggravating alcoholic liver

injury (Mandrekar and Szabo, 2009). Despite abstinence, alcoholic liver injury will

continue, which currently poses great challenges to the control and reverse of alcoholic

liver disease.

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 Multiple signaling networks underlying the pathogenesis of ALD have been

extensively explored, while the epigenetics mechanism is brought up more recently. The
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concept of epigenetics is related to the regulation of chromatin compaction and gene

expression without involvement of DNA sequence changes. Epigenetic regulation

mainly includes DNA methylation, histone modifications, and microRNAs-based

mechanisms, which can be inherited during cellular and developmental processes

(Berger et al., 2009). Abundant epigenetic research on alcohol damage has revealed that

many epigenetic variations are involved in signaling networks related to the initiation

and progression of ALD.

Currently, there are no FDA approved drugs for the treatment of ALD, the

mainstream therapy for ALD is still abstinence from alcohol, nutritional support and

corticosteroids(Mathurin and Bataller, 2015). In addition, many medicines are of

limited use because of liver toxicities. Therefore, it’s imperative to find new therapeutic

targets and less harmful compounds for ALD. Since the epigenetic alterations could be

altered by the environmental factors, and the reversal potential regulation of epigenetic

alterations might not add further burden to damaged liver, treatment of the ALD

through epigenetic modification is novel and hopeful(Shivashankara et al., 2012,

Shankar et al., 2013).

Epigenetics modifications and alcohol damage

Alcohol and its metabolites can change epigenetic modifications by altering DNA

methylation, histone modifications and miRNAs expression, which can affect alcohol

damage by involving various pathways.

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 DNA methylation alterations by alcohol

DNA methylation is one of the epigenetic modifications in eukaryotic organisms, which

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is catalyzed by DNA methyltransferases (DNMTs). DNMTs can transfer the methyl of

S-adenosyl methionine (SAMe) to 5 carbon atoms of cytosine in double stranded DNA,

forming 5-methyl cytosine (5-mC) (Varela-Rey et al., 2013). It is generally believed that

hypermethylation is related to gene silencing, while hypomethylation has the opposite

effect. However, DNA methylation is a dynamic process, closely related to specific gene

methylation or demethylation, so it can regulate diverse physiological and pathophysiological

processes. As shown in Figure 1, alcohol can alter DNA methylation in three ways,

including through the inhibition of DNMT activity directly, interfering in one carbon

metabolism by changing the intracellular redox state or limiting the intake of folic acid,

which decreases SAMe level ultimately.

Effect of alcohol on methylation is various, long-term alcohol abuse can cause

DNMT activity decrease, lower SAM level and global DNA hypomethylation in peripheral

blood or liver(Bönsch et al., 2006). Moreover, the low methylation status was correlated

with the occurrence of hepatic steatosis. Study in Dnmt1N/+ mice showed that reduced

Dnmtase activity protected mice from alcoholic hepatosteatosis by dysregulating genes

involved in lipid metabolism and oxidative stress(Huban et al.,2012). When considering

fibrosis, Zeybel and his colleagues demonstrated that the methylation in CpGs of peroxisome

proliferator activated receptor (PPAR) -α, which possesses an antifibrogenic effect, was

significantly increased in ALD compared to normal liver tissues (Zeybel et al., 2015).

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 Histone modification by alcohol

Histones are subject to diverse posttranslational modifications, mainly including

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acetylation, methylation, and phosphorylation. Histone modification alterations after

alcohol exposure have been discovered in multiple organs, including the liver. Histone

acetylation in mice liver cell lines can be induced after alcohol feeding (Park et al.,

2003). Pal-Bhadra and Lee later found the H3 methylation on lysine (Pal-Bhadra et al.,

2007) and phosphorylation on serine respectively(Lee and Shukla, 2007), creating a

new area in the study of alcohol-associated histone modifications. Additionally,

different histone epigenetic modifications behave in time-dependent manners. H3

phosphorylation generally occurs first in alcohol-induced cultured hepatocytes, which is

followed by acetylation and finally methylation (Shukla and Lim, 2013, Aroor et al.,

2010).

