Ethanol-induced Liver Injury Research Articles

Effect of autophagy inhibitor chloroquine on acute alcoholinduced liver disease

To investigate the role of autophagy inhibitor chloroquine (CQ) in acute ethanol-induced liver injury and its mechenism. Twenty-one C57BL/6 male mice were randomly divided into three groups:control group, ethanol group, CQ + ethanol group (n=7). Mice in ethanol group were administered 33% (v/v) ethanol at a dose of 4.5 g/kg body weight. Ethanol-induced liver steatosis in each group was detected by hematoxylin and eosin staining. Hepatic lipid accumulation was detected by staining with Oil red O. Hepatic tissue triglyceride (TG) levels, serum aspartate aminotransferase(AST) and alanine aminotransferase(ALT) were determined by biochemical assays. Protein expression of microtubule-associated protein 1 light chain 3(LC3) and nuclear factorκB p65(NF-κB p65) were measured by Western blot and immunofluorescence. Pro-inflammatory factors tumor necrosis factor-α(TNF-α)、interleukin 6(IL-6) were detected by ELISA. Compared with control group, ethanol induced liver injury proved by accumulation of hepatic lipids, TG levels, AST and ALT activities were significantly increased by ethanol, protein expression of LC3-Ⅱ was also markedly increased by ethanol. Compared with ethanol group, addition of CQ increased furtherthe level of LC3-Ⅱexpression, and TG amount, serum AST and ALT activities, and the expression of NF-κB p65, TNF-αand IL-6. Acute ethanol-intake could induce liver steatosis and inflammation, and autophagy inhibitor CQ exacerbatedethanol-induced liver injury, suggested that autophagy might be protective effect in acute ethanol-induced liver disease.

Expression of cytokines in rats with ethanol-induced liver injury treated with Zhige oral solution

Expression of cytokines in rats with ethanol-induced liver injury treated with Zhige oral solution

Protective Effects of Taraxasterol against Ethanol-Induced Liver Injury by Regulating CYP2E1/Nrf2/HO-1 and NF-κB Signaling Pathways in Mice.

Taraxasterol, a pentacyclic-triterpene compound, is one of the main active components isolated from the traditional Chinese medicinal herb Taraxacum. The objective of this study is to evaluate the protective effects of taraxasterol and its possible underlying mechanisms against ethanol-induced liver injury in mice. ICR mice were fed with Lieber-DeCarli diet containing 5% ethanol for 10 d and then challenged with a single dose of 20% ethanol (5 g/kg BW) by intragastric administration. The mice were intragastrically treated daily with taraxasterol (2.5, 5, and 10 mg/kg). Tiopronin was used as a positive control. The liver index was calculated, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in sera were detected. The contents of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) and the activity of superoxide dismutase (SOD) in the livers were measured. The histopathological changes of liver tissues were observed by hematoxylin and eosin (H&E) staining. The protein expression levels of hepatic cytochrome P450 2E1 (CYP2E1), nuclear factor erythroid 2-related factor 2 (Nrf2), antioxidant protein heme oxygenase-1 (HO-1), and nuclear factor-kappa B (NF-κB) signaling pathway in liver tissues were detected by immunohistochemistry and Western blot methods. Taraxasterol significantly reduced the ethanol-induced increases of liver index, ALT, AST, and TG levels in sera and TG and MDA contents in the livers and hepatic ROS production and suppressed the ethanol-induced decreases of hepatic GSH level and SOD activity. Taraxasterol also significantly inhibited the secretion of proinflammatory cytokines TNF-α and IL-6 induced by ethanol. In addition, taraxasterol improved the liver histopathological changes in mice with ethanol-induced liver injury. Further studies revealed that taraxasterol significantly inhibited the ethanol-induced upregulation of CYP2E1, increased the ethanol-induced downregulation of Nrf2 and HO-1, and inhibited the degradation of inhibitory kappa Bα (IκBα) and the expression level of NF-κB p65 in liver tissues of ethanol-induced mice. These findings suggest that taraxasterol possesses the potential protective effects against ethanol-induced liver injury in mice by exerting antioxidative stress and anti-inflammatory response via CYP2E1/Nrf2/HO-1 and NF-κB signaling pathways.

