Effect Of Bacillus Amyloliquefaciens SC06 On Gut Microbiota And Bile Acid Metabolism In Ob/Ob Mice

Backgrouds:Numerous studies have been confirmed the links between obesity,intestinal flora and bile acid metabolism.In recent years,evidence has accumulated that the gut microbiota is an important environmental factor contributing to obesity.Bile acid(BA)is synthesized from cholesterol in the liver and stored in the gallbladder duct,and then secreted into the small intestine to to facilitate fat digestion and absorption,meanwhile their antibacterial activity exerted strong selective forces on for the intestinal microbiota,while BA pool can be also changed by the intestinal flora through enzymes.Probiotics are defined as ’live microorganisms that,when administered in adequate amounts,confer a health benefit on the host’.Most of the studies carried in experimental animals have clearly demonstrated that administration of probiotics can be effective in the prevention and treatment of obesity.And,specific strains belonging to Lactobacillus and Bifidobacterium species have been widely used as probiotic treatment in well-established-animal models of obesity.However,apart from them,Bacillus species(Bacillus spp.)represented another potential probiotic in dealing with obesity,but few reports focus on regulating intestinal flora structure and bile acid metabolism.In the current study,we explored the effects of Bacillus amyloliquefaciens SC06 on male ob/ob(Leptin-Deficiency)C57BL/6 mice by using 16S rRNA Sequencing,LC-MS/MS analysis and Spearman correlation analysis.Methods:24 male C57BL/6J mice and ob/ob mice on the C57BL/6J background(5 weeks old,n=8 per group)were purchased from Slac Laboratory Animal Co.,Ltd.(Shanghai,China)and used 1 week after receipt to stabilize all metabolic conditions.Thereafter,animals were divided into three groups,wild type C57BL/6J mice were fed with normal diet regarded as normal control groups(NC group),ob/ob mice were fed a normal diet(OBC group)and a normal diet gavaged with SC06(OBB group,2×10^8 cfu/day)for 8 weeks.Results:1、SC06 ameliorated fat mass percentage,hepatic steatosis and liver lipid metabolism disorders in ob/ob mice.Compared with the NC group,OBC group had a significant increase in body weight,daily food intake and liver and fat mass weight,and exhibited the markedly TG increase and HDL-C decrease in secrum.Examination of hematoxylin-eosin(H&E)-stained sections of white adipose tissue(WAT)indicated a larger size of adipocytes in ob/ob mice than NC group;moreover,liver HE and oil-red O sections from mice of OBCgroup exhibited visible intracellular vacuolization and marked lipid accumulation.In the meantime,compared with the OBC group,the significant decreased body weight and fat mass in OBB group were observed at the 8th week.Besides,SC06 treatment reduced LDL-C and enhanced HDL-C notably in ob/ob mice liver,as well as the degree of hepatic steatosis.2、SC06 treatment showed apparent changes in gut microbial ecology.Compared with the OBC group,OBB group showed a significantly lower a diversity index.The principal coordinates analysis(PCoA)analysis showed that three groups were clearly clustered into separate classes.The calculated relative abundance of bacteria at the phylum level showed that compared with NC group,the significant increased relative abundance of Bacteroidetes,Patescibacteria and Epsilonbacteraeota,and the decreased Firmicutes,Proteobacteria and Actinobacteria was observed in OBC group,while only two phylum levels in ob/ob mice(Proteobacteria and Patescibacteria)were notably altered by SC06 administration.LEfSe analysis showed that at the genus level,compared with the NC group,there were 30 bacteria showed higher abundance in the OBC group,while 35 species of bacteria were less abundant.What’s more,19 species were higher in the OBC group,while 22 species were higher in the OBB group.3、Effects of SC06 treatment on cecal metabolite profiles in ob/ob miceIn total,165 metabolites increased significantly in OBC group,while 16 metabolites reduced significantly compared with NC group;metabolites were significantly increased in OBC group,including 1,2,3-Benzenetriol,L-Malic acid,2(1H)-Pyridinone,2’-Deoxyuridine,Xanthine,Larixinic Acid and Cholic acid et.al,while 3,4-Dihydroxybenzoic acid and trans-3-Coumaric acid were decreased.A volcano plot showed the metabolites differentially expressed between the OBC and OBB groups,28 metabolites increased and 75 metabolites decreased significantly and in the OBB group.Metabolites enriched in OBB group,including 1-Stearoyl-2-arachidonoyl-sn-glycerol and L-Saccharopine et.al,while 2(1H)-Pyridinone,2’-Deoxyuridine,Xanthine,Larixinic Acid and Cholic acid et.al decreased significantly by SC06 treatment.4、Correlation analysis between the cecal microbiota and metabolites As it showed,those bacterial genera members increased in OBB group were always negatively correlated against the metabolite decreased,and conversely bacteria decreased always exhibited a positive correlation with metabolites.These results suggest close association between microbial dysbiosis in OBB group resulting in subsequent changes to metabolite profiles.5、Effects of SC06 treatment on bile acids metabolism in ob/ob miceCompared with NC group,ob/ob mice groups have reduced bile acid metabolism.In the meantim,SC06 downregulated bile acid synthesis(CYP7A1 and CYP8B1)and export(BSEP)and reabsorption(Otap1b2 and Otaplal)in liver and increase ileum reabsorption(OSTα,OSTβ and IBABP)into the blood in obese mice by activating FXR-SHP/FGF15 signaling.These results suggested the existence of an altered negative feedback loop of bile acid synthesis from the gut to the hepatocytes,which was regulated by FXR,and further supporting a link between gut microbiota,FXR and bile acid metabolism.Conclusions:SC06 ameliorated fat mass percentage,hepatic steatosis and liver lipid metabolism disorders by shaping the gut microbial composition,changing metabolites and regulating bile acid metabolism depending on the FXR signaling pathway in male ob/ob mice.