Mouse Model Of Alcoholic Liver Disease Research Articles

The effect of STAT1, miR-99b, and MAP2K1 in alcoholic liver disease (ALD) mouse model and hepatocyte.

Alcoholic liver disease (ALD) serves as the leading cause of chronic liver diseases-related morbidity and mortality, which threatens the life of millions of patients in the world. However, the molecular mechanisms underlying ALD progression remain unclear. Here, we applied microarray analysis and experimental approaches to identify miRNAs and related regulatory signaling that associated with ALD. Microarray analysis identified that the expression of miR-99b was elevated in the ALD mouse model. The AML-12 cells were treated with EtOH and the expression of miR-99b was enhanced in the cells. The expression of miR-99b was positively correlated with ALT levels in the ALD mice. The microarray analysis identified the abnormally expressed mRNAs in ALD mice and the overlap analysis was performed with based on the differently expressed mRNAs and the transcriptional factors of miR-99b, in which STAT1 was identified. The elevated expression of STAT1 was validated in ALD mice. Meanwhile, the treatment of EtOH induced the expression of STAT1 in the AML-12 cells. The expression of STAT1 was positively correlated with ALT levels in the ALD mice. The positive correlation of STAT1 and miR-99b expression was identified in bioinformatics analysis and ALD mice. The expression of miR-99b and pri-miR-99b was promoted by the overexpression of STAT1 in AML-12 cells. ChIP analysis confirmed the enrichment of STAT1 on miR-99b promoter in AML-12 cells. Next, we found that the expression of mitogen-activated protein kinase kinase 1 (MAP2K1) was negatively associated with miR-99b. The expression of MAP2K1 was downregulated in ALD mice. Consistently, the expression of MAP2K1 was reduced by the treatment of EtOH in AML-12 cells. The expression of MAP2K1 was negative correlated with ALT levels in the ALD mice. We identified the binding site of MAP2K1 and miR-99b. Meanwhile, the treatment of miR-99b mimic repressed the luciferase activity of MAP2K1 in AML-12 cells. The expression of MAP2K1 was suppressed by miR-99b in the cells. We observed that the expression of MAP2K1 was inhibited by the overexpression of STAT1 in AML-12 cells. Meanwhile, the apoptosis of AML-12 cells was induced by the treatment of EtOH, while miR-99b mimic promoted but the overexpression of MAP2K1 attenuated the effect of EtOH in the cells. In conclusion, we identified the correlation and effect of STAT1, miR-99b, and MAP2K1 in ALD mouse model and hepatocyte. STAT1, miR-99b, and MAP2K1 may serve as potential therapeutic target of ALD.

Ethanol extracts of Isochrysis zhanjiangensis alleviate acute alcoholic liver injury and modulate intestinal bacteria dysbiosis in mice.

Alcoholic liver disease (ALD) is responsible for 3.3 million deaths per annum. Efficacious therapeutic modalities or drug treatments for ALD have not yet been found, so it is urgent to seek new agents for preventing ALD and its related disease. Many experiments have indicated that modulating the gut microbiota and regulating the toll-like receptor 4 (TLR4)/nuclear transcription factor-κB (NF-κB) inflammatory pathway can provide a new target for prevention and treatment of ALD. Marine microalgae have their natural metabolic pathways to synthesize various of bioactive compounds as promising candidates for hepatoprotection. In this study, we investigated ethanol extracts from Isochrysis zhanjiangensis (EEIZ) to evaluate their ability to alleviate acute alcoholic liver injury, regulate TLR4/NF-κB inflammatory pathway and modulate intestinal bacteria dysbiosis in mice for ALD treatment. In the acute ALD mouse model, EEIZ reduced levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, triacylglyceride, total cholesterol and low-density lipoprotein, while increasing the level of high-density lipoprotein. Besides, TLR4, myeloid differentiation factor 88, NF-κB and tumor necrosis factor-α expression levels in liver tissue were effectively downregulated by EEIZ. Furthermore, treatment with EEIZ enhanced intestinal homeostasis and significantly alleviated the damage caused by alcohol. EEIZ showed effective hepatoprotective activity against alcohol-induced acute liver injury in mice as it could alleviate hepatocyte damage, suppress the TLR4/NF-κB inflammatory pathway and regulate the intestinal flora structure. EEIZ could be a good candidate for preventing acute alcoholic liver injury. © 2024 Society of Chemical Industry.

Kefir Ameliorates Alcohol-Induced Liver Injury Through Modulating Gut Microbiota and Fecal Bile Acid Profile in Mice.