Histone acetylation is regulated by histone acetyltransferase (HATs) and histone

deacetylase (HDACs) that acetylate and deacetylate histone respectively. According to

the functional domain, HATs can be divided into three subfamilies, GNAT, MYST and

p300/CBP, whose function is to transfer the acetyl group on acetyl coenzyme A to lysine

residues (Zhang and Dent, 2005). High acetylation of histone is usually related to gene

activation, which requires access to transcription factors. HDACs include four classes, of

which sirtuin 1 (SIRT1) of Class Ⅲ focused especially. In vivo, the acetic acid produced

in alcohol metabolism can be transformed into acetyl coenzyme A (acetyl-CoA) by

acetyl-CoA synthetase (AceCS1) under the activation of SIRT1, as shown in Figure 4. The

acetyl-coA can acetylate histones in the presence of HATs, and the acetylated histones

can be deacetylated under the catalysis of SIRT1(Underwood et al., 2002, Yeung et al.,

2004). Thus, SIRT1 plays a vital role in the balance between gene silencing and

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 activation. Moreover, SIRT1 can interact with microRNAs, such as miR34a, and is

involved in signaling pathways related to ALD, especially AFLD, as shown in Figure2

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(You et al., 2015, Liangpunsakul, 2015). Plenty of research has shown that alcohol

exposure can alter histone modifications under the mediation of various enzymes,

which further affects the expression of their target genes and induces a series of

biological effects (Table 1).

Histone methylation occurs primarily on the lysine and arginine residues of H3 and

H4, and SAMe is the main donor, just like in DNA methylation(Lu and Mato, 2012).

Influence of methylation in lysine on transcription is associated with which lysine is

methylated. Generally, methylation occurs in H3K4, H3K36, H3K79 related to

transcription activation, while H3K9, H3K27, and H4K20 are associated with

transcription repression (Bassett and Barnett, 2014). For instance, study in rat

hepatocytes treated with ethanol showed that H3K9 methylation was associated with

downregulation of genes like L-serine dehydratase and Cytochrome P 450 2C11 while

H3K4 methylation with upregulation of alcohol dehydrogenases(Pal-Bhadra et al.,

2007). Interestingly, lysine can be methylated as well as acetylated, such as in H3K9,

and transcriptional regulation caused by these two modifications may have opposite

effects (Strahl and Allis, 2000). Histone demethylation is mainly catalyzed by the HDMs

enzyme, however, there are few studies about HDMs in ALD.

The phosphorylation of histone is catalyzed by MAPK, largely in serine 10, 28 of H3

after alcohol exposure, and is generally related to transcriptional activation (Lee and

Shukla, 2007). Acetyl-CoA generated after alcohol metabolism can participate in the

tricarboxylic acid cycle, which provides ATP or GTP, whose phosphate can further be

transferred to N-terminal amino acid residues under the mediation of MAPK(Bhopale et

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 al., 2017). Different alcoholic drinking patterns have diverse effects on the epigenetic

modification of histone. For instance, binge drinking can significantly induce histone
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H3K9 acetylation and H3S10 phosphorylation (H3AcK9PS10), while chronic drinking

does not have the same effect. This has been verified in cell lines and mice, providing

potential targets for the treatment of binge drinking-induced ALD (James et al., 2012).

MicroRNAs modification by alcohol

MiRNAs are small noncoding RNAs, approximately 21-nucleotide long, which are

involved in posttranscriptional gene expression regulation. The pri-miRs formed in the

nucleus can be cleaved by RNAases Drosha and Dicer successively when transferred to

cytoplasm, then combined with Ago2, GW182, forming RNA-induced silencing complex

(RISC). Mature miRNAs can cleave mRNA or repress mRNA translation through their

combination with 3'-UTR mRNA, thus inhibiting the expression of target mRNA. One

miRNA generally has diverse target genes and one gene is usually regulated by various

miRNAs at the same time, constituting a complex regulatory network. Therefore,

miRNAs can regulate most biological or pathological processes. In alcoholic liver disease,

miRNAs can contribute to the progression of liver homeostasis, as shown in Figure2, hepatic

fibrosis, as shown in Figure3, and liver inflammation, as shown in Figure4. MiRNAs not only

exist inside of cells, but are also stable in the circulatory system, such as blood, which

provides great opportunities for investigation of novel biomarkers for ALD (Starkey

