Changes Of Bile Acid Targeted Metabonomics And Gut Microbiotaand Their Effects On The Progression Of Nonalcoholic Fatty Liver Disease

BackgroundNonalcoholic fatty liver disease（NAFLD）is a complex disease spectrum including nonalcoholic fatty liver（NAFL）,nonalcoholic steatohepatitis（NASH）,and NASH-related cirrhosis.In terms of hepatic manifestation,NASH has a greater risk of progression than NAFL and is more likely to progress to cirrhosis or even liver cancer.Moreover,evidence suggests that NAFLD is associated with a number of extrahepatic complications,such as type 2 diabetes mellitus（T2DM）,cardiovascular events,and colorectal cancers.Although studies have examined changes in bile acids and gut microbiota in NAFLD/NASH,there have been no systematic studies on the interaction between bile acids and intestinal microbiota in different clinical phenotypes of NAFLD,and no studies have explored the effects of changes in bile acids and gut flora on the progression of NAFLD disease.Therefore,the purpose of this study is to investigate the differences in bile acids and gut microbiota of different NAFLD phenotypes,analyze the relationship between bile acids and intestinal microbiota,and explore the possible role of bile acids and intestinal flora in different NAFLD phenotypes.MethodsWe established NAFL,NASH,NAFL+T2DM,and CCl₄ mouse models to represent common NAFLD clinical phenotypes.Bile acid-targeted metabolomics was used to analyze bile acid changes in different parts including serum,liver and caecal contents.16s rDNA was used to detect microbial flora in caecal contents,and Spearman correlation of bile acid and gut microbiota of phylum and genus levels were analyzed.ResultsWe found significant changes in bile acids in different parts of different phenotypes of NAFLD.Compared with NC group,GCA,TUDCA and TDCA were significantly increased in the serum in NAFL group,but NorDCA and 7-ketoLCA were significantly decreased;in the liver,alloLCA was significantly increased,but GCDCA,GUDCA,HCA,NorCA,NorDCA,6,7-diketoLCA,7-ketoLCA was significantly reduced;in cecal contents,NorDCA and DCA were significantly increased,but HCA,alloLCA,6,7-diketoLCA,and NorCA were significantly decreased.Compared with NC group,CA,CDCA,UDCA,and NorDCA were significantly increased in the serum in NASH group,but THDCA was significantly decreased;in the liver,CA,GCDCA,DCA,GUDCA,NorCA,and NorDCA were significantly increased,butωMCA and 6,7-diketoLCA were significantly decreased;in cecal contents,NorDCA and NorCA were significantly increased,but TωMCA and HDCA were significantly decreased.Compared with NC group,GCA,DCA,TUDCA,THDCA,TDCA were significantly increased in the serum in NAFL+T2DM group,but 7-ketoLCA was significantly decreased;in the liver,DCA,UDCA,muroCA were significantly increased,but CA,6,7-diketoLCA,7-ketoLCA was significantly reduced;in cecal contents,CA,CDCA,TUDCA,muroCA,HDCA,DCA and LCA were significantly elevated,but 6,7-diketoLCA was significantly reduced.Compared with NC group,CA,CDCA,UDCA,TCA,GCA,and TUDCA were significantly elevated in the serum in CCl₄ group;in the liver,GCA,TCA,THDCA,UDCA,HDCA,TUDCA were significantly elevated;in cecal contents,GHDCA,THDCA,TUDCA were significantly elevated,but GCA was significantly reduced.Intestinal microbial analysis showed that intestinal flora of NC group,NAFL group,NASH group and NAFL+T2DM group mainly consisted of Firmicutes,Bacteroides and Verrucomicrobia.The dominant groups of NC group and CCl₄ are Firmicutes,Bacteroides and Proteobacteria.At the phylum level,compared with NC group,the abundance of Firmicutes and Actinobacteria in NAFL group significantly increased,while the abundance of Bacteroides and Proteobacteria was significantly reduced.The abundance of Actinobacteria in NASH group was significantly increased.In NAFL+T2DM group,the abundance of Firmicutes,Proteobacteria and Actinobacteria were significantly increased,and the abundance of Bacteroides was significantly reduced.Compared with NC group,the abundance of Proteobacteria in CCl₄ group was significantly increased,and the abundance of Spirochaetes was decreased.At the genus level,the dominant flora of different phenotypes of NAFLD changed significantly.Compared with NC group,Clostridium IV of NAFL group was significantly increased;Bacteroides,Parabacteroides,Eubacterium,and Staphylococcus in NASH group were significantly elevated,while Barnesiella was significantly reduced;in NAFL+T2DM group,Oligella,Oscillibacter and Halomonas were significantly elevated,while Clostridium XVⅢ and Clostridium XlVb were significantly reduced.Compared with NC group,the abundance of the genus Rusococcus,Butyricicoccus,Intestinimonas,and Clostridium XlVa in CCl₄group was significantly increased.In addition,the abundance of Helicobacter,Prevobacterium,Desulfovibrio,and Streptococcus in CCl₄ group increased significantly,while the abundance of Saccharibacteria_genera_incertae_sedis and Odoribacter decreased significantly.In terms of the correlation between bile acids of caecal contents and intestinal flora,the abundance of Actinobacteria was positively correlated with NorDCA and DCA,and negatively correlated with 6,7-diketoLCA and HCA.6,7-diketoLCA was significantly positively correlated with Bacteroidetes and was significantly negatively correlated with Firmicutes.Proteobacteria was positively correlated with LCA,NorCA and TUDCA,and Candidatus Saccharibacteria was significantly negatively correlated with LCA.At the genus level,Bacteroides was significantly negatively correlated with TωMCA andβMCA.Parabacteroides were significantly negatively correlated withαMCA,βUDCA,βMCA,TβMCA and TωMCA,while Prevotella was significantly negatively correlated with GCA.Akkermansia is significantly positively correlated with NorCA and NorDCA.Ruminococcus is significantly negatively correlated with NorDCA,TDCA,TCA,and DCA.Ruminococcus was significantly negatively correlated withβ-MCA,while Escherichia/Shigella was positively correlated withαMCA andβMCA.Eubacterium was significantly negatively correlated with TωMCA,αMCA,βMCA,andωMCA.ConclusionDifferent phenotypes of NAFLD disease are associated with changes in bile acids of different parts（serum,liver and caecal contents）and intestinal microbiota dysbiosis.The intestinal flora may work with bile acids to promote the development of NAFLD disease.