High-throughput system metabolomics method reveals new mechanistic insights of chlorogenic acid by using liquid chromatography coupled to high resolution mass spectrometry.

It has increasingly been recognized that metabolism is highly interconnected with disease, and system metabolomics studies have aimed to discover metabolic biomarkers and analyze the pathways of metabolome phenotypes. To better understand the metabolic alteration related with disease, a urine metabolic profile using a high-throughput system metabolomics technology approach was applied to probe the underlying molecular mechanisms of alcohol-induced liver injury and the therapeutic effects of chlorogenic acid (CA). In this study, endogenous low-molecular-weight metabolites were characterized using liquid chromatography coupled with mass spectrometry (LC-MS). The acquired data was parsed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to identify potential biomarkers. A total of 19 biomarkers were identified in a model of alcohol-induced liver injury rats, and it was found that chlorogenic acid had a regulatory effect on 14 of them, associated with multiple metabolic pathways. Comprehensive pathway analysis suggests that CA has the ability to regulate abnormal metabolic states. In addition, accessory examinations such as biochemical analysis and histopathological observations were also performed that showed similar results. As a natural product agent against ethanol-induced liver injury, CA was validated in the rebalancing of a wide range of metabolic disorders. High-throughput system metabolomics represents a powerful approach for revealing new mechanistic insights of natural products.

Patchouli oil isolated from the leaves of Pogostemon cablin ameliorates ethanol-induced acute liver injury in rats via inhibition of oxidative stress and lipid accumulation.

Excessive alcohol consumption can cause serious hepatic injury which is associated with oxidative stress and fatty metabolic disturbance. Patchouli oil (PO) is a sort of food supplement with high medicinal value in hepatoprotection, but its ability against ethanol-induced liver failure has not been demonstrated. Thus, this study aimed to investigate the potential hepatoprotection of PO through an ethanol-induced hepatotoxicity rat model. Our results showed that PO pretreatment could dramatically decrease the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in serum, paralleled by an improvement of histopathology alterations. Additionally, PO could markedly suppress the content of reactive oxygen species (ROS), tumor necrosis factor alpha (TNF-α), free fatty acid (FFA), and triglyceride (TG), while enhancing the activities of glutathione (GSH), glutathione reductase (GR), and superoxide dismutase (SOD) as well as the ratio of glutathione to oxidized glutathione (GSH/GSSG) in liver. The protective effect of PO against oxidative stress was interrelated with restraining the mRNA and protein expression of hepatic microsomal enzyme cytochrome P450 2E1 (CYP2E1). What's more, PO pretreatment could also accelerate lipometabolism via up-regulating expressions of adenosine monophosphate-activated protein kinase (AMPK), peroxisome proliferator-activated receptor α (PPAR-α), and carnitine palmitoyltransferase 1 (CPT-1) and down-regulating expressions of nuclear factor-kappaB (NF-κB) p65, sterol regulatory element-binding protein 1 (SREBP-1c), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD-1). To conclude, PO showed potent effect against ethanol-induced hepatotoxicity by relieving oxidative stress and preventing lipid accumulation.

Targeting heme oxygenase-1 by quercetin ameliorates alcohol-induced acute liver injury via inhibiting NLRP3 inflammasome activation.