Lewis et al., 2011). A large number of studies have shown that a variety of miRNAs are

abnormal in alcohol-induced liver injury, indicating that the expression of miRNAs

could be altered by alcohol and have an important role in ALD (Dolganiuc et al., 2009,

Bala and Szabo, 2012, Szabo and Satishchandran, 2015). However, research about

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 relation between other noncoding RNAs, such as long non-coding RNAs, and ALD is

obviously less. Only one research study directed by Yang Z and his colleagues showed
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that lncRNA AK054921 and AK12865 were potential serum biomarkers of alcohol

induced alcoholic liver disease and survival predictors of patients with alcoholic

cirrhosis(Yang et al., 2017). Epigenetic alterations in AFLD, alcoholic liver fibrosis and

alcoholic liver inflammation were shown in Figure 2, Figure 3 and Figure 4 respectively.

Presently, miRNAs related to ALD mainly focused on miR-155, miR-34a, miR-132, and

miR-122, other miRNAs and their effects were shown in Table 2.

It has been demonstrated by a series of studies that miR-155 plays an important

role in ALD (Hartmann and Tacke, 2016). MiR-155 expression level upregulation can be

observed in hepatocytes and Kupffer cells (KCs) in ethanol-fed mice, alcohol-exposed

RAW264.7 macrophages (Bala and Szabo, 2012, Bala et al., 2011) . The increase of

miR-155 in macrophages was primarily related to LPS. LPS in the liver can combine

with Toll-Like Receptor-4 (TLR4) to form complexes and enter KCs and upregulate

miR-155 under the mediation of NF-κB, which promotes the release of inflammatory

factor TNF-α(Bala et al., 2011, Hritz et al., 2008). Moreover, upregulation of miR-155

can also enhance the sensitivity of KCs to LPS, thus aggravating the inflammatory

reaction(Bala and Szabo, 2012). Studies in miR-155 gene knockout mice found that

when compared to the control group, alcohol-induced liver injury, steatosis,

inflammation, and fibrosis were significantly alleviated in miR-155 KO mice (Bala et al.,

2016). A series of responses caused by miR-155 KO were pointed out to be probably

related to the PPAR signal pathway. PPARα and PPAR-γ, as targets of miR-155, are

involved in the process of fat metabolism, oxidative stress, inflammatory reactions, and

fibrosis (Bala et al., 2016). In hepatocytes, the upregulation of miR-155 caused by

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 alcohol exposure can inhibit PPARα expression, thereby inducing expression of genes

related to fat metabolism and fatty acid uptake, such as FABP4, LXRa, ACC1, and LDLR,
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as shown in Figure 2. Moreover, miR-155 can also inhibit the combination of PPAR and

PPRE, which promotes the expression of MCP-1 and aggravates steatohepatitis. In

macrophages, upregulation of miR-155 induced by alcohol exposure not only

downregulates the antifibrosis gene PPAR-γ expression, but also represses the

fibrosis-related genes, like STAT3, which regulate the process of liver fibrosis, as shown

in Figure 3. Moreover, miR-155 can induce macrophages polarized into the M1

phenotype by inhibiting the expression of C/EBP and STAT3, and releasing

inflammatory factors such as IL-1 and TNF, which promote inflammatory reactions, as

shown in Figure 4. (Bala et al., 2016, Hartmann and Tacke, 2016).

MiR-34a is a member of the miR-34 family, which has important effects on tumor

growth inhibition involving multiple aspects of cell cycles and cell apoptosis. through

targeting tumor suppressor gene p53 on its promoter region (Hermeking, 2009).