Alcoholic liver disease (ALD) is the leading cause of liver-related deaths worldwide. Kefir has been studied for its properties of anti-obesity, rebuilding intestinal homeostasis, and alleviating non-alcoholic fatty liver disease. However, the possible role of kefir in the prevention or treatment of ALD has not been carefully considered. Here, it evaluated the protective effects of kefir supplementation on alcohol-induced liver injury. C57BL/6J mice are fed to Lieber-DeCarli liquid diet containing alcohol to build ALD mouse model, followed by oral administration with kefir. Results indicate that kefir treatment improves liver pathological changes, decreases the expression levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and inflammatory markers, and increases antioxidant levels. Kefir supplementation also restores the intestinal barrier and altered microbial composition, indicates as increases of Blautia, Bacteroides, and Parasutterella and decreases in the Firmicutes/Bacteroidetes (F/B) ratio and populations of Psychrobacter, Bacillus, and Monoglobus. Moreover, kefir supplementation decreases the levels of total bile acids (BAs) and primary BAs and increases the secondary/primary BA ratio. Gut microbes play a key role in the conversion of primary to secondary fecal BAs. Kefir can ameliorate ALD through regulating the composition of the gut microbiota.

The protective effect of Nostoc commune Vauch. polysaccharide on alcohol-induced acute alcoholic liver disease and gut microbiota disturbance in mice.

In recent years, the incidence of alcoholic liver disease (ALD) has gradually increased, the development of ALD is attached great attentions. Nostoc commune Vauch. polysaccharide (NCVP) is beneficial to maintain the gut health, but the protective effect of NCVP on the liver has not been reported yet. To study the protective effect and the underlying mechanisms of NCVP on ALD, a mouse model of acute ALD was established. We built an acute ALD mouse model and explored the protective effect of NCVP through the detection of cytokines, histological examination, determination of short chain fatty acids, and 16S rRNA analysis of gut microbiota. NCVP had hepatoprotective effects on acute alcohol-induced mice by improving antioxidant capacity, reducing oxidative stress and the serum cytokine levels (IL-1β, IL-6, and TNF-α). Simultaneously, histopathological changes in liver indicated that NCVP could inhibit local hepatocyte necrosis, cytoplasmic vacuolation and inflammatory cell infiltration induced by alcohol. NCVP also increased the level of total short-chain fatty acids of acute ALD mice. In addition, NCVP could significantly decrease the Firmicutes/Bacteroidetes ratio and the abundance of Patescibacteria, Helicobacter, and Actinomycetes and increase the abundance of Lachospiraceae, Prevotellaceae-UCG-003, Lactobacillaceae, and Desulfovibrio. Our study proved that NCVP had in vivo hepatoprotective effect on acute ALD mice and provided scientific evidences that NCVP might be a promising drug candidate for the prevention and treatment of ALD.

Porphyromonas gingivalis exacerbates alcoholic liver disease by altering gut microbiota composition and host immune response in mice.

Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, increases the risk of systemic diseases. P. gingivalis infection is closely associated with alcoholic liver disease (ALD), but the underlying mechanism remains unclear. We aimed to investigate the role of P. gingivalis in the pathogenesis of ALD. An ALD mouse model was established using a Lieber-DeCarli liquid diet, and C57BL/6 mice were treated with P. gingivalis to detect the pathological indicators of ALD. Oral administration of P. gingivalis exacerbated alcohol-induced alterations in the gut microbiota, leading to gut barrier dysfunction and inflammatory response and disruption of the T-helper 17 cell/T-regulatory cell ratio in the colon of ALD mice. Furthermore, P. gingivalis worsened liver inflammation in ALD mice by increasing the protein expression of toll-like receptor 4 (TLR4) and p65, increasing the mRNA expression of interleukins-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) and up-regulating the transforming growth factor-beta 1 (TGF-β1) and galectin-3 (Gal-3) production. These results indicate that P. gingivalis accelerates the pathogenesis of ALD via the oral-gut-liver axis, necessitating a new treatment strategy for patients with ALD complicated by periodontitis.