Alcoholic hepatitis (AH) is characterized by inflammation and necrosis of liver tissue caused by excessive alcohol consumption and it even causes organ failure sometimes, in which oxidative stress plays an important role. Quercetin is a bioactive flavonoid in the class of polyphenols with a free-radical scavenging ability and anti-inflammatory activity. Recently it has aroused great interest because of its potential benefits in the prevention and intervention of cancer, cardiovascular abnormalities, neurodegenerative diseases, metabolic disorders and liver fibrosis. However, its role in alcoholic liver injury is still unclear and needs to be elucidated. Through database analysis and serum measurements, we found that there was a decline in the level of heme oxygenase-1 (HO-1) in patients with acute alcoholic hepatitis compared to healthy controls. Quercetin could elevate the expression of nuclear factor E2-related factor 2(Nrf2)/HO-1 and ameliorate ethanol-induced acute liver injury in rats. Moreover, this protective effect of quercetin could be diminished when combined with the HO-1 inhibitor ZnppIX which demonstrated a critical role of HO-1 in quercetin's hepatoprotection. The underlying mechanism of quercetin's benefit on the liver may be explained by its anti-oxidant properties and inhibitory effect on the ROS/NF-κB/NLRP3 inflammasome/IL-1β and IL-18 pathway by inducing HO-1. Meanwhile, quercetin also upregulated the anti-inflammatory factor IL-10, while it was found uncorrelated with HO-1 expression. In conclusion, quercetin can preserve the function of the liver in acute alcoholic injury by upregulating the expression of IL-10 and HO-1 and thus inhibiting NLRP3 inflammasome activation and inflammatory factor secretion. In other words, quercetin looks promising as an alternative treatment and HO-1 may be a potential target in the crosstalk of inflammation and oxidative stress in alcoholic liver damage.

Promotion of autophagosome-lysosome fusion via salvianolic acid A-mediated SIRT1 up-regulation ameliorates alcoholic liver disease.

Salvianolic acid A (SalA) is a water-soluble phenolic carboxylic acid extracted from Salvia miltiorrhiza that has extensive pharmacological activities and plays an essential role in liver disease treatment. However, the mechanism of SalA in treating alcoholic liver disease (ALD) remains unclear. Here, we studied the protective effects of SalA on chronic ethanol-induced liver injury involving Sirtuin 1 (SIRT1)-mediated autophagy activation. The results showed that SalA pretreatment reduced the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and cholesterol (TC) in vivo and enhanced hepatic cell viability while mitigating apoptosis and hepatic steatosis in vitro. Furthermore, SalA protected against chronic ethanol-induced liver injury by restoring autophagosome–lysosome fusion, as indicated by the increased expression levels of LC3-II, cathepsin B, lysosomal-associated membrane protein 2 (LAMP-2), and RAB7 and the decreased expression of SQSTM1. More importantly, pretreatment with SalA significantly up-regulated the expression of SIRT1, an NAD+-dependent deacetylase. These increased levels of SIRT1 stimulated autophagy under conditions of chronic ethanol exposure. Interestingly, SIRT1 siRNA abrogated SalA-induced autophagosome–lysosome fusion. This finding indicates that the protective effects of SalA are associated with SIRT1 activation. Collectively, our study demonstrates that SalA pretreatment protects against chronic ethanol-induced liver injury via the SIRT1-mediated restoration of autophagosome–lysosome fusion.

Hepatoprotective effect of esculetin on ethanol-induced liver injury in human HepG2 cells and C57BL/6J mice

The objective of this study was to investigate the protective effect of esculetin against ethanol-induced liver injury in both HepG2 cells and mice. Treatment with esculetin significantly prevented ethanol-induced cytotoxicity in HepG2 cells by reducing the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). This protective effect was likely associated with antioxidant potential of esculetin, as evidenced by the attenuation of reactive oxygen species (ROS) and malondialdehyde (MDA) production and restoration of the depleted glutathione (GSH) levels in ethanol-induced HepG2 cells. Esculetin also dramatically increased the nuclear translocation of NF-E2-related factor 2 (Nrf2) and antioxidant response element (ARE) reporter gene activity, which led to the upregulation of ARE-responsive enzymes. In animal studies, esculetin markedly prevented ethanol-induced liver injury by reducing GSH depletion, MDA production, and the lipid contents in liver. Moreover, esculetin lowered the levels of serum ALT, serum AST, and the content of serum lipids.

