Effects And Mechanisms Of Cholesterol-lowering Probiotics On Intestinal Barrier Function And Gut Microbiota In Mice With Non-alcoholic Fatty Liver Disease

Background and ObjectivesNon-alcoholic fatty liver disease（NAFLD）is one of the most important causes of liver disease worldwide and will probably emerge as the leading cause of end-stage liver disease in the coming decades^[1,2].In 2018,it is estimated that about 25%of the world population has NAFLD^[3].NAFLD is the hepatic manifestation of metabolic syndrome and is highly prevalent in obese and diabetic subjects^[4].NAFLD represents a disease spectrum,ranging from simple steatosis in the absence of inflammation to nonalcoholic steatohepatitis（NASH）,liver cirrhosis and hepatocarcinoma（HCC）^[5].With the improvement of the life quality,the incidence of nonalcoholic fatty liver disease has increased year by year in China.Currently,there is no approved drug regimen to treat NASH.Diet and lifestyle interventions are mainstay in the treatment of NAFLD,and weight-loss≥7%confers histological improvements of NASH^[6].However,lifestyle interventions are notoriously difficult to maintain^[7].A large mount of evidence suggests that gut dysbiosis can cause intestinal inflammation as well as chronic inflammatory liver disease.To be specific,a close anatomical and functional link between gastrointestinal tract and the liver was naturally existed via the hepatic portal system,which is termed as gut-liver axis.It permits gut bacteria and their metabolic products to transfer into the liver on account of this direct relationship between the two organs and accelerates the progress of NAFLD^[8].Fat in diet,couple with other factors,may give rise to gut dysbiosis,as well as intestinal barrier injury and increased intestinal permeability thus resulting the gut bacteria and bacteria-derived products translocation into the mesenteric portal bloodstream.Subsequently the inflammatory cytokines were induced to produce,which participate in the development of NAFLD.In our previous study,cholesterol-lowering probiotics were found to reduce liver lipid deposition,inhibit liver inflammation and improve systemic metabolic status in NAFLD rats^[9].We hypothesize that cholesterol-lowering probiotics ameliorate NAFLD via gut-liver cross-talk:the cholesterol-lowering probiotics reduce gut inflammation,augments intestinal barrier function;decreases intestinal fat transport,and modulates gut microbiota.MethodsTwenty-four male Ldlr^-/-mice were randomly divided into three groupsincluding control group,model group and probiotics intervention group.Mice in the control group received normal diet.The mice model of NAFLD was established by feeding mice with chronic high fat diet（45%of calories derived from fat diet）for 12weeks.Mice in the probiotics intervention group were given high fat diet together with cholesterol-lowering probiotics through oral gavage.The expression of liver and intestinal inflammatory genes and liver cholesterol synthesis gene was determined by q-PCR.Western blot assay was used to detect the protein expression of intestinal tight junction and HMGCR.Tissue inflammation was evaluated by HE staining.The gut microbiota was analyzed by 16S rRNA gene sequencing.Results1.Changes in body weight,fasting blood glucose,glucose tolerance,and blood lipid levels in mice:Compared with the control group,the weight of the model group increased significantly（P<0.01）.Compared with the model group,the intervention group showed reduced the weight gain induced by the high-fat diet（P<0.05）.Compared with the control group,the fasting blood glucose of the model group increased significantly,the glucose tolerance impaired（P<0.01）.Compared with the model group,the fasting blood glucose of the mice decreased significantly after the intervention of cholesterol-lowering probiotics,and the glucose tolerance improved（P<0.01）.Compared with the control group,the levels of total cholesterol and low-density lipoprotein were significantly increased in the model group（P<0.01）.Compared with the model group,the levels of total cholesterol and low-density lipoprotein in the mice were significantly decreased after the cholesterol-lowering probiotics intervention（P<0.01）.2.Liver and intestinal pathological changes in mice:Compared with the control group,HE staining showed that the liver cells were disordered in the model group,the inflammatory cells infiltrated significantly,and a large number of fat droplets were deposited.After the cholesterol-lowering probiotics intervention,liver inflammatory cell infiltration and hepatic steatosis is reduced.Intestinal HE staining showed small intestine villus injury in the model group,and cholesterol-lowering probiotics could improve the injury after intervention.3.HMGCR expression level in mouse liver:There was no significant difference in liver HMGCR mRNA expression between the model group and the control group.Compared with the model group,the expression of HMGCR mRNA in the liver of the intervention group was significantly decreased（P<0.01）.There was no significant difference in liver HMGCR protein expression between the model group and the control group;Compared with the model group,the liver HMGCR protein expression level in the intervention group was significantly decreased（P<0.01）.4.Compared with the control group,the expression levels of liver TLR4mRNA（P<0.01）,CCL2 mRNA（P<0.05）,CCL4 mRNA（P<0.01）,IL1αmRNA（P<0.01）and TNFαmRNA（P<0.01）were significantly higher in the model group;compared with the model group,TLR4 mRNA（P<0.05）,CCL2 mRNA（P<0.05）,CCL4 mRNA（P<0.01）,IL-1αmRNA（P<0.01）,TNFαmRNA（P<0.01）expression levels in the liver of the intervention group were significantly reduced.Compared with the control group,the expression levels of ileum CCL2 mRNA（P<0.05）,IL-1βmRNA（P<0.01）and IL6 mRNA（P<0.05）in the model group were significantly increased;compared with the model group,the expression levels of ileum CCL2 mRNA（P<0.05）,IL1βmRNA（P<0.01）,and IL6 mRNA（P<0.05）were significantly decreased in the intervention group.5.Compared with the control group,the expression of the ileal tight junction proteins Claudin1（P<0.01）,Claudin3（P<0.05）,and Zo1（P<0.01）were significantly decreased in the model group.Compared with the model group,the expression ofClaudin1(P<0.05,Claudin3（P<0.05）and Zo1（P<0.01）were significantly increased in the intervention group.6.Changes in the intestinal microbiota of mice:Compared with the control group,the Shannon index of the model group decreased（P<0.01）;compared with the model group,the Shannon index of the intervention group increased（P<0.05）.PCA analysis of mouse intestinal microbiota showed that the intestinal microbiota composition of the mice in each treatment group was significantly aggregated,and the intestinal microbiota structure of the probiotics intervention group was more similar to that of the control group.Analysis of species composition in intestinal microbiota of mice showed that at the level of the Phylum,the model group showed a reduction in the proportion of Bacteroidetes and proteobacteria,and an increased level of Firmicutes,the ratio of Bacteroidetes/Firmicutes in the model group was decreased compared with that in the control group,while cholesterol-lowering probiotics partly restored the ratio.ConclusionsCholesterol-lowering probiotics may improve the liver and intestinal inflammation by reducing liver cholesterol synthesis,improvement of intestinal mucosal barrier function,and regulate intestinal microbiota,thereby improving the NAFLD in mice.