| Background:Non-alcoholic fatty liver disease(NAFLD)refers to a chronic liver disease characterized by diffuse fat infiltration and fat accumulation in liver parenchyma cells without a history of excessive drinking.NAFLD is often accompanied by obesity and insulin resistance,but not all obese patients will develop NAFLD,mainly due to the intestinal flora plays an important role.Probiotics and traditional Chinese medicine have been reported to regulate intestinal microecological balance and reduce the body’s cholesterol level.Salvia miltiorrhiza polysaccharide is a kind of natural polymer compounds extracted from traditional Chinese medicine Salvia miltiorrhiza,which has a wide range of biological functions including anti-inflammation,immunomodulation,anti-oxidation and anti-aging.Probiotics combined with plant polysaccharides have been reported to treat NAFLD,but there has been no report of salvia polysaccharides combined with probiotics on NAFLD,and it is unclear whether salvia polysaccharides can enhance the effect of probiotics on NAFLD.Objective:1.The purpose of this study was to screen for probiotics that can efficiently reduce cholesterol and triglyceride.2.To investigate the effect of probiotics combined with salvia miltiorrhiza polysaccharide on lipid metabolism disorder and liver pathological changes in NAFLD mice.3.To investigate the effect of probiotics combined with salvia miltiorrhiza polysaccharide on inflammation,gut microbiota and intestinal barrier integrity in NAFLD mice.Methods:1.The bacterial strains were isolated from fecal samples of 20 healthy people.Ammonium iron sulfate method and ELISA kit were used to detect the efficiency of reducing cholesterol and TG,respectively,and the intestinal strains resistant to digestive tract were screened out.Then,the screened strains were sequenced for 16S rDNA and compared with BLAST on NCBI to determine the type of strain.2.SPF C57BL/6N mice were randomly divided into normal diet(ND),high fat diet(HFD),probiotics treatment(L.plantarum+B.bifidum,LB)and probiotics combined with salvia miltiorrhiza polysaccharide treatment groups(L.plantarum++50 mg/kg salvia miltiorrhiza polysaccharide,LBM).3.After the experiment is over,the body weight,body length and liver weight of the mice were measured,and the Lee’s index and liver index were calculated.The serum levels of TG,total cholesterol(TC),high density lipoprotein cholesterol(HDL-c),low density lipoprotein cholesterol(LDL-c),glutamic oxaloacetic transaminase(AST)and glutamic pyruvic transaminase(ALT)were measured by automatic biochemical analyzer.4.The pathological changes of liver and intestinal mucosa in each group were observed by Hematoxylin and eosin(HE)staining.5.ELISA and qRT-PCR were used to detect serum and liver TNF-α and other inflammatory factors.6.qRT-PCR and Western blot were used to detect the expression levels of lipid metabolism-related factors,TLR4/NF-κB,PI3K/Akt/mTOR.7.The fecal samples of mice were sequenced by 16S rDNA sequencing technique,and the composition of intestinal flora and the difference of species abundance in each group were analyzed.8.The concentration of short-chain fatty acids(include butyric acid and acetic acid)in stool samples of each group mice by High Performance Liquid Chromatography(HPLC)Results:1.The results showed that Bifidobacterium bifidum and Lactobacillus plantarum had the ability to decrease TC and TG,and had strong acid resistance,anti-bile salt,anti-pepsin,antitrypsin and anti-cell adhesion properties.2.Compared with ND group,the Lee’s index,liver index,TG,TC,LDL-c,ALT,AST,HDL-c,HOMA-IR,LPS,TNF-α,IL-6 and IL-1β were significantly increased in HFD group,while the LB and LBM could significantly decrease these parameters.3.Obvious swelling,steatosis and inflammatory cell infiltration were observed in HFD group.LB and LBM intervention can effectively improve the hepatic steatosis and inflammatory cell infiltration,and the effect of IBM intervention is better.4.Compared with ND group,the mRNA expression levels of lipid metabolic factors in HFD group were significantly decreased.Both LB and LBM intervention could block this inhibition.5.Compared with ND group,the mRNA and protein expression levels of TLR4 and NF-κB in liver of HFD group were significantly increased.Compared with HFD group,the mRNA and protein expression levels of TLR4 and NF-κB in LB and LBM groups was decreased,and the expression of TLR4 and NF-κB in LBM group was lower than that in LB group.6.PI3K/Akt/mTOR pathways were activated in HFD group.The phosphorylation levels of PI3K,Akt and mTOR proteins were significantly reduced in LB and LBM groups.The improvement effect of the LBM group is more significant than that of the LB group.7.Compared with ND group,the abundance of Bacteroides was decreased,while the abundance of Firmicutes and Proteobacteria was increased in the intestinal tract of HFD-fed group.LB and LBM intervention reduced the relative abundance of Proteus and Oscillospira,and increased the abundance of Bacteroides,Lactobacillus and Bifidobacterium,and the intervention effect was better in the LBM group.8.Compared with ND group,the concentration of butyric acid and acetic acid was decreased in mice intestinal tract of HFD group.Compared with HFD group,the the concentration of butyric acid and acetic acid in LB and LBM groups was inreased,.However,the concentration of butyric acid and acetic acid in LBM group was more than that in LB group.9.Compared with the HFD group,the mRNA and protein expression levels of ZO-1 and Occludin were increased in the LB and LBM groups,and these changes were more obvious in the LBM group.Conclusion:Combination of the probiotics and salvia miltiorrhiza polysaccharide can regulate the liver function,and decreased serum levels of lipids in mice of NAFLD.These beneficial effects of combination were related to the inhibition of TLR4/NF-κB and PI3K/Akt/mTOR pathway in the liver,the regulation of intestinal flora composition and the improvement of intestinal mucosal barrier function. |
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