HEPATOPROTECTIVE ACTIVITY OF ETHANOLIC EXTRACT OF TERMINALIA CHEBULA FRUIT AGAINST ETHANOL INDUCED HEPATOTOXICITY IN RATS

Objective: The purpose of this study was to evaluate the effect of Terminalia chebula fruit extract on liver antioxidant enzymes in ethanol-induced hepatotoxicity in rats.Method: Rats were divided into six different groups each having six. Group 1 served as a control, Group 2 received 40% ethanol (2 ml/100 g, oral), in sterile water, Groups 4, 5, and 6 served as extract treatment groups and received 50, 100, and 200 mg/kg, orally, ethanolic fruit extract of T. chebula (TCE) and Group 3 served as standard group and received silymarin 25 mg/kg orally. All the treatment protocols followed 21 days, and after which rats were sacrificed, the liver was taken for antioxidant and histological studies, respectively.Results: The ethanol-treated group rats (G2) showed variable decrease in antioxidant parameter (catalase, glutathione, and glutathione reductase) levels. Administration of ethanolic TCE significantly prevented ethanol-induced elevation in the levels of malondialdehyde lipid peroxidation and decreased antioxidant parameters in experimental groups of rats. The effect of extract was compared with a standard drug, silymarin. The changes in antioxidant parameters were supported by histological profile.Conclusion: It is concluded that the ethanolic fruit TCE protects against ethanol-induced oxidative liver injury in rats.

HIMH0021 attenuates ethanol-induced liver injury and steatosis in mice.

Chronic alcohol consumption causes alcohol-induced lipogenesis and promotes hepatic injury by preventing the oxidation of hepatocellular fatty acids through the suppression of the activation of AMP-activated protein kinase (AMPK). HIMH0021, an active flavonoid compound, which is a component of the Acer tegmentosum extract, has been shown to protect against liver damage caused by alcohol consumption. Therefore, in this study, we aimed to determine whether HIMH0021 could regulate alcoholic fatty liver and liver injury in mice. Oral administration of 10 days of Lieber-DeCarli ethanol plus a single binge of 30% ethanol (chronic-plus-binge model) induced steatosis and liver injury and inflammation in mice, which appears similar to the condition observed in human patients with alcohol-related diseases. HIMH0021, which was isolated from the active methanol extract of A. tegmentosum, inhibited alcohol-induced steatosis and attenuated the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) during hepatocellular alcohol metabolism, both of which promote lipogenesis as well as liver inflammation. Treatment with HIMH0021 conferred protection against lipogenesis and liver injury, inhibited the expression of cytochrome P4502E1, and increased serum adiponectin levels in the mice subjected to chronic-plus-binge feeding. Furthermore, in hepatocytes, HIMH0021 activated fatty acid oxidation by activating pAMPK, which comprises pACC and CPT1a. These findings suggested that HIMH0021 could be used to target a TNFα-related pathway for treating patients with alcoholic hepatitis.

Lychee (Litchi chinensis Sonn.) Pulp Phenolic Extract Provides Protection against Alcoholic Liver Injury in Mice by Alleviating Intestinal Microbiota Dysbiosis, Intestinal Barrier Dysfunction, and Liver Inflammation.