Hydrogen gas ameliorates acute alcoholic liver injury via anti-inflammatory and antioxidant effects and regulation of intestinal microbiota

Alcoholic liver disease (ALD) is a globally prevalent liver-related disorder characterized by severe oxidative stress and inflammatory liver damage, for which no effective treatment is currently available. Hydrogen gas (H2) has been demonstrated to be an efficient antioxidant in various diseases in animals as well as humans. However, the protective effects of H2 on ALD and its underlying mechanisms remain to be elucidated. The present study demonstrated that H2 inhalation ameliorated liver injury, and attenuated liver oxidative stress, inflammation, and steatosis in an ALD mouse model. Moreover, H2 inhalation improved gut microbiota, including increasing the abundance of Lachnospiraceae and Clostridia, and decreasing the abundance of Prevotellaceae and Muribaculaceae, and also improved intestinal barrier integrity. Mechanistically, H2 inhalation blocked activation of the LPS/TLR4/NF-κB pathway in liver. Notably, it was further demonstrated that the reshaped gut microbiota may accelerate alcohol metabolism, regulate lipid homeostasis and maintain immune balance by bacterial functional potential prediction (PICRUSt). Fecal microbiota transplantation from mice that had undergone H2 inhalation significantly alleviated acute alcoholic liver injury. In summary, the present study showed that H2 inhalation alleviated liver injury by reducing oxidative stress and inflammation, while also improving intestinal flora and enhancing the intestinal barrier. H2 inhalation may serve as an effective intervention for preventing and treating ALD in a clinical context.

Regulation of NOX/p38 MAPK/PPARα pathways and miR-155 expression by boswellic acids reduces hepatic injury in experimentally-induced alcoholic liver disease mouse model: novel mechanistic insight

Alcoholic liver disease (ALD) refers to hepatic ailments induced by excessive alcohol intake. The pathogenesis of ALD comprises a complex interplay between various mechanistic pathways, among which inflammation and oxidative stress are key players. Boswellic acids (BAs), found in Boswellia serrata, have shown hepatoprotective effects owing to their antioxidant and anti-inflammatory activities, nevertheless, their therapeutic potential against ALD has not been previously investigated. Hence, this study was performed to depict the possible protective effect of BAs and detect their underlying mechanism of action in an experimentally-induced ALD mouse model. Male BALB/c mice were equally categorized into six groups: control, BAs-treated, ALD, and ALD that received BAs at three-dose levels (125, 250, and 500 mg/kg) by oral gavage for 14 days. Results showed that the high dose of BAs had the most protective impact against ALD according to histopathology examination, blood alcohol concentration (BAC), and liver function enzymes. Mechanistic investigations revealed that BAs (500 mg/kg) caused a significant decrease in cytochrome P450 2E1(CYP2E1), nicotine adenine dinucleotide phosphate oxidase (NOX) 1/2/4, p38 mitogen-activated protein kinase (MAPK), and sterol regulatory element-binding protein-1c (SREBP-1c) levels, and the expression of miR-155, yet increased peroxisome proliferator-activated receptor alpha (PPARα) levels. This led to an improvement in lipid profile and reduced hepatic inflammation, oxidative stress, and apoptosis indices. In summary, our study concludes that BAs can protect against ethanol-induced hepatic injury, via modulating NOX/p38 MAPK/PPARα pathways and miR-155 expression.

MTORC1 inhibition uncouples lipolysis and thermogenesis in white adipose tissue to contribute to alcoholic liver disease.

Adipose tissue thermogenic activities use fatty acids from lipolysis for heat generation. Therefore, a tight coupling between lipolysis and thermogenesis is physiologically imperative in maintaining not only body temperature but also lipids homeostasis. Adipose tissue dysfunction contributes to alcoholic liver disease (ALD). Here, studies were conducted to examine how alcohol intake affects adipose tissue thermogenic activities and whether altered adipose tissue thermogenesis contributes to ALD. Both the Lieber-DeCarli and the NIAAA mouse models of ALD were used. Denervation surgery in epididymal fat pads was performed. CL316,243, a selective β3-adrenoceptor agonist, SR59230A, a selective β3 adrenoceptor (ADRB3) antagonist, and rapamycin, a selective mechanistic target of rapamycin complex 1 (mTORC1) inhibitor, were administrated through i.p. injection. Adipocyte-specific Prdm16 knockout mice were subjected to alcohol-containing diet chronically. Chronic alcohol consumption, which enhances adipose tissue lipolysis, inhibits thermogenic activities of beige adipocytes in inguinal white adipose tissue (WAT), leading to an uncoupling status between lipolysis and thermogenesis in WAT at both basal and ADRB3 stimulation states. CL316,243 administration exacerbates liver pathologies of ALD. Alcohol intake inhibits mTORC1 activities in WAT. In mice, mTORC1 inhibition by rapamycin inhibits the thermogenesis of iWAT, whereas enhancing WAT lipolysis. Further investigations using adipocyte-specific Prdm16 knockout mice revealed that functional deficiency of beige adipocytes aggravates liver pathologies of ALD, suggesting that the inhibitory effect of alcohol on WAT browning/thermogenesis contributes to ALD pathogenesis. Chronic alcohol consumption induces an "uncoupling status" between lipolysis and browning/thermogenesis in WAT by inhibiting mTORC1 activation. Diminished WAT browning/thermogenesis, concomitant with enhanced lipolysis, contributes to ALD pathogenesis.

