Soyasaponin A₂ Prevents Methionine And Choline Deficient Diet-induced Nonalcoholic Fatty Liver Disease By Regulating The Liver-Bile Acid-Gut Microbiota Metabolic Axis In Mice

Background and objectivesNonalcoholic fatty liver disease(NAFLD)is a major public health problem that seriously threats human health.The latest research shows that the liver-bile acid-gut microbiota axis(LBGMA)plays an important role in the pathogenesis and progression of NAFLD.Soyasaponins have a wide range of health-promoting effect including hepatoprotection,hypocholesterolemia,and anti-inflammation,etc.which indicates its potentials to prevent NAFLD.However,no study is available regarding the direct preventive abilities of soyasaponins against NAFLD.This study aims to investigate the preventive bioactivities of soyasaponin A2(SS-A2)against methionine-choline deficiency diet(MCD)-induced NAFLD and further clarify its mechanism by targeting LBGMA.Accomplishment of this study will provide theoretical basis for using phytochemical soyasaponins to prevent NAFLDMethods1.One hundred and twenty 6-week-old male C57BL/6 mice were randomly divided into 6 groups.Each group had 20 mice that kept in two cages with 10 mice in each cage.Mice in the 1st group were used as control group and fed methionine and choline supplemented(MCS)diet.Mice in the 2nd group were used as positive control group and fed MCD.Mice in the 3rd group were used as medicine intervention group and fed MCD containing 0.4 mg/kg.BW obeticholic acid(OCA).Mice in the 4th,5th and 6th group were fed MCD supplemented with 1 mg/kg·BW(LSS group),50 mg/kg-BW(LSS group)and 100 mg/kg·BW(HSS group)of SS-A2,respectively.Half mice(n=10)from each group were intervened for 4 weeks and the other half mice from each group were intervened for 16 weeks.At the end of each intervention,mice were killed after anesthetization.Samples of blood,liver tissues and terminal ileum tissues along with its containings were collected.2.Liver tissues were stained by using HE and Masson staining and evaluated by using the NAFLD activity score(NAS)and liver fibrosis score.The levels of total cholesterol(TC)and triglyceride(TG)in serum,liver and feces and of high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),aspartate aminotransferase(AST),tumor necrosis factor a(TNF-?)and interleukin-6(IL-6)in serum were determined by commercial kits.3.The microbiota in cecal contents was detected by 16S rDNA high thoughput sequencing method.4.The concentrations of bile acids in liver,serum and colon contents were determined by UPLC-MS/MS.5.The protein expression levels of farnesoid X receptor(FXR),bile salt export pump(B SEP),sodium/taurocholate cotransporting polypeptide(NTCP)and cholesterol 7?-hydroxylase(CYP7A1)in liver and the protein levels of FXR,fibroblast growth factor 15(FGF-15)and apical sodium-dependent BA transporter(ASBT)in the terminal ileum were determined by western blot.6.Data were analyzed by two independent sample T test or one-way analysis of variance among groups by using SPSS 20.0 statistical software.The data of bile acids were analyzed by Kruskal-Wallis test.The relationship between intestinal microbiota and bile acids were analyzed by Spearman correlation analysis.Results are presented as average ± SEM.Ap value of less than 0.05 indicates statistical significance.Results1.The liver tissue NAS score in the MCD group were 3.70±0.09,while lobular inflammation and NAS scores of OCA group were all lower than MCD group at 4 weeks of intervention(p<0.05).The hepatic steatosis,lobular inflammation,ballooning and NAS scores of LSS,MSS and HSS groups were all lower than MCD group(p<0.05).The liver tissue NAS score in the MCD group were 5.44±0.11,while the scores of hepatic steatosis,lobular inflammation,ballooning,NAS and liver fibrosis of OCA,LSS,MSS and HSS groups were all lower than MCD group at 16 weeks of intervention(p<0.05).At 4 and 16 weeks of intervention,the levels of liver TG and serum ALT of OCA,LSS,MSS and HSS groups were all lower than MCD group(p<0.05).The levels of serum TNF-? of MSS and HSS groups were lower than MCD group at 16 weeks of intervention(p<0.05).2.At 4 weeks of intervention,the relative abundance of Desulfovibrionaceae of HSS group was higher than MCD group(p<0.05).The relative abundance of Erysipelotrichaceae and Faecalibaculum of OCA,LSS and HSS groups,and the relative abundance of Candidatus Saccharimonas of the LSS and MSS groups were lower than MCD group(p<0.05).At 16 weeks of intervention,the relative abundance of Actinobacteria of OCA,MSS and HSS groups were lower than MCD group(p<0.05),while the relative abundance of Protebacteria of OCA group was higher than MCD group(p<0.05).Compared with the MCD group,LSS group increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes,Lactobacillaceae and Lactobacillus(p<0.05).The relative abundance of Erysipelotrichaceae and Faecalibaculum of OCA,LSS,MSS and HSS groups were lower than MCD group(p<0.05)and the relative abundance of Desulfovibrionaceae and Desulfovibrio increased significantly in OCA and MSS groups(p<0.05).The relative abundance of unidentified-Costridiales(at family and genus levels)increased significantly in OCA and HSS groups(p<0.05).3.At 16 weeks of intervention,OCA and SS-A2 groups reduced the accumulation of most serum bile acids in MCD-induced NAFLD mice(p<0.05).Compared with the MCD group,OCA and HSS groups increased the levels of liver and serum THDCA and promoted the excretion of most bile acids in the intestine(p<0.05).4.At 16 weeks of intervention,gut microbiota was correlated with bile acids in most contents of liver,serum and colon contents,and the relative abundance of Faecalibaculum was negatively correlated with serum THDCA(p<0.05).5.At 16 weeks of intervention,compared with the MCD group,LSS and HSS groups improved terminal ileum FXR,FGF15 expression,and OCA,LSS,MSS and HSS groups improved ASBT expression(p<0.05).Conclusion1.SS-A2 preventive intervention in MCD induced NAFLD mice for 4 or 16 weeks,can improve the liver hepatic steatosis,lobular inflammation,ballooning and liver fibrosis,and reduce the liver TG,serum ALT and TNF-?,which indicate that SS-A2 have the bioactivity of preventing NAFLD.2.SS-A2 preventive intervention in MCD induced NAFLD mice for 4 or 16 weeks,regulate gut microbiota of NAFLD mice varied with intervention time and intervention dose.At 4 weeks of preventive intervention,can reduce the relative abundance of Erysipelotrichaceae,Candidatus Saccharimonas and Faecalibaculum,and increase the relative abundance of Desulfovibrionaceae.At 16 weeks of preventive intervention,can increase the relative abundance of Bacteroidetes,Desulfovibrionaceae,Desulfovibrio and unidentified-Costridiales(at family and genus levels),and reduce the relative abundance of Actinobacteria,Firmicutes,Erysipelotrichaceae,Lactobacillaceae,Faecalibaculum and Lactobacillus which indicates that SS-A2 can prevent NAFLD by regulating gut microbiota.3.SS-A2 preventive intervention in MCD induced NAFLD mice for 16 weeks,can reduce the accumulation of serum bile acid,and increase the levels of liver and serum THDCA and excretion of bile acids in the intestine.Faecalibaculum was negatively correlated with serum THDCA.SS-A2 can improve terminal ileum FXR,FGF15 and ASBT expression.It is suggested that SS-A2 can play a biological role in preventing NAFLD by regulating LBGMA.