Liver injury is the most common consequence of alcohol abuse, which is promoted by the inflammatory response triggered by gut-derived endotoxins produced as a consequence of intestinal microbiota dysbiosis and barrier dysfunction. The aim of this study was to investigate whether modulation of intestinal microbiota and barrier function, and liver inflammation contributes to the hepatoprotective effect of lychee pulp phenolic extract (LPPE) in alcohol-fed mice. Mice were treated with an ethanol-containing liquid diet alone or in combination with LPPE for 8 weeks. LPPE supplementation alleviated ethanol-induced liver injury and downregulated key markers of inflammation. Moreover, LPPE supplementation reversed the ethanol-induced alteration of intestinal microbiota composition and increased the expression of intestinal tight junction proteins, mucus protecting proteins, and antimicrobial proteins. Furthermore, in addition to decreasing serum endotoxin level, LPPE supplementation suppressed CD14 and toll-like receptor 4 expression, and repressed the activation of nuclear factor-κB p65 in the liver. These data suggest that intestinal microbiota dysbiosis, intestinal barrier dysfunction, and liver inflammation are improved by LPPE, and therefore, the intake of LPPE or Litchi pulp may be an effective strategy to alleviate the susceptibility to alcohol-induced hepatic diseases.

Acanthoic acid protectsagainst ethanol-induced liver injury: Possible role of AMPK activation and IRAK4 inhibition

The aim of this study was to investigate the effects of acanthoic acid (AA) on the regulation of inflammatory response, lipid accumulation, and fibrosis via AMPK- IRAK4 signaling against chronic alcohol consumption in mice. Ethanol-induced liver injury was induced in male mice by Lieber-DeCarli diet for 28d. And mice in AA groups were gavaged with AA (20 or 40mg/kg) for 28d. AA treatment significantly decreased serum AST and TG, hepatic TG levels, serum ethanol and LPS levels compared with chronic ethanol administration. AA ameliorated histological changes, lipid droplets, hepatic fibrosis, and inflammation induced by ethanol. AA significantly increased the expressions of p-LKB1, p-AMPK, and SIRT1 caused by chronic ethanol administration, and attenuated the increasing protein expressions of IRAK1 and IRAK4.siRNA against AMPKα1 blocked AMPKα1 and increased IRAK4 protein expressions, compared with control-siRNA-transfected group, while AA treatment significantly decreased IRAK4 expressions compared with AMPKα1-siRNA-transfected group. AMPK-siRNA also blocked the decreased effect of AA on inflammatory factors. AA decreased over-expression of IRAK4 and inflammation under ethanol plus LPS challenge. AA recruited LKB1-AMPK phosphorylation and activated SIRT1 to regulate alcoholic liver injury, especially, inhibited IRAK1/4 signaling pathway to regulate lipid metabolism, hepatic fibrosis and inflammation caused by alcohol consumption.

Lactobacillus plantarum LC27 and Bifidobacterium longum LC67 simultaneously alleviate ethanol-induced gastritis and hepatic injury in mice

Some probiotics attenuate ethanol-induced gastritis or liver injury. However, it is not clear whether probiotics could simultaneously alleviate ethanol-induced gastritis and liver injury. Therefore, we isolated anti-inflammatory probiotics Bifidobacterium longum LC67 and Lactobacillus plantarum LC27 in vitro and investigated their inhibitory effects against ethanol-induced gastritis and liver injury in mice. Oral administration of LC27 or LC67 suppressed ethanol-induced hemorrhagic ulcerative lesions area, myeloperoxidase activity, tumor necrosis factor (TNF) expression, CXCL4, and NF-κB activation in the stomach as well as alanine aminotransferase, aspartate transaminase, and TNF levels in the blood. Treatment with LC27 or LC67 increased ethanol-suppressed alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities in the liver. The mixture of LC27 and LC67 additively attenuated ethanol-induced gastritis and liver injury in mice. These findings suggest that LC27 and LC67 can simultaneously attenuate ethanol-induced gastritis and liver injury by inhibiting inflammatory responses and increasing ADH and ALDH activities.

Prophylactic tributyrin treatment mitigates chronic-binge ethanol-induced intestinal barrier and liver injury.