Targetable Brg1-CXCL14 axis contributes to alcoholic liver injury by driving neutrophil trafficking.

Alcoholic liver disease (ALD) accounts for a large fraction of patients with cirrhosis and hepatocellular carcinoma. In the present study we investigated the involvement of Brahma-related gene 1 (Brg1) in ALD pathogenesis and implication in ALD intervention. We report that Brg1 expression was elevated in mouse models of ALD, in hepatocyte exposed to alcohol, and in human ALD specimens. Manipulation of Brg1 expression in hepatocytes influenced the development of ALD in mice. Flow cytometry showed that Brg1 deficiency specifically attenuated hepatic infiltration of Ly6G+ neutrophils in the ALD mice. RNA-seq identified C-X-C motif chemokine ligand 14 (CXCL14) as a potential target for Brg1. CXCL14 knockdown alleviated whereas CXCL14 over-expression enhanced ALD pathogenesis in mice. Importantly, pharmaceutical inhibition of Brg1 with a small-molecule compound PFI-3 or administration of an antagonist to the CXCL14 receptor ameliorated ALD pathogenesis in mice. Finally, a positive correlation between Brg1 expression, CXCL14 expression, and neutrophil infiltration was detected in ALD patients. In conclusion, our data provide proof-of-concept for targeting the Brg1-CXCL14 axis in ALD intervention.

Kluyveromyces marxianus supplementation ameliorates alcohol-induced liver injury associated with the modulation of gut microbiota in mice.

The aim of this study was to evaluate the intervention effect of the potential probiotic Kluyveromyces marxianus YG-4 isolated from Tibetan kefir grains on alcoholic liver disease (ALD). Eight-week-old male C57BL/6J mice were fed with a Lieber-DeCarli (LDC) diet containing ethanol with a progressively increasing concentration from 1% to 4% (vol/vol) to establish an ALD mouse model. Our results suggested that K. marxianus treatment improved ALD, as demonstrated by the reduction of serum ALT and AST levels and the suppression of TLR4/NF-κB-mediated inflammatory response in the liver. K. marxianus administration significantly elevated antioxidant activities of SOD, CAT and GSH-Px, and reduced the MDA level in mice. K. marxianus supplementation repaired the gut barrier by increasing tight junction proteins and the number of goblet cells in the colon of ALD mice. In addition, treatment with K. marxianus restored alcohol-induced gut dysbiosis. Specifically, K. marxianus administration depleted the abundance of Lactobacillus, Coriobacteriaceae_UCG-002 and Candida, while increased that of Allobaculum, Dubosiella and Epicoccum in mice. Our findings open new possibilities for K. marxianus application in ALD treatment.

AMPK-Regulated Autophagy Contributes to Ursolic Acid Supplementation-Alleviated Hepatic Steatosis and Liver Injury in Chronic Alcohol-Fed Mice.

Alcoholic liver disease (ALD) is a chronic liver disease caused by long-term heavy consumption of alcohol. The pathogenesis of ALD is complex, and there is no effective clinical treatment at present. Ursolic acid (UA), a general triterpenoid with multiple biological roles, is widely distributed in plants. This study aims to explore the therapeutic effect and potential mechanisms of UA that protect against liver injury and hepatic steatosis in an ALD mouse model. In this study, we analyzed the lipid accumulation and the effect of UA treatment in a mouse model of ALD; AML12 and HepG2 cells were used to study the biological effect and potential mechanisms of UA on ethanol-induced hepatotoxicity. The morphologic and histological detections showed that UA significantly reduced alcohol-induced liver injury and hepatic steatosis. In addition, UA dramatically ameliorated alcohol-induced metabolic disorders, oxidative stress, and inflammation. Furthermore, UA treatment activated autophagy via the AMPK-ACC pathway to protect hepatocytes from lipotoxicity. Thus, these findings demonstrate that UA treatment alleviates alcoholic-induced liver injury by activating autophagy through the AMPK-ACC pathway. Therefore, UA may represent a promising candidate for the treatment of ALD.

A novel reactive aldehyde species inhibitor prevents the deleterious effects of ethanol in an animal model of alcoholic liver disease.