Upregulation of miR-34a was shown in alcohol-induced liver injury and played an

important and complex role in ALD (Meng et al., 2012, Dippold et al., 2013). Expression

of miR-34a was found significantly increased, while p53 was significantly decreased, in

the liver tissue of AH patients with Mallory body formation in comparison to normal

tissues, suggesting that regulation of miR-34a by p53 was weakened in the process of

Mallory body generation, as shown in Figure 4 (Liu et al., 2015). Intriguingly, miR-34

can be methylated, because its 5’-promoter region is embedded in a CpG island. Studies

in N-Heps and HiBECs cells showed that the hypomethylation of miR-34a in promoter

region induced by ethanol exposure was associated with the high expression of

miR-34a. These changes in the expression of miR-34a can regulate the survival or

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 remodeling of hepatocytes by CASP2, SIRT1 or MMP-2, MMP-9 respectively, as shown in

Figure 3(Meng et al., 2012). Ying Wan’s study has indicated that antisense miR-34a
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could reverse fibrosis induced by HSCs activation under LPS mediation by inducing

cellular senescence in alcohol induced liver fibrosis(Wan et al., 2015). In addition, the

metabolic stress conditions, miR-34a can also regulate plasma lipoprotein metabolism

and affect the pathogenesis of NAFLD through the miR-34a-HNF4a signaling pathway,

however, the pathway has not been verified in AFLD(Xu et al., 2015).

In alcohol-fed mice, the expression of miR-132 in KCs is significantly increased

compared with controls, but has no significant differences in hepatocytes(Bala and

Szabo, 2012). However, the study about miR-132 is more focused on its inhibition of

P300 expression and regulation of antiviral (herpes virus, human cytomegalovirus)

reactions (Lagos et al., 2010). MiR-212 and miR-132 are the products of the same gene,

thus they have abundant homology. However, unlike miR-132, the upregulation of

miR-212 expression in alcohol-exposed mice mainly occurred in the small intestine,

suggesting its important role in maintaining the permeability of intestinal wall. Studies

on intestinal specimens from ALD patients and Caco-2 cell models found that alcohol

can induce miR-212 expression while downregulating the expression of zonula

occludens-1 proteins (ZO-1), indicating that miR-212 may affect intestinal permeability

through ZO-1, as shown in Figure 4(Tang et al., 2008).

Along with miR-212, miR-122a can also affect intestinal permeability by targeting

transmembrane tight junction protein, occludin, as shown in Figure 4 (Bala and Szabo,

2012). Probiotics can be used to reduce intestinal permeability by downregulating

miR-122a expression, thus promoting the expression of occludin protein, preventing the

development of ALD (Li et al., 2016a). MiR-122 is one of the liver-specific miRNAs

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 expressing abundantly in the liver, especially in hepatocytes, which can affect liver

steatosis (Sud et al., 2015), fibrosis (Li et al., 2013), inflammation(Momen-Heravi et al.,
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2015a). In hepatocytes, miR-122 can regulate the synthesis of fatty acids and

cholesterol by targeting genes related to fatty acid generation, such as ACC2, FASN, as

shown in Figure 2 (Esau et al., 2006). Additionally, studies in HepG2 cells have shown

that miR-370 can indirectly promote fat synthesis by enhancing the expression of

miR-122 (Iliopoulos et al., 2010). A mouse model study found that miR-122

downregulation in the alcohol-induced liver injury can activate the expression of HIF-1,

which contributes to the acceleration of early hepatobiliary cancer (Ambade et al.,

2016). In addition, in alcohol-induced liver injury, miR-122 upregulation in plasma

correlates with ALT and other liver injury-related indicators, suggesting that miR-122

could possibly be a marker for liver injury diagnosis (Ambade et al., 2016, Bala et al.,

2012).

MicroRNA alterations in Circulation

Many studies have observed the presence of microRNAs in circulation, such as in blood

and body fluids, in various ways, including associations with exosomes or macro

vesicles, combining into HDL (Weber et al., 2010, Valadi et al., 2007). Extracellular

microRNAs may play the role of signal transduction between cells and organs.

Moreover, because of their cell or organ source specificity, circulating miRNAs are

expected to become biomarkers of diseases, and have attracted the attention of a great

many scholars.