Impaired gut-liver axis is a potential factor contributing to alcoholic liver disease. Ethanol depletes intestinal integrity and causes gut dysbiosis. Butyrate, a fermentation byproduct of gut microbiota, is altered negatively following chronic ethanol exposure. This study aimed to determine whether prophylactic tributyrin could protect the intestinal barrier and liver in mice during combined chronic-binge ethanol exposure. C57BL/6J mice exposed to 5% v/v ethanol-containing diet for 10days received a single ethanol gavage (5g/kg) 9h before euthanasia. Control mice were isocalorically pair-fed maltose dextrin for ethanol. Diets were supplemented (5mM) with tributyrin or glycerol. Intestine and liver disease activity was assessed histologically. Protein and mRNA expression of tight junction (TJ) proteins, toll-like receptors, and tumor necrosis factor-alpha were assessed. Caco-2 monolayers with or without ethanol exposure and/or sodium butyrate were used to test butyrate's direct effects on intestinal integrity. Chronic-binge ethanol feeding impaired intestinal TJ protein co-localization staining; however, tributyrin co-treatment mitigated these effects. Ethanol depleted TJ and transepithelial electrical resistance in Caco-2 monolayers, but butyrate co-treatment reduced these effects. Hepatic toll-like receptor mRNA expression and tumor necrosis factor-alpha protein expression was induced by ethanol; however, the response was significantly dampened in mice co-treated with tributyrin. Tributyrin altered localization of both neutrophils and single hepatocyte death: Leukocytes and apoptotic hepatocytes localized predominantly around the portal tract in ethanol-only treated mice, whereas localization predominated around the central vein in ethanol-tributyrin mice. Prophylactic tributyrin supplementation mitigated effects of combined chronic-binge ethanol exposure on disruption of intestinal TJ localization and intestinal permeability and liver injury.

Omega-3 polyunsaturated fatty acids ameliorate ethanol-induced adipose hyperlipolysis: A mechanism for hepatoprotective effect against alcoholic liver disease

Alcohol exposure induces adipose hyperlipolysis and causes excess fatty acid influx into the liver, leading to alcoholic steatosis. The impacts of omega-3 polyunsaturated fatty acids (n-3 PUFA) on ethanol-induced fatty liver are well documented. However, the role of n-3 PUFA in ethanol-induced adipose lipolysis has not been sufficiently addressed. In this study, the fat-1 transgenic mice that synthesizes endogenous n-3 from n-6 PUFA and their wild type littermates with an exogenous n-3 PUFA enriched diet were subjected to a chronic ethanol feeding plus a single binge as model to induce liver injury with adipose lipolysis. Additionally, the differentiated adipocytes from 3T3-L1 cells were treated with docosahexaenoic acid or eicosapentaenoic acid for mechanism studies. Our results demonstrated that endogenous and exogenous n-3 PUFA enrichment ameliorates ethanol-stimulated adipose lipolysis by increasing PDE3B activity and reducing cAMP accumulation in adipocyte, which was associated with activation of GPR120 and regulation of Ca2+/CaMKKβ/AMPK signaling, resultantly blocking fatty acid trafficking from adipose tissue to the liver, which contributing to ameliorating ethanol-induced adipose dysfunction and liver injury. Our findings identify that endogenous and exogenous n-3 PUFA enrichment ameliorated alcoholic liver injury by activation of GPR120 to suppress ethanol-stimulated adipose lipolysis, which provides the new insight to the hepatoprotective effect of n-3 PUFA against alcoholic liver disease.

Triticum aestivum sprout-derived polysaccharide exerts hepatoprotective effects against ethanol-induced liver damage by enhancing the antioxidant system in mice.

