Effects And Mechanisms Of Hepatic Lipid Regulation In Mice By Lactobacillus Plantarum

The incidence of non-alcoholic fatty liver disease is increasing year by year with a trend of younger age.It has been reported that the prevalence rate of adults in China is currently 15%-25%,which has become the largest liver disease beyond viral hepatitis.So far,non-alcoholic fatty liver is still short of some specific drugs.Although there are some drugs that can protect the liver,reduce enzyme and blood lipid,most of which are accompanied by adverse side effects.Safer and healthier non-drug therapies have been proposed successively,including probiotics.Lactobacillus plantarum have been reported that they can promote intestinal flora balance,alleviate metabolic syndrome,immune regulation and other