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 MicroRNA expression among different types of liver injuries are various, and the

existing form of extracellular mode is also different, suggesting that microRNAs in

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circulation are associated with the pathogenesis of diseases and promising in disease

diagnosis (Bala et al., 2012). Alcohol exposure can promote the secretion of exosomes

carrying miR-27a from monocytes, overexpression of miR-27a can induce the activation

of naive monocytes into M2 macrophages by activating the ERK signaling pathway

(Saha et al., 2015, Saha et al., 2016). MiR-122 in exosomes from alcohol-exposed

hepatocytes or sera of alcohol-fed mice can enhance the sensitivity of monocytes to LPS

and promote the release of inflammatory factors by inhibiting the HO-1 signaling

pathway, as shown in Figure 4 (Momen-Heravi et al., 2015a). Studies in mice and human

beings have also found that drinking can induce a significant increase of exosomes and

related miRNAs in blood, which can then lead to the occurrence of inflammation. Gyongyi

Szabo’s study in mice showed that alcohol feeding can increase EVs (mainly exosomes) and

miRNAs it package in plasma, which then verified in the plasma of patients with AH, and

further ROC analysis found that miR-192 showed promising value in the diagnosis of

AH(Momen-Heravi et al., 2015b). Ariel E. Feldstein and his colleagues found that

hepatocytes were the main source of EVs in mice with early alcoholic fatty liver disease, and

these EVs contain a miRNA barcode that is specific for different liver injury types. This

phenomenon has been validated in comparison between the early reversible ASH patients and

non-alcoholic patients(Eguchi et al., 2016). These differences of circulating miRNAs can not

only be used as biomarkers for diagnosis, but also provide a new strategy for the tracking and

treatment of ALD.

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 Reversing epigenetic alterations for ALD therapy

Epigenetic modifications caused by alcohol have complicated effects on ALD,

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considering the reversal potential of epigenetic regulation, relevant research results

may be translated into effective clinical treatments, providing novel ways for the

prevention and treatment of ALD.

In vivo studies showed that increasing methyl demand could further aggravate

alcohol-induced liver damage (Kharbanda et al., 2014).DNA methylation alterations can

be reversed by supplement with dietary methyl donors (Medici et al., 2014). Thus,

increasing the intake of food or drugs containing methyl groups may be effective in

slowing ALD progression (Day and Kempson, 2016). However, despite successful

animal-testing trials, the therapeutic protection of methyl donors SAMe and betaine

against alcoholic liver injury is still clinically controversial. Therefore, systematic,

randomized trials are still required to verify effects of these methyl donors on ALD

(Medici and Halsted, 2013).

Resveratrol, a polyphenol principally from grapes, has shown considerable

promise in liver injury prevention and therapy(Jiang et al., 2017). In vivo and in vitro

research has proven the therapeutic effects of resveratrol on alcohol-induced liver

injury through histone modifications involving AMPK and SIRT1 (Bishayee et al., 2010).

Additionally, resveratrol could downregulate miR-21, which is upregulated in ALD and

related to cell apoptosis (Francis et al., 2014), remodeling and liver regeneration

(Dippold et al., 2012) in human SW480 colon cancer cells. Therapeutic effect of

resveratrol on other disease through epigenetic regulation also reported(Zhang et al.,

2011, Dai et al., 2015). Therefore, resveratrol could possibly reverse or cure ALD

through epigenetic regulation, which needs further investigation.

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 In addition, common traditional compounds with potential of epigenetic

modification may be used for the therapy of ALD. Statins are widely used in the
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treatment of hyper lipemia and coronary artery disease, which also closely related to

lipid metabolism, oxidative stress and inflammation(Zheng et al., 2017, Chen et al.,

2012). Recent study has reported that statins are associated with reduced total

mortality among patients with ALD(Stokkeland et al., 2017). However, the underlying

mechanism is unknown. Previous researches have indicated that statins, such as

simvastatin, are related to the expression of miR-34a and SIRT1 (Tabuchi et al., 2012,

Kilic et al., 2015), both of which are critical in the development of ALD. Whether statins

can ameliorate ALD through epigenetic modification deserves further research. In a

word, treatment of ALD through epigenetic modification has provided a novel idea for

the therapy of ALD.

Prospective

The relationship between epigenetic modifications and alcohol damage is complicated.

