The Role Of Gut Microbiota-bile Acid Axis In Liver Fibrosis Based On Microbiomics And Targeted Metabolomics

Objective:This study investigated the changes in the gut microbiota-bile acid axis in rats with liver fibrosis by constructing a rat model of liver fibrosis and combining microbiomics and bile acid-targeted metabolomics analysis methods.We also investigated the effect of the alteration of the gut microbiota-bile acid axis on liver fibrosis by using fecal bacteria transplantation and probiotic preparations to regulate the gut microbiota and thus alter the gut microbiota-bile acid axis,in order to reveal the role of the gut microbiota-bile acid axis in liver fibrosis from both forward and reverse and to provide new strategies for the treatment of liver fibrosis.Methods:Firstly,50 male Sprague Dawley rats were randomly divided into control and experimental groups,and the experimental rats have injected with carbon tetrachloride-olive oil solution subcutaneously and intraperitoneally twice a week for4 weeks to construct a rat model of liver fibrosis.After 4 weeks,the rats in the experimental group with successful modeling were then randomly divided into the model group,the fecal microbiota transplantation group,and the probiotics group.The rats in the fecal microbiota transplantation group were gavaged with a microbiota solution made from the fresh feces of the control rats,and the rats in the probiotics group were gavaged with Bifidobacterium tetrasodium solution,while the rats in the control and model groups were gavaged with equal amounts of saline once a day for12 weeks.Feces,serum,and liver tissue were collected from rats after 12 weeks.The microbiota composition of the rat intestine was determined by 16s r RNA sequencing,and the levels of 22 common bile acids in rat serum were determined by ultra-performance liquid chromatography-mass spectrometry.Four indexes of serum liver fiber were determined by ELISA,combined with HE staining and Masson staining of liver tissue to evaluate the degree of liver fibrosis in rats.Results:1.Gut microbiota characteristics of liver fibrosis rats:compared with the control group,the gut microbiota composition of rats in the model group was significantly altered,and there was a significant difference in the beta diversity of gut microbiota between the two groups.Lef Se analysis showed that in the model group rats,Clostridia,Clostridiales,Clostridiaceae＿1,Erysipelotrichaceae＿incertae＿sedis,Clostridium＿sensu＿stricto,Terrisporobacter,Enterobacteriaceae,Cellulosilyticum Escherichia＿Shigella,and Sutterella significantly increased in abundance,while Parasutterella,Burkholderiales,Betaproteobacteria,Anaerostipes,Bifidobacterium,Bifidobacteriaceae,Bifidobacteriales,Olsenella,Actinobacteria,and Allobaculum were significantly reduced in abundance.2.Characteristics of serum bile acid metabolism in rats with liver fibrosis:compared with the control group,serum bile acid metabolism was significantly altered in the model rats,and the two groups were clearly separated in the OPLS-DA plot.In the serum of the model group rats,the concentrations of 19 bile acids were significantly increased(P<0.05).Among them,10 bile acids,including Tα-MCA,TCDCA,GCDCA,GCA,TCA,THDCA,TUDCA,GHDCA,GUDCA,andα-MCA,had VIP values>1 in the OPLS-DA model,and the Fold Change between the control and model groups was>2.3.Correlation between gut microbiota and serum bile acids in rats with liver fibrosis:20 microbiotas screened in LEf Se analysis that was significantly different in control and model groups were subjected to Spearman correlation analysis with 10bile acids that were significantly different in serum.The results showed that the gut microbiota and bile acids in liver fibrosis rats were significantly correlated.Among them,TCA was significantly and strongly negatively correlated with Anaerostipes and Actinobacteria(r_s were-0.8016 and-0.8000,P were 0.0031 and 0.0051,respectively),and TCDCA were significantly and strongly negatively correlated with Bifidobacterium,Bifidobacteriales,and Bifidobacteriaceae(-0.8105 for all three r_sand 0.0002 for all three P).4.Regulation of gut microbiota in rats with liver fibrosis by fecal microbiota transplantation and probiotics preparation:fecal microbiota transplantation and probiotic preparation can improve the disordered gut microbiota in rats with liver fibrosis.Theβ-diversity of gut microbiota was significantly different between the fecal microbiota transplantation group and the model group,and theβ-diversity of gut microbiota was also significantly different between the probiotics group and the model group.Fecal microbiota transplantation increased Actinobacteria,Bifidobacteriales,Bifidobacteriaceae,Bifidobacterium,Allobaculum and Olsenella reduced gut microbiota in rats with liver fibrosis,and reduced Clostridia,Clostridiales,Turicibacter and Enterorhabdus increased gut microbiota in rats with liver fibrosis.Probiotics preparations increased Verrucomicrobia,Carnobacteriaceae,Akkermansia,Erysipelotrichaceae,Atopostipes and Rothia,reduced gut microbiota in rats with liver fibrosis,reduced Firmicutes,Clostridia,Clostridiales,Cellulosilyticum Turicibacter and Enterorhabdus increased gut microbiota in rats with liver fibrosis.5.Effect of fecal microbiota transplantation and probiotics preparation on bile acid metabolism in liver fibrosis rats:fecal microbiota transplantation and probiotics preparation can improve the disordered bile acid metabolism in liver fibrosis rats.In the OPLS-DA model,the fecal microbiota transplantation group was completely separated from the model group,and the probiotics group was also separated from the model group.Among them,fecal microbiota transplantation reduced significantly elevated 13 bile acids in the serum of liver fibrosis rats,including CA,CDCA,UDCA,α-MCA,β-MCA,DCA,HDCA,GUDCA,GHDCA,TCDCA,TUDCA,Tα-MCA and THDCA,and probiotic preparation reduced 14 bile acids,including CDCA,β-MCA,GCA GCDCA,GUDCA,GDCA,GHDCA,TCA,TCDCA,TUDCA,Tα-MCA,TDCA,TLCA and THDCA in the serum of liver fibrosis rats.6.Effects of fecal microbiota transplantation and probiotic preparation on the degree of liver fibrosis in rats after improving the gut microbiota-bile acid axis:HE staining and Masson staining of liver tissues showed that the degree of liver fibrosis in rats in the fecal microbiota transplantation and probiotic groups were reduced compared with the model group.In addition,four indexes of serum liver fiber were reduced in the rats in the fecal microbiota transplantation and probiotics groups compared with the model group.Conclusion:The gut microbiota-bile acid axis has an important role in liver fibrosis.The gut microbiota-bile acid axis was significantly disturbed in rats with liver fibrosis,and fecal microbiota transplantation and probiotic preparations could improve the disturbed gut microbiota-bile acid axis in rats with liver fibrosis and thus reduce liver fibrosis.