Effects Of Cholesterol-lowering Probiotics On Gut Microbiota And Bile Acid Pool In Mice With Non-alcoholic Fatty Liver Disease

ObjectivesNon-alcoholic fatty liver disease（NAFLD）is a chronic liver disease caused by excessive drinking and other clear liver injury factors such as viruses and drugs.The main pathological feature is lipid deposition in liver cells.NAFLD includes non-alcoholic fatty liver（NAFL）,non-alcoholic steatohepatitis（NASH）,non-alcoholic steatohepatitis fibrosis,cirrhosis,and partial progress to hepatocellular carcinoma.The global prevalence of NAFLD is 20%-30%,and it gradually brings serious medical burden to the society.With the improvement of economic level and the change of lifestyle,the prevalence of NAFLD in China has increased from 18%to29%in the past decade.The diagnosis and treatment of NAFLD has been increasingly attracted attention,among which probiotics has also become an important treatment method.Our research group previously studied the lipid-lowering effects of probiotics DM9054（Lactobacillus rhamnosus GG）and86066（Lactobacillus plantarum,WCSF1）,they could improve intestinal barrier function,regulate FXR pathway,liver bile acid metabolism and transport.However,the effects of lipid-lowering probiotics on gut microbiota and bile acid pool in NAFLD animal models constructed by FXR^-/-mice are still unclear.Therefore,this study is intended to explore its further association and mechanism.Methods（1）The experimental mice were previously generated in C57BL/6 background using CRISPR/Cas9 gene editing technology.The homozygous FXR^-/-mice were obtained by crossing FXR^+/-mice with C57BL/6 mice.The genotype of mice was identified by agarose gel electrophoresis and western blot.（2）Thirty male C57BL/6（WT）mice and 27 FXR^-/-mice were randomly divided into three groups:normal diet group,high-fat diet group and high-fat diet combined with lipid-lowering probiotics gavage group,namely ND-WT,ND-FXR^-/-,HFD-WT,HFD-FXR^-/-,HFD+P-WT and HFD+P-FXR^-/-.The normal diet group and the high-fat diet group were given normal saline gavage.Lipid-lowering probiotics are Lactobacillus rhamnose LGG and Lactobacillus plantarum WCFS1.（3）All mice were sacrificed 16 weeks after feeding in SPF animal room.Intraperitoneal（i.p.）glucose tolerance test（IPGTT）was performed by i.p.injection of glucose solution one week before the execution.The weight gain and glucose tolerance of mice in each group were compared.（4）The liver function indexes;serum lipids and serum total bile acid levels of mice were determined by automatic biochemical analyzer;HE staining was performed to evaluate the changes.（5）Analyzed the changes of gut microbiota and the metabolism of bile acid pool by sequencing 16S r DNA genes and bile acids.Results1.Mice gene identification:Agarose gel electrophoresis showed that 27 gene knockout mice’s genotypes were FXR^-/-;Western Blot showed that FXR protein were expressed at extremely low levels in 27 knockout mice.2.Body weight change and glucose tolerance level of mice:compared with ND-WT and ND-FXR^-/-groups,the gain of HFD-WT and HFD-FXR^-/-groups was significant,with statistical difference（P<0.05）.Compared with HFD-WT and HFD-FXR^-/-groups,the weight of HFD+P-WT and HFD+P-FXR^-/-groups showed a significant downward（P<0.01）.Compared with ND-WT and ND-FXR^-/-group,the blood glucose of HFD-WT and HFD-FXR^-/-group was significantly increased,and the glucose tolerance was impaired,with statistical difference（P<0.05）.Compared with HFD-WT and HFD-FXR^-/-groups,the blood glucose of HFD+P-WT and HFD+P-FXR^-/-groups was significantly decreased,and the glucose tolerance level tended to be normal,with statistical significance（P<0.05）.3.Serum biochemical indicators and HE staining conditions:Compared with ND-WT and ND-FXR^-/-groups,the levels of TC,and TG in HFD-WT and HFD-FXR^-/-groups were obviously increased,the level of LDL-C was significantly reduced（P<0.05）;Compared with HFD-WT and HFD-FXR^-/-groups,The levels of TC,TG,and HDL-C in the HFD+P-WT and HFD+P-FXR^-/-group were significantly reduced,and the level of LDL-C was significantly increased（P<0.05）.Compared with the ND group,the liver structure of the mice in the HFD group was abnormal,fatty degeneration was obvious,and a large number of lipid droplets and a small amount of inflammatory cell infiltration appeared in the pathological section.After the intervention of probiotics gavage,the liver damage was significantly improved,the degree of steatosis,the number of lipid droplets and inflammatory cells were reduced.4.Changes of gut microbiota in mice:Compared with the ND group,the fecal microbial Alpha diversity of mice in the HFD group decreased,while the abundance of Firmicutes and Actinomycetes increased（P<0.01,P<0.05）,the abundance of Bacteroidetes decreased（P<0.05）,and the abundance of Lactobacillus significantly decreased（P<0.01）.Compared with the HFD group,the fecal microbial Alpha diversity of mice in the HFD+P group increased,but the abundance of Firmicutes decreased（P<0.01）.The PCo A of the gut microbiota showed obvious aggregation in each group.The ND group and the HFD+P group tended to be close,indicted that the two groups were similar,while HFD group was farther away from the other two groups.5.Metabolomics analysis of bile acid pool in mice:Compared with the ND group,the fecal bile acid content of mice in the HFD group was increased,with statistical significance（P<0.01）;Compared with the mice in the HFD group,the fecal bile acid content in the HFD+P group decreased,and the difference was statistically significant（P<0.01）.And the proportion of fecal bile acids tended to be normal after probiotics intervention under high fat diet.ConclusionsCompared with normal mice,FXR^-/-mice were more easily induced into the animal model of NAFLD under the condition of high fat diet,and their liver fat infiltration,degrees of gut microbiota disturbance and bile acid metabolism abnormality were more significant.The intervention of cholesterol-lowering probiotics could significantly improve the disorder of glucose and lipid metabolisms and the damage of liver function in NAFLD mice induced by high fat diet.Cholesterol-lowering probiotics may promote the normalization of bile acid pool composition by regulating gut microbiota,thus inhibiting the occurrence and development of NAFLD.