Ethanol, ethanol metabolites and ROS produced during metabolic processes can

regulate DNA methylation, histone modifications and miRNAs expression. These

epigenetic mechanisms regulate gene expression at the transcriptional or the

posttranscriptional level, which will eventually be involved in various signaling

pathways and affect the initiation and progression of ALD. However, there has not been

a study of epigenetic status changes with the development of alcoholic liver disease,

which needs more research. A great deal of researches have provided new biomarkers

and therapy targets for the diagnosis and treatment of ALD. Treatment of ALD with

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 common harmless compounds through epigenetic modification is feasible and

promising.
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Acknowledgments

This work was supported by the national science and technology plan (Grants

2017ZX09304014), the National Scientific Foundation of China (Grants 81302850),

the Changsha Science and Technology Plan (Kq1602014) and the Natural Science

Foundation of Hunan Province (S2018JJ2629). The funders had no role in the study

design, data collection and analysis, decision to publish or preparation of the

manuscript.

Declaration of interest

The authors have declared that no competing interests exist.

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 Abbreviations

ALD Alcoholic liver disease

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ROS Reactive oxygen species

HCC Hepatocellular carcinoma

GBD Global Burden of Disease

DALYS Disability-adjusted life years

ADH Alcohol dehydrogenase

CYP2E1 Cytochrome P450 2E1

ALDH Acetaldehyde dehydrogenase

ER Endoplasmic reticulum

AFLD Alcoholic fatty liver disease

LPS Lipopolysaccharides

BMI Body Mass Index

DNMTs DNA methyltransferases

5-mC 5-methyl cytosine

SAMe S-Adenosyl methionine

HATs Histone acetyltransferase

HDACs Histone deacetylase

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 SIRT1 Sirtuin 1

Acetyl-CoA Acetyl coenzyme A

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AceCS1 Acetyl-CoA synthetase

HMTs Histone methyl transferases

RISC RNA-induced silencing complex

ASO Antisense oligonucleotides

KCs Kupffer cells

TLR Toll-Like Receptor

ZO-1 Zonula occludens-1 proteins

GLUL Glutamine synthetase gene

IBS Irritable bowel syndrome

EVs Extracellular vehicles

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 Figure Legends
Accepted Article
Figure 1. Association between alcohol and DNA methylation

Alcohol can affect DNA methylation via inhibiting DNMT activity and decreasing SAMe

levels. DNMT activity can be directly inhibited by alcohol and its metabolites or

S-adenosyl homocysteine (SAH) which is formed simultaneously with DNA methylation.

SAH can further be converted into high homocysteine by SAH hydrolase (SAHH).

Alcohol metabolism can generate a large amount of ROS, which then needs to consume

the glutathione (GSH) transited from high homocysteine, resulting in decreased levels of

methionine and further reduced supply of SAMe. Additionally, the transformation

process of high homocysteine into methionine requires the participation of folic acid,

the intake of which may be inhibited after alcohol exposure. Therefore, all of these

abovementioned factors can affect DNMT activity, levels of SAMe, and folic acid supply

can affect DNA methylation.

Figure 2. Epigenetic alterations in AFLD

AFLD mainly manifests as increased fat synthesis and decreased fatty acid (FA)

oxidation in the hepatocytes. Alcohol exposure can induce acetaldehyde accumulation,

ER-stress and CYP2E1 activation which may be regulated by miR-378 (Mohri et al.,

2010). These changes can further lead to AFLD through the upregulation of sterol

regulatory element-binding protein-1 (SREBP-1) and direct or indirect inhibition of

AMP-activated protein kinase (AMPK) and PPAR-α. For the SREBP-1 pathway,

intracellular ER-stress can upregulate the expression of miR-34a (Meng et al., 2012) and

miR-217 (Yin et al., 2012),which inhibits SIRT1 and induces SREBP-1. This can increase

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 activity of lipogenic enzyme by increasing H3K9 acetylation of lipin-1 (Shen et al., 2010,

Yin et al., 2014). In binge drinking, the inhibition of SIRT1 can also induce the activation
Accepted Article
of ADH, which can be inhibited by resveratrol (Liangpunsakul, 2015). On the one hand,

ROS increases can directly or indirectly inhibit AMPK, which not only results in

activation of acetyl-CoA carboxylase (ACC), but also upregulates SREBP-1. The

activation of ACCs not only increase fat synthesis but also decrease FA oxidation by

downregulating carnitine palmitoyl transferase 1 (CPT-1) (Gao and Bataller, 2011). In