Triticum aestivum sprout-derived polysaccharide (TASP) has anti-diabetic properties, but no information is available in regards to its protective effect against ethanol-induced hepatic injury. This study aimed to investigate the mechanism behind the protective role of TASP against ethanol-induced liver injury invivo. Male C57BL/6 mice were administered ethanol with or without TASP for 10consecutive days by oral gavage. Silymarin was administered in the same manner as a positive control. TASP reduced ethanol-induced hepatic lipid accumulation and serum alanine aminotransferase(ALT) and aspartate aminotransferase (AST) levels. TASP also prevented glutathione(GSH) depletion and increased the superoxide dismutase(SOD) in liver tissue. In addition, TASP significantly inhibited ethanol-induced cytochrome P4502E1(CYP2E1) activation, and upregulated the expressions of nuclear factor erythroid 2-related factor2(Nrf2) and hemeoxygenase-1(HO-1), and downregulated NADPH oxidase genes in ethanol fed mice. Furthermore, the upregulation of Nrf2 was found to be regulated by a phosphatidylinositol 3-kinase(PI3K)/Akt pathway. TASP also attenuated hepatic injury by modulation of caspase-3 and apoptosis-associated mitochondrial proteins including B-cell lymphoma-2(Bcl-2) and Bcl-2-associatedX (Bax) in liver tissues of mice. The study demonstrated that TASP treatment protects against ethanol-induced hepatic injury via multiple pathways by inhibiting steatosis and improving antioxidant marker levels during hepatic injury. Such properties provide a basis for therapeutic agents against alcohol-induced liver injury.

Fungus-derived hydroxyl radicals kill hepatic cells by enhancing nuclear transglutaminase.

We previously reported the importance of induced nuclear transglutaminase (TG) 2 activity, which results in hepatic cell death, in ethanol-induced liver injury. Here, we show that co-incubation of either human hepatic cells or mouse primary hepatocytes derived from wild-type but not TG2−/− mice with pathogenic fungi Candida albicans and C. glabrata, but not baker’s yeast Saccharomyces cerevisiae, induced cell death in host cells by enhancing cellular, particularly nuclear, TG activity. Further pharmacological and genetic approaches demonstrated that this phenomenon was mediated partly by the production of reactive oxygen species (ROS) such as hydroxyl radicals, as detected by a fluorescent probe and electron spin resonance. A ROS scavenger, N-acetyl cysteine, blocked enhanced TG activity primarily in the nuclei and inhibited cell death. In contrast, deletion of C. glabrata nox-1, which encodes a ROS-generating enzyme, resulted in a strain that failed to induce the same phenomena. A similar induction of hepatic ROS and TG activities was observed in C. albicans-infected mice. An antioxidant corn peptide fraction inhibited these phenomena in hepatic cells. These results address the impact of ROS-generating pathogens in inducing nuclear TG2-related liver injuries, which provides novel therapeutic targets for preventing and curing alcoholic liver disease.

Hepatocyte-derived macrophage migration inhibitory factor mediates alcohol-induced liver injury in mice and patients

Macrophage migration inhibitory factor (MIF) is a multi-potent cytokine that contributes to the inflammatory response to injury. MIF is expressed by multiple cell types; however, the cellular source and actions of MIF in alcoholic liver disease (ALD) are not well known. Here we tested the hypothesis that non-myeloid cells, specifically hepatocytes, are an important cellular source of MIF in ALD. MIF expression was measured in HuH7 and differentiated THP-1 cells in response to ethanol. Ethanol-induced liver injury was assessed in C57BL/6 (WT) and Mif-/- bone marrow chimeras. MIF was measured in peripheral and suprahepatic serum, as well as visualized by immunohistochemistry in liver biopsies, from patients with alcoholic hepatitis (AH). HuH7 hepatocytes, but not THP-1 macrophages, released MIF in response to ethanol challenge in culture. In chimeric mice expressing MIF in non-myeloid cells (Mif-/-→WT), chronic ethanol feeding increased ALT/AST, hepatic steatosis, and expression of cytokine/chemokine mRNA. In contrast, chimeric mice not expressing MIF in non-myeloid cells (WT→Mif-/-) were protected from ethanol-induced liver injury. Immunohistochemical staining of liver biopsies from patients with AH revealed a predominant localization of MIF to hepatocytes. Interestingly, the concentration of MIF in suprahepatic serum, but not peripheral serum, was positively correlated with clinical indicators of disease severity and with an increased risk of mortality in patients with AH. Taken together, these data provide evidence that hepatocyte-derived MIF is critical in the pathogenesis of ALD in mice and likely contributes to liver injury in patients with AH. Lay summary: Alcoholic liver disease is a major cause of preventable mortality worldwide, and lacks specific pharmacological therapies. Recent studies have recognized that macrophage migration inhibitor factor (MIF) has a critical role in the inflammatory response to liver damage. However, the cells that produce this protein are still unknown. Our present findings reveal that hepatocytes, the main cell type in the liver, are primarily responsible for MIF production in response to alcohol, which promotes liver injury. Our study suggests that drugs inhibiting MIF production could be beneficial in treating patients with liver disease due to excessive alcohol consumption.

