The Role And Mechanism Of Rifaximin In The Occurrence And Progression Of Non-alcoholic Fatty Liver Disease

Background and purpose:With the increasing number of obese people,non-alcoholic fatty liver disease（NAFLD）has become the most common liver disease in the world,including non-alcoholic fatty liver（NAFL）,non-alcoholic steatohepatitis（NASH）,non-alcoholic fatty liver fibrosis and cirrhosis.Recent studies have found that NAFLD is closely related to type 2 diabetes,metabolic syndrome,and cardiovascular and cerebrovascular diseases.NAFLD has become the most common cause of chronic liver disease in Western countries and will become the most common indication for liver transplantation in the future.Due to the lack of specificity in the early diagnosis of NAFLD,there is still no uniform standard for the treatment of NAFLD.This study aims to explore the role of Rifaximin in the development and progression of NAFLD and its internal pathophysiological mechanism.Methods:1.High-fat diet（HFD）or methionine/choline deficient diet（MCD）induced the formation of NAFL or NASH in mice,and then Rifaximin was administered at different starting times or different treatment.Liver HE staining,oil red O staining,Sirius red staining,IHC staining,q PCR and Western blot methods were used to detect liver lipid deposition,red collagen deposition,lipid metabolism,liver fibrosis,and liver inflammation.2.Enzyme coupling method was used to detect serum ALT,AST,triglyceride,and total cholesterol levels in mice.Glucose tolerance test（GTT）and Insulin tolerance test（ITT）tests were used to detect glucose tolerance and insulin tolerance in NAFL mice.3.The feces of NAFL and NASH mice were subjected to 16S r RNA microbial diversity sequencing to analyze the influence of Rifaximin on the intestinal microbiota of NAFL and NASH mice.4.Intestinal microbiota-bile acid correlation analysis was used to analyze the correlation between gut microbiota and ileal bile acids in NASH mice.The bile acid full spectrum analysis was used to detect the bile acids levels in the terminal ileum of NASH mice.Intestinal bile acid-Fxr-Fgf15 signaling pathway and liver HNF1α-Fxr signaling pathway were detected in NASH mice.5.Moreover,intestinal decontamination NASH mice were involved to elucidate the molecular mechanisms of Rifaximin in improving NASH.6.A liver-specific HNF1αknockout NASH model was constructed and treated with Rifaximin to observe the effect of Rifaximin on HNF1α^HKO NASH mice.Results:1.A NAFL mouse model was established after 8 weeks of HFD feeding.After 4or 8 weeks of Rifaximin treatment,the body weight and epididymal fat weight of the mice in the Rifaximin group were obviously reduced,and the lipid deposition in liver was significantly improved compared to the NAFL group（P<0.05 or P<0.01）.Compared with the NAFL group,Rifaximin treatment significantly inhibited liver lipid metabolism,liver inflammation,and mononuclear macrophage infiltration,and reduced triglycerides and total cholesterol levels in serum and liver in the Rifaximin group（P<0.05 or P<0.01 or P<0.001）.In addition,Rifaximin improved glucose tolerance and insulin tolerance in the Rifaximin group compared to the NAFL group（P<0.05 or P<0.01）.2.A NASH mouse model was established after 2 weeks of MCD feeding.After 2or 4 weeks of Rifaximin treatment,compared with the NASH group,Rifaximin treatment significantly inhibited liver lipid metabolism,liver inflammation,and mononuclear macrophage infiltration,and lowered liver triglycerides and total cholesterol levels in the Rifaximin group（P<0.05 or P<0.01 or P<0.001）.Compared with the NASH group,Rifaximin treatment markedly reduced serum AST and ALT levels,improved liver function,and inhibited the procession of liver fibrosis in the Rifaximin group（P<0.05 or P<0.01）.Rifaximin preventive treatment also significantly improved liver lipid metabolism in the NASH mice induced by MCD feeding,reduced liver inflammation and inhibited liver fibrosis procession（P<0.05 or P<0.01）.3.16S r RNA microbial diversity sequencing of NAFL mice showed that compared with the NAFL group,abundance of the fecal microbiota in the Rifaximin group was significantly reduced,and species composition of intestinal microbiota changed significantly after Rifaximin treatment（P<0.05 or P<0.01 or P<0.001）.Compared with the NAFL group,Bacteroides increased significantly in the Rifaximin group,while Firmicutes and Proteobacteria decreased obviously（P<0.05 or P<0.01）.4.16S r RNA microbial diversity sequencing of NASH mice showed that compared with the NASH group,types and abundance of the fecal microbiota in the Rifaximin group were both significantly reduced,and species composition of intestinal microbiota changed significantly after Rifaximin treatment（P<0.05 or P<0.01 or P<0.001）.Compared with the NASH group,the Helicobacter,Faecalibaculum and Muribaculaceae in the Rifaximin group were significantly reduced,while Parabacteroides was markedly increased（P<0.05 or P<0.01）.Among them,the most obvious changes in the intestinal microbiota of NASH mice were Helicobacter hepaticus and Parabacteroides distasonis after Rifaximin treatment（P<0.001）.5.The correlation analysis between Helicobacter hepaticus and Parabacteroides distasonis and ileal bile acids showed that Helicobacter hepaticus was positively correlated with deoxycholic acid（DCA）,while Parabacteroides distasonis was negatively correlated with DCA（P<0.05）.Bile acids full spectrum analysis of terminal ileum of NASH mice showed that Rifaximin treatment significantly reduced the level of DCA in the terminal ileum（P<0.001）.Moreover,Rifaximin markedly inhibited intestinal microbiota-DCA-Fxr signaling pathway and activated liver HNF1α-Fxr signaling pathway in NASH mice（P<0.05 or P<0.01 or P<0.001）.6.Furthermore,we treated the NASH mice with combined antibiotics to decontaminate the intestinal microbiota,and then treated them with Rifaximin.The results showed that Rifaximin could not significantly improve liver lipid deposition,liver inflammation,and liver fibrosis progression in intestinal decontamination NASH mice（P>0.05）.7.Cre-lox P recombination system was used to knockout hepatocyte HNF1αin mice,and hepatocyte-specific HNF1αknockout mouse was successfully established.HNF1α^HKO mice spontaneously formed NASH,and then were treated with Rifaximin.The results showed that Rifaximin could not obviously improve liver lipid deposition,liver inflammation,and liver fibrosis progression in HNF1α^HKO NASH mice（P>0.05）.Conclusion:Rifaximin treatment significantly improved the development and progression of NAFL and NASH in mice.Rifaximin also obviously inhibited liver inflammation and liver fibrosis procession in NASH mice.Rifaximin exerts pathophysiological effects by inhibiting the intestinal microbiota-DCA-Fxr signaling pathway and activating the liver HNF1α-Fxr signaling pathway.