NAFLD, miR-122 has proven to be vital to the activation of ACCs, whereas there is no

current research into AFLD (Esau et al., 2006). On the other hand, ROS increases can

inhibit the PPARα pathway, which can also be inhibited by the upregulation of miR-155

in hepatocytes, though its mechanism has not been validated (Bala et al., 2016). The

inhibition of PPARα can result in FA oxidation inhibition via downregulating the

expression of FA oxidation genes. In addition, alcohol exposure can also decrease

adiponectin release from adipose tissue, which upregulates TNF-α in the liver, which is

involved in PPARα inhibition (You et al., 2005). Alcohol exposure also causes autophagy,

which can be induced after miR-26a overexpression, resulting in decreased lipid

droplets accumulation (Han et al., 2015).

Figure 3. Epigenetic alterations in alcoholic liver fibrosis.

Alcoholic liver fibrosis primarily manifests as the extracellular matrix and collagen

deposition in HSCs, which is directly or indirectly induced by stimulation from alcohol,

acetaldehyde, and LPS. Alcohol exposure can directly induce MLL1 activation and

increase H3K4me3, resulting in extracellular matrix (ECM) deposition in HSCs (Page et

al., 2015). It can also inhibit DNMTs in hepatocytes, which leads to upregulation of

miR-34a and MMP-2, MMP-9, resulting in the remolding of liver cells (Meng et al., 2012).

This article is protected by copyright. All rights reserved.

 The accumulation of LPS in the macrophages, on the one hand, can induce miR-155

expression under mediation of NF-κB. This inhibits the PPAR-γ pathway while
Accepted Article
upregulating C/EBPβ expression, resulting in the release of cytokines TNF-α and IL-1β,

which activates HSCs (Bala et al., 2016). On the other hand, it can increase miR-21

expression in hepatocytes under the mediation of IL-6/Stat3, which then inhibits

apoptosis and fibrosis (Francis et al., 2014). Both the upregulation of miR-21 and

miR-155 can be reversed by resveratrol in other disease, though haven’t proved in ALD.

In addition, LPS can activate HSCs directly via TLR-4 (Seki et al., 2007). MiR-122 is also

important to fibrosis formation, which can negatively regulate collagen and ECM

deposition in HSCs (Li et al., 2013). However, its effect on alcoholic liver fibrosis

requires further research.

Figure 4. Epigenetic alterations in alcoholic liver inflammation

Alcohol-induced liver inflammation is induced by excessive release of proinflammatory

cytokines or chemokines, manifesting as hepatocytic MDBs information and neutrophil

infiltration. Alcohol can induce inflammation via metabolites and LPS stimulation. The

acetyl-CoA synthesized in alcohol metabolism and SIRT1 disruption because of miR-217

upregulation (Yin et al., 2015) can increase histone acetylation of genes related to

proinflammatory reaction, which stimulate proinflammatory cytokine release from KCs

(Kendrick et al., 2010). LPS accumulation in the liver because of increased intestinal

permeability can also aggravate inflammation. Alcohol and its metabolites can induce

miR-122a (Bala and Szabo, 2012) and miR-212 (Tang et al., 2008) expression, which

promotes intestinal permeability via targeting tight junction proteins. Increasing LPS in

KCs through TLR-4 can induce miR-155 (Bala et al., 2016) expression, resulting in

proinflammatory cytokine release. However, the upregulation of miR-182 in KCs is

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 unknown (Blaya et al., 2016). Increasing release of cytokines can further activate

chemokine secretion from HSCs and hepatocytes. Moreover, hepatocyte-derived

Accepted Article
exosomes containing miR-122 can enhance the sensitivity of KCs to LPS (Momen-Heravi

et al., 2015a). In addition, the upregulation of miR-182 (Blaya et al., 2016)and miR-214

(Dong et al., 2014) can also lead to liver injury in hepatocytes. Additionally,

upregulation of miR-34a due to decreasing SAMe levels and downregulation of miR-483

in hepatocytes are related to MDBs formation (Liu et al., 2015). Upregulation of

miR-223 in neutrophils after alcohol exposure can ameliorate neutrophil infiltration

and liver injury (Li et al., 2016b), which can be aggravated by MCP-1 after miR-199

downregulation in the liver endothelium (Yeligar et al., 2009).