Hepatoprotective effect of chitosan-caffeic acid conjugate against ethanol-treated mice

The chitosan-caffeic acid (CCA) conjugate shows a hepatoprotective effect against oxidative stress-induced hepatic damage in cultured hepatocytes. The objective of this study is the verification of the hepatoprotective effect of the CCA in vivo against ethanol-induced liver injury in mice. The administration of ethanol resulted in the increase of the serum-aminotransferase activities (AST and ALT), triglycerides, total cholesterol, and lipid peroxidation. The CCA co-administration, however, significantly (p<0.05) ameliorated these serum biomarkers. The antioxidant-enzyme activities in the liver tissue, including those of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), were significantly decreased by a chronic ethanol administration, whereas the hepatic lipid-peroxidation level was increased. Moreover, the chronic ethanol administration elevated the gene expression of pro-inflammatory cytokines such as tumor-necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the liver tissue. The CCA co-administration, however, significantly (p<0.05) increased the activities of the SOD, CAT, and GPx and caused the down-regulation of the TNF-α- and IL-6-gene expressions in the liver tissue. An histopathologic evaluation also supported the hepatoprotective effect of the CCA against ethanol-induced hepatotoxicity in the mice.

C-C chemokine receptor type 5 deficiency exacerbates alcoholic fatty liver disease through pro-inflammatory cytokines and chemokines-induced hepatic inflammation.

Chemokines and chemokine receptors implicated with alcoholic liver disease. Studies have shown that inflammation and oxidative stress induce fat molecules aggregation in liver. We evaluated the relationship between alcoholic fatty liver disease and C-C chemokine receptor 5 (CCR5) and impact of inflammation and oxidative stress in fat molecule deposition. Lieber-DeCarli diet containing ethanol or isocaloric control diets were fed to wild-type and CCR5 knockout mice for 10days and gavaged with a single dose of ethanol or isocaloric maltose dextrin at 11th day. Cytokine, chemokine, and reactive oxygen species levels were measured in liver tissues to study the role of CCR5 in alcoholic fatty liver disease. C-C chemokine receptor type 5 knockout mice exacerbated ethanol-induced liver injury. Serum levels of aspartate aminotransferase and alanine aminotransferase were higher in CCR5 knockout mice than wild-type mice, and CCR5 knockout mice showed more severe lipid accumulation in liver tissue than wild-type mice after ethanol feeding. Increased expressions of pro-inflammatory cytokines TNF-α and IL-6 and chemokines CCL2, CCL3, CCL4, and CCL5 result in exacerbation of hepatitis in CCR5 knockout mice after ethanol feeding. Oxidative stress induced by reactive oxygen species was more severe in CCR5 knockout mice, and increasing level of fatty acid import and decreasing level of lipid degradation resulted in lipid accumulation in ethanol-fed CCR5 knockout mice. Deficiency of CCR5 exacerbates alcoholic fatty liver disease by hepatic inflammation induced by pro-inflammatory cytokines and chemokines and oxidative stress.