Current treatment strategies for alcoholic liver disease (ALD) are limited by the lack of agents specifically targeting the metabolic breakdown products of ethanol. Reactive aldehyde species (RASP) inhibitors have been developed that have the capability to sequester these aldehyde byproducts, potentially limiting toxicity. The purpose of this study was to determine if the RASP inhibitor ADX-629 could target these metabolic breakdown products in a mouse model of ALD. A chronic/binge mouse model of ALD was used to determine the efficacy of ADX-629 treatment. Mice were fed an alcohol-containing (5%) liquid or control diet for 10days and treated by oral gavage with ADX-629 30min prior to administering a bolus gavage of 31.5% ethanol. Test groups included: Control - no ADX, Control+ADX, Ethanol - no ADX and Ethanol+ADX. Compared to ethanol-fed mice receiving sham treatment, ethanol mice treated with ADX-629 demonstrated significant decreases (p<0.05) in liver acetaldehyde (AA), liver malondialdehyde-acetaldehyde (MAA), circulating anti-MAA antibody, liver/serum triglycerides (p<0.01) levels, and overall fat accumulation in the liver as determined by Oil Red O and bodipy staining (p<0.0001). Serum levels of pro-inflammatory cytokines IFN-γ and MCP-1 levels were decreased following ADX-629 treatment (p<0.01). These findings demonstrate that the use of this unique RASP inhibitor (ADX-629) is effective in the treatment of ALD. Given the ubiquitous nature of aldehydes in the context of tissue inflammation and damage, ADX-629 and other RASP inhibitors may have additional applications in disease states.

Non-Alcoholic Components in Huangjiu as Potential Factors Regulating the Intestinal Barrier and Gut Microbiota in Mouse Model of Alcoholic Liver Injury.

Different alcoholic beverages and drinking patterns might exert divergent impacts on alcoholic liver disease (ALD) progression. Whether the abundant non-alcoholic components (NAC) in fermented wine could alleviate ethanol (EtOH)-induced adverse influences on the liver remains unknown. Hence, the chronic ALD mouse model was established to compare the effects of Huangjiu (a typical fermented wine) and EtOH feeding on the liver, intestinal barrier, gut microbiota, and intestinal short-chain fatty acids (SCFAs) content. Although Huangjiu intake led to slight hepatic steatosis, it mitigated oxidative stress, inflammation, and intestinal damage relative to EtOH intake. In comparison with EtOH feeding, Huangjiu significantly improved the intestinal barrier integrity and reduced hepatic lipopolysaccharide levels by up-regulating the expression of intestinal tight junction proteins (ZO-1 and occludin) and antimicrobial activity peptides (Reg3β and Reg3γ). The administration of Huangjiu NAC partially restored alcohol-induced gut microbiota dysbiosis via recovering the abundance of Lactobacillus, Faecalibaculum, and Akkermansia. Moreover, mice receiving Huangjiu showed higher SCFAs levels (such as acetic acid and butyric acid) than those receiving EtOH. Huangjiu consumption resulted in lower hepatotoxicity than pure EtOH, at the same alcohol dose. The NAC in Huangjiu might attenuate the progression of ALD by regulating intestinal barrier function and microbiota-meditated gut ecology.

Glial cell line-derived neurotrophic factor contributes to alcoholic-induced liver injury by regulating the NF-κB pathway.

Alcoholic liver disease (ALD) is associated with high morbidity and mortality worldwide. The pathogenesis of ALD is not completely understood. Although accumulating evidence suggests an important role of glial cell line-derived neurotrophic factor (GDNF) in several diseases, there are no data concerning its role in ALD. This study compared patients with ALD with control subjects and used a mouse model and a cell culture model to investigate the function of GDNF in ALD and its mechanism of action in hepatocyte injury. Serum levels of GDNF were measured in 25 patients with ALD and 25 healthy control subjects. A 4-week Lieber-DeCarli ethanol (EtOH) liquid diet combined with the Gao-Binge model was used in the mouse study. Mouse primary hepatocytes and Huh-7 cells were used for cell experiments. The parameters of liver injury, inflammatory cytokines, and lipid metabolism were measured. Patients with alcoholic hepatitis had higher serum GDNF than control subjects. Expression of GDNF mRNA and protein was markedly increased in mice in the chronic-plus-binge ALD mouse model. The level of GDNF mRNA was upregulated in primary hepatic stellate cells isolated from ethanol-fed mouse liver. Ethanol induced GDNF expression in LX2 cells. The levels of inflammatory cytokines (tumor necrosis factor α, interleukin 1β, and monocyte chemotactic protein 1) were significantly increased after GDNF stimulation in primary hepatocytes and Huh-7 cells. After GDNF stimulation, levels of both p-AKT and p-NF-κB were significantly increased in primary hepatocytes and Huh-7 cells. The NF-κB activity induced by GDNF was significantly decreased by an NF-κB inhibitor, which limited hepatocyte injury and inflammation. The concentration of GDNF is increased in the circulation of ALD patients. GDNF promotes alcohol-induced liver injury and inflammation via the activation of NF-κB, which mediates hepatocyte injury and inflammatory cytokine expression. Based on these findings, GDNF is a potential therapeutic target for preventing or ameliorating liver injury in ALD.