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ccepted Articl Table1 Modification of Histones Related to ALD and Their Effects

Histones Modification and Enzyme Target genes and expression Effect Reference

H3K9 Acetylation, HATs ADH, Activating Speed up ethanol metabolism (Park et al., 2005, Park et al.,

2012)

H3K9 Acetylation, SIRT1 Lipogenic enzymes, activating Increase fat generation (Shen et al., 2010), (Yin et al.,

2014)

H3K4 Methylation, MLL1 Elastin, activating Deposition of ECM proteins (Page et al., 2015)

H3 Acetylation, p300/CBP TGF-β1, haven’t proved in ALD Fibrosis and HSC activation (Ding et al., 2013)

H3K9 Methylation, JMJD1A PPAR-γ, haven’t proved in ALD Fibrosis and HSC activation (Jiang et al., 2015)

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ccepted Articl Table 2 MicroRNAs Related to ALD and Their Effects

microRNAs Dysregulation Target genes Effect Reference

miR-21 increased FASLG, DR5 decrease apoptosis (Francis et al., 2014),

(Dippold et al., 2012)

miR-26a decreased DUSP4/5 reduce autophagy and induce hepatic steatosis (Han et al., 2015)

miR-182 Decreased (in induce steatohepatitis (Dolganiuc et al., 2009)

alcohol-feeding mice)

miR-182 Increased (in AH IL-6 induce inflammation, related to AH severity (Blaya et al., 2016)

patients)

miR-199 decreased HIF-1α, ET1 increase infiltration of neutrophils (Yeligar et al., 2009)

miR-214 increased GSR, POR induce oxidative stress (Dong et al., 2014)

miR-217 increased SIRT, lipin-1 induce fat accumulation and inflammation (Yin et al., 2012, Yin et al.,

2015)

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ccepted Articl
miR-223 Decreased (in peripheral IL-6 Exacerbate neutrophil infiltration and liver (Li et al., 2016b)

blood neutrophils) injury

miR-378 --- CYP2E1, p110α, lipid homeostasis, hepatic stellate activation (Mohri et al., 2010, Liu et al.,

Gli3 2014, Hyun et al., 2016)

miR-483-3p decreased BRCA Induce MDB formation and inflammation (Liu et al., 2015)

miR-570 --- 24 genes (AAK1, cell proliferation and invasion, CYP2E1activity (Liu et al., 2013, Guo et al.,

etc) 2015, Nakano et al., 2015)

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 NAD+ NADH
ADH
Accepted Article
Ethanol Acetaldehyde Acetic acid
CYP2E1 ALDH
NADPH+H++O2 NADP++H2O NAD+ NADH+H+
ROS ROS

GSH
Folic acid

MTR
Methionine
Homocysteine
MAT
SAHH
SAMe
SAH

DNMTs
DNA DNA-CH3

Figure 1

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 Fat synthesis ↑ FA oxidation ↓ FA accumulation
Accepted Article
Hepatocytes
Lipin-1AC ↑ CPT-1 ↓
Autophag ↑
SREBP-1 ↑ ACC ↑ PPAR- α ↓

SIRT 1 ↓ miR-122 miR-155 ↑ miR-26a

?
miR-217, miR-34a ↑ AMPK ↓ Resvertrol
TNF-α ↑
miR-378

Acetaldehyde, CYP2E1-ROS, LPS

Alcohol Adiponetine ↓

Figure 2

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 Hepatic
stellate cell
ELASTINME ↑ Activation miR-122
Accepted Article
MLL 1 ↑

Apoptosis Extracellular
TLR4 matrix deposition
Cell remodelling ROS TNF- α, IL-1 β ↑

Resvertrol PPAR-Υ ↓ C/EBP β ↑

Kupffer
? ? miR-155 ↑ cell
MMP2,MMP9 ↑ miR-21 ↑ NF- κB

miR-34a ↑
STAT 3 IL-6 TLR4
DNMT

Hepatocytes
Alcohol LPS ↑

Figure 3

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Accepted Article

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