Crosstalk between gut microbiota and host lipid metabolism in a mouse model of alcoholic liver injury by chronic baijiu or ethanol feeding

This study demonstrates that compounds in baijiu, a traditional Chinese alcoholic beverage, can attenuate the development of ethanol-induced liver injury by regulating the crosstalk between gut microbiota and host lipid metabolism.

Synbiotics Alleviate Hepatic Damage, Intestinal Injury and Muscular Beclin-1 Elevation in Rats after Chronic Ethanol Administration

The purpose of this study was to investigate the beneficial effects of synbiotics on liver damage, intestinal health, and muscle loss, and their relevance in rats with chronic ethanol feeding. Thirty Wistar rats fed with a control liquid diet were divided into control and synbiotics groups, which were respectively provided with water or synbiotics solution (1.5 g/kg body weight/day) for 2 weeks. From the 3rd to 8th week, the control group was divided into a C group (control liquid diet + water) and an E group (ethanol liquid diet + water). The synbiotics group was separated in to three groups, SC, ASE, and PSE. The SC group was given a control liquid diet with synbiotics solution; the ASE group was given ethanol liquid diet with synbiotics solution, and the PSE group was given ethanol liquid diet and water. As the results, the E group exhibited liver damage, including increased AST and ALT activities, hepatic fatty changes, and higher CYP2E1 expression. Intestinal mRNA expressions of occludin and claudin-1 were significantly decreased and the plasma endotoxin level was significantly higher in the E group. In muscles, beclin-1 was significantly increased in the E group. Compared to the E group, the PSE and ASE groups had lower plasma ALT activities, hepatic fatty changes, and CYP2E1 expression. The PSE and ASE groups had significantly higher intestinal occludin and claudin-1 mRNA expressions and lower muscular beclin-1 expression when compared to the E group. In conclusion, synbiotics supplementation might reduce protein expression of muscle protein degradation biomarkers such as beclin-1 in rats with chronic ethanol feeding, which is speculated to be linked to the improvement of intestinal tight junction and the reduction of liver damage.

MyD88 in Hepatic Stellate Cells Promotes the Development of Alcoholic Fatty Liver via the AKT Pathway

Myeloid differentiation primary response gene 88 (MyD88), an adaptor protein in the TLRs signaling pathway, is expressed in various liver cells including hepatocytes, Kupffer cells and hepatic stellate cells (HSCs). However, the specific role of MyD88 in HSCs in alcoholic fatty liver (AFL) has not been well investigated. Here, to study the role of MyD88 in HSCs in the development of AFL and its related molecular mechanism, chronic-binge ethanol mice models were established in mice with specific MyD88 knockout in quiescent (MyD88GFAP−KO) and activated HSCs (MyD88SMA−KO), respectively. The results showed that the expression of MyD88 in HSCs was significantly increased in ethanol-induced liver tissues of wild-type mice. MyD88 deficiency in quiescent HSCs inhibited inflammation and lipogenesis, but in activated HSCs it only inhibited inflammation under the ethanol feeding condition. Further mechanism studies found that MyD88 promoted the osteopontin (OPN) secretion of HSCs, which further activated the AKT signaling pathway of hepatocytes and upregulated lipogenic gene expression to promote fat accumulation. In addition, OPN also promotes inflammation by activating p-STAT1. Thus, MyD88 may represent a potential candidate target for the prevention and targeted therapy in AFL, and MyD88 inhibitor can be also applied in inhibiting adipogenesis.

Improvement Lipophagy by Restoring Rab7 Cycle: Protective Effects of Quercetin on Ethanol-Induced Liver Steatosis

Background: Chronic alcohol consumption induces lipophagy retard which contributes the pathogenesis of liver steatosis. Lipophagy-related Rab7 responsible for the fusion between autophagosomes and lysosomes has been presumed as a crucial regulator on the progression of alcohol liver disease (ALD) despite elusive mechanisms. More importantly, whether hepatoprotective quercetin targets Rab7-associated lipophagy disorder or not, still remains uncertain. Results: ALD mice were induced by chronic-plus-single-binge ethanol feeding that manifested by hampering autophagosomes formation with lipid droplets (LDs) and fuse with lysosomes compared with the normal control, which was normalized partially by quercetin. GST-RILP pulldown assay of Rab7 indicated an improved GTP-Rab7 as quercetin treatment for ethanol-feeding mice. Lipophagy was blocked when HepG2 cells were transfected with siRNA-Rab7, which was reversed through co-transfection of Rab7Wt plasmid. Rab7-specific inhibitor CID1067700 aggravated ethanol-induced steatosis and autophagic flux disruption visualized by immunofluorescence co-localization analysis and mCherry-GFP-LC3 transfection. HepG2 cells overexpressing Rab7Wt show a stronger alleviation on alcohol-induced lipophagy dysfunction than Rab7Q67L. Furthermore, TBC1D5 responsible for Rab7 inactivation was downregulated after alcohol administration, but regained by quercetin. Rab7 circulation retarded by ethanol and corrected by quercetin was further revealed by fluorescence recovery after photobleaching (FRAP). Conclusions: Chronic alcoholic not only inactivates Rab7 but also retards TBC1D5-mediated Rab7 turnover, synergistically obstructing lipophagy flux and promoting ethanol-induced steatosis. Quercetin attenuates adipohepatic degeneration by normalizing ethanol-imposed Rab7 disorders and subsequent lipophagy disturbance, highlighting a novel mechanism and promising prospect of quercetin-like phytochemicals against the crucial first hit from the alcohol.