Alcohol Abstinence Rescues Hepatic Steatosis and Liver Injury via Improving Metabolic Reprogramming in Chronic Alcohol-Fed Mice.

Background: Alcoholic liver disease (ALD) caused by chronic ethanol overconsumption is a common type of liver disease with a severe mortality burden throughout the world. The pathogenesis of ALD is complex, and no effective clinical treatment for the disease has advanced so far. Prolonged alcohol abstinence is the most effective therapy to attenuate the clinical course of ALD and even reverse liver damage. However, the molecular mechanisms involved in alcohol abstinence-improved recovery from alcoholic fatty liver remain unclear. This study aims to systematically evaluate the beneficial effect of alcohol abstinence on pathological changes in ALD.Methods: Using the Lieber-DeCarli mouse model of ALD, we analysed whether 1-week alcohol withdrawal reversed alcohol-induced detrimental alterations, including oxidative stress, liver injury, lipids metabolism, and hepatic inflammation, by detecting biomarkers and potential targets.Results: Alcohol withdrawal ameliorated alcohol-induced hepatic steatosis by improving liver lipid metabolism reprogramming via upregulating phosphorylated 5′-AMP -activated protein kinase (p-AMPK), peroxisome proliferator-activated receptor-α (PPAR-α), and carnitine palmitoyltransferase-1 (CPT-1), and downregulating fatty acid synthase (FAS) and diacylglycerol acyltransferase-2 (DGAT-2). The activities of antioxidant enzymes, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-px), were significantly enhanced by alcohol withdrawal. Importantly, the abstinence recovered alcohol-fed induced liver injury, as evidenced by the improvements in haematoxylin and eosin (H&E) staining, plasma alanine aminotransferase (ALT) levels, and liver weight/body weight ratio. Alcohol-stimulated toll-like receptor 4/mitogen-activated protein kinases (TLR4/MAPKs) were significantly reversed by alcohol withdrawal, which might mechanistically contribute to the amelioration of liver injury. Accordingly, the hepatic inflammatory factor represented by tumour necrosis factor-alpha (TNF-α) was improved by alcohol abstinence.Conclusion: In summary, we reported that alcohol withdrawal effectively restored hepatic lipid metabolism and reversed liver injury and inflammation by improving metabolism reprogramming. These findings enhanced our understanding of the biological mechanisms involved in the beneficial role of alcohol abstinence as an effective treatment for ALD.

Comprehensive Analysis of the Expression Profiles of Hepatic lncRNAs in the Mouse Model of Alcoholic Liver Disease.

Background and Aim: The worldwide prevalence of alcoholic liver disease (ALD) due to escalating alcohol consumption has presented an unprecedented pressure on human health. A few studies have determined long non-coding RNAs (lncRNAs) involved in the pathogenesis of liver diseases. However, the roles of lncRNAs in ALD development is still poorly understood.Methods: An ALD mouse model was established and confirmed. Expression profiles of lncRNAs were obtained by whole transcriptome sequencing. The altered lncRNAs in ALD mice were further verified by qRT-PCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to enrich the functions of these lncRNAs. In combination with miRNA and mRNA profiles, we constructed concise endogenous RNA (ceRNA) networks. The function of the most up/downregulated lnRNA was further verified and investigated in both ALD model and AML-12 cells.Results: Totally, five downregulated lncRNAs were obtained and verified in ALD mice. The GO term and KEGG pathway analyses revealed that the identified lncRNAs were associated with alcohol-induced hepatic oxidative damage, cellular inflammation, and lipid metabolism. Combination the differentially modulated miRNAs and mRNAs with ceRNA network analysis, we constructed five ceRNA networks and obtained 30 miRNAs and 25 mRNAs that may participate in ALD. Further, we verified and investigate the function of the most downregulated lnc_1700023H06Rik. Depletion lnc_1700023H06Rik reduced genes encoding for lipid metabolism, especially mRNA Acat2 (ENSMUST00000159697) and Pgrmc2 (ENSMUST00000058578) both in vivo and in vitro. Knocking down lnc_1700023H06Rik induced triglyceride accumulation and lactate dehydrogenase leakage in AML12 cells, consisting with that in alcohol-treated cells.Conclusion: The five remarkably downregulated lncRNAs in ALD mouse model were identified as novel biomarkers, highlighting the key role of lncRNAs in the development of ALD. The effect of lnc_1700023H06Rik plays a pivotal role in lipid deposition and its pathological pathway in ALD needs further investigation.