Oral administration of PEGylated TLR7 ligand ameliorates alcohol-associated liver disease via the induction of IL-22

Effective therapies for alcohol-associated liver disease (ALD) are limited; therefore, the discovery of new therapeutic agents is greatly warranted. Toll-like receptor 7 (TLR7) is a pattern recognition receptor for single-stranded RNA, and its activation prevents liver fibrosis. We examined liver and intestinal damage inTlr7−/−mice to determine the role of TLR7 in ALD pathogenesis. In an alcoholic hepatitis (AH) mouse model, hepatic steatosis, injury, and inflammation were induced by chronic binge ethanol feeding in mice, andTlr7deficiency exacerbated these effects. Because these results demonstrated that endogenous TLR7 signaling activation is protective in the AH mouse model, we hypothesized that TLR7 activation may be an effective therapeutic strategy for ALD. Therefore, we investigated the therapeutic effect of TLR7 agonistic agent, 1Z1, in the AH mouse model. Oral administration of 1Z1 was well tolerated and prevented intestinal barrier disruption and bacterial translocation, which thus suppressed ethanol-induced hepatic injury, steatosis, and inflammation. Furthermore, 1Z1 treatment up-regulated the expression of antimicrobial peptides, Reg3b and Reg3g, in the intestinal epithelium, which modulated the microbiome by decreasing and increasing the amount ofBacteroidesandLactobacillus,respectively. Additionally, 1Z1 up-regulated intestinal interleukin (IL)-22 expression. IL-22 deficiency abolished the protective effects of 1Z1 in ethanol-induced liver and intestinal damage, suggesting intestinal IL-22 as a crucial mediator for 1Z1-mediated protection in the AH mouse model. Collectively, our results indicate that TLR7 signaling exerts protective effects in the AH mouse model and that a TLR7 ligand, 1Z1, holds therapeutic potential for the treatment of AH.

Engineered Butyrate-producing Bacillus Subtilis Alleviated Ethanol-induced Intestinal and Liver Damage in Mice

Ethanol-induced intestinal and liver injury are closely associated with intestinal dysbiosis and altered short-chain fatty acid (SCFA) metabolites which is crucial for intestinal health. Bacillus subtilis (BS) strains with biotherapeutic potential can benefit the host through maintaining intestinal homeostasis and regulating systematic immunity via producing small molecules, although these molecules do not include butyrate. To combine the advantages of butyrate and BS, we evaluated the bioactivity of an engineered butyrate-producing Bacillus subtilis (BPBS) strain against ethanol exposure in a chronic-binge ethanol feeding mouse model. Our findings suggested that prophylactic BPBS supplementation restored eubiosis of the gut microbiota and intestinal barrier function, which obviously reduced bacterial translocation of microbial products especially lipopolysaccharide (LPS) to the circulatory system. Additionally, the decrease of serum LPS is responsible for the relief of hepatic inflammation via the Toll-like receptor 4 (TLR4) pathway, resulting in improved hepatic structure and function. Collectively, these results demonstrated that engineered BPBS intervention imparted novel hepatoprotective functions by improving intestinal barrier function and reducing systematic inflammation under ethanol exposure, as well as paving the way for further exploration of engineered probiotics in improving human health care.

DNMT3b-mediated methylation of ZSWIM3 enhances inflammation in alcohol-induced liver injury via regulating TRAF2-mediated NF-κB pathway

The regulation of macrophages during inflammatory responses is a crucial process in alcoholic liver disease (ALD) and aberrant macrophage DNA methylation is associated with inflammation. Our preliminary screening results of macrophage methylation in the present study demonstrated the zinc finger SWI2/SNF2 and MuDR (SWIM)-domain containing 3 (ZSWIM3) were hypermethylated in the 5′ untranslated region (5′-UTR) region. ZSWIM3, a novel zinc finger-chelate domain of SWIM, is predicted to function in DNA-binding and protein-binding interactions. Its expression was found to be consistently decreased in macrophages isolated from livers of ethyl alcohol (EtOH)-fed mice and in EtOH+lipopolysaccharide (LPS)-induced RAW264.7 cells. Over-expression of ZSWIM3 was found to attenuate chronic+binge ethanol feeding-induced liver injury and inhibit inflammatory responses in vivo. Enforced expression of ZSWIM3 in vitro was also found to have anti-inflammatory effects. Aberrant expression of ZSWIM3 in alcohol-induced liver injury (ALI) was found to be associated with hypermethylation. Analysis of CpG prediction indicated the presence of two methylated sites in the ZSWIM3 promoter region and methylation inhibitor and DNA methyltransferases (DNMTs)-siRNA transfection were found to restore down-regulated ZSWIM3. Chromatin immunoprecipitation (ChIP) assay and molecular docking affirmed the role of DNMT 3b (DNMT3b) as a principal regulator of ZSWIM3 expression. Mechanistically, ZSWIM3 might affect inflammation by binding with tumor necrosis factor receptor-associated factor 2 (TRAF2), which further mediates the activation of the nuclear transcription factor κB (NF-κB) pathway. The present study, therefore, provides detailed insights into the possible structure and function of ZSWIM3 and thus, contributes new substantial research in the elucidation of the pathogenesis of ALI.