Comparative Transcriptome Analysis Provides Potential Insights Into The Mechanism Of Camel Milk In Regulating Alcoholic Liver Disease In Mice

Alcoholic liver disease (ALD) is a general term used to refer to these alcohol-related liver damage. In this study, we investigated the hepatoprotective effect of camel milk (CM) in an ALD mouse model and its underlying mechanism at the transcriptome level. Male C57BL/6NCr were divided into 3 groups: normal diet (NC) ; normal diet, then ethanol (ET); and normal diet, then ethanol and camel milk (ET+CM). Comparative hepatic transcriptome analysis among the groups was performed by Illumina RNA sequencing. The result showed that a total of 526.76+19.87, 563.04+17.84, and 513.56+20.41 million clean reads were obtained for the NC, ET, and ET+CM groups, respectively. Compared with the Et group, 423 differentially expressed genes (DEGs) (including 160 upregulated and 263 downregulated genes) were identified in the NC group, and 186 differentially expressed genes (including 62 upregulated and 124 downregulated genes) were identified in the ET+CM group. The enrichment analyses revealed that the NOD-like receptor signaling pathway, the Toll-like receptor signaling pathway, the MAPK signaling pathway, and mTOR signaling pathway enriched the most differentially expressed genes. The findings of this study provide insights into the development of nutrition-related therapies for alcoholic liver disease (ALD) with camel milk.

Effects of Dihydroxylated Fatty Acid Mediators on Ethanol‐Induced Liver Injury in a Mouse Model of Alcoholic Liver Disease

BackgroundAlcoholic liver disease (ALD) is the most common chronic liver disease and a major contributor to liver disease burden worldwide, and a disease for which there is no FDA‐approved treatment. The mechanisms of ALD pathogenesis are not completely understood. Data from our group indicate that dihydroxylated fatty acids (dihydroxy‐FAs) are increased in the livers of human alcoholic hepatitis patients. Dihydroxy‐FA lipid mediators are produced via cytochrome P450/epoxide hydrolase‐mediated metabolism of polyunsaturated fatty acids and are generally pro‐inflammatory. Despite their increase in human ALD, the role of dihydroxy‐FAs in ALD has not been explored.HypothesisExogenous administration of dihydroxy‐FAs will exacerbate liver injury in a mouse model of ALD.MethodsMale C57BL/6 mice were provided ethanol (EtOH) using a chronic‐binge Lieber DeCarli ad libitum EtOH feeding model. Mice were administered either 8,9‐DiHETE (arachidonic acid metabolite), a combination of 9,10‐DiHOME and 12,13‐DiHOME (linoleic acid metabolites), or a vehicle control by i.p. injection once daily for two days prior to sacrifice. Liver injury, inflammation, and steatosis were measured. To further elucidate the effects of dihydroxy‐FAs on liver cells, primary mouse hepatocytes or HepG2 cells were treated with 8,9‐DiHETE, 9,10‐DiHOME, 12,13‐DiHOME, or a vehicle control. Effects on baseline or palmitic acid (PA)‐induced cell death were measured by MTT assay and apoptotic cell death was identified by Annexin‐V staining (cellomics).ResultsIn vivo, 8,9‐DiHETE treatment modestly while 9,10/12,13‐DiHOME significantly increased liver injury as determined by elevated plasma ALT levels (1.3‐fold and 2.2‐fold p=0.04, respectively). While 8,9‐DiHETE had no effect on steatosis, 9,10/12,13‐DiHOME decreased liver triglyceride/free fatty acid levels compared to vehicle control. Dihydroxy‐FA treatment decreased the hepatic expression of several pro‐inflammatory chemokines at the mRNA level, including Mip2a and Mcp1. In vitro, 8,9‐DiHETE treatment of HepG2 cells or primary mouse hepatocytes significantly exacerbated PA‐induced cell death as determined by MTT assay, whereas 9,10‐DiHOME, and 12,13‐DiHOME had no effect on PA‐induced or baseline cell death or apoptosis.ConclusionsDifferent dihydroxy‐FAs have diverse effects in vivo, in a mouse model of ALD, and in vitro. In vivo, dihydroxy‐FAs exacerbated liver injury and was associated with down‐regulated several pro‐inflammatory cytokines. In vitro, 8,9‐DiHETE increased hepatocyte cell death, while 9,10/12,13‐DiHOME showed no effects. Future studies are warranted to dissect the effects of individual dihydroxy‐FAs in ALD.Support or Funding InformationThis work was funded by grants from the National Institutes of Health and the Department of Veterans Affairs.

Noninvasive 40-Hz Light Flicker Rescues Circadian Behavior and Abnormal Lipid Metabolism Induced by Acute Ethanol Exposure via Improving SIRT1 and the Circadian Clock in the Liver-Brain Axis.

Sirtuin 1 (SIRT1) is a protein deacetylase with important cellular functions, as it regulates numerous processes, including the circadian rhythm in peripheral tissues. Efforts are ongoing to reveal how Sirt1 can be used to treat diseases, such as alcoholic liver disease (ALD), Alzheimer's disease, and liver fibrosis. We have recently shown that noninvasive exposure to 40-Hz light flicker activates hypothalamic SIRT1 gene expression, thereby regulating the central circadian clock. This study investigated the effects of 40-Hz light flicker in a mouse model of ALD. RNA sequencing (RNA-seq) analysis was performed to explore the potential pathways affected by 40-Hz light flicker. We found that 40-Hz light flicker significantly decreased the acute ethanol-induced increases in serum alanine aminotransferase (ALT) and serum triglyceride (TG) levels and reduced fat-droplet accumulation in mouse livers. Additionally, 40-Hz light flicker significantly suppressed ethanol-induced increases in sterol regulatory element binding protein 1 (SREBP-1) and fatty acid synthase (Fasn) levels. Furthermore, the ethanol induced significant decreases in both Sirt1 levels and phosphorylation of adenosine monophosphate-activated protein kinase subunit (AMPKα), compared with those in the control group. Strikingly, pretreatment with 40-Hz light flicker ameliorated such ethanol-induced decreases in SIRT1 levels and AMPKα phosphorylation. In addition, ethanol-induced increases in levels of brain and muscle arnt-like protein-1 (BMAL1), circadian locomotor output cycles kaput (CLOCK), and period 2 (PER2) were reversed by 40-Hz light flicker. RNA-seq analysis revealed significant differences in expression of genes related to the AMPK signalling. Moreover, ethanol consumption altered mRNA levels of Sirt1 and circadian genes in the suprachiasmatic nucleus (SCN), indicating that ethanol influenced central pacemaker genes; however, 40-Hz light flicker reversed these ethanol-induced changes. Taken together, our findings demonstrate that 40-Hz light flicker rapidly influence the SCN and exhibits inhibitory properties on hepatic lipogenesis, indicating that 40-Hz light flicker has therapeutic potential for preventing alcoholic liver steatosis.

RNA Sequencing Reveals a Comprehensive Circular RNA Expression Profile in a Mouse Model of Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is the most common liver disease and a severe mortality burden in the world. However, ALD is rarely detected at its early stages. Thus, exploration of an early event for ALD may help the prognosis and further therapy of ALD. Several circRNAs were proven as novel molecular biomarkers for the progression of chronic nonalcoholic fatty liver disease. Whether circRNAs are involved explicitly in ALD remains unknown. The expression profile of circRNAs in an ALD mouse model was depicted by circRNA sequencing. The dysregulated circRNAs were verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Bioinformatics and circRNA/microRNA (miRNA) crosstalk analyses were applied to predict the potential functions of circRNAs. Finally, the miRNA expression was confirmed by miRNA sequencing. Compared with the control group, 6 members of circRNAs were up-regulated, and 4 were down-regulated in the ALD model. GO enrichment analyses revealed these circRNAs were predominantly enriched in the endoplasmic reticulum, arachidonic acid metabolism, and cytochrome P450 metabolism pathway. Among these circRNAs, the differential expression of 5 circRNAs was validated and consistent with qRT-PCR, and only the up-regulated mou_circ_1657 is included in the circBase. Further, the crosstalk analysis of circRNA-miRNA revealed 7 miRNAs were targeted by mou_circ_1657, of which miR-19-5b was the only miRNA that was down-regulated in the ALD mice according to the miRNA sequencing data, suggesting it needs further attention in ALD. This study demonstrates that a cluster of circRNAs is aberrantly expressed in the livers of ALD mice. mou_circ_1657/miR-19-5b may play a critical role in the development of ALD. Our study provides new insight into the future investigation and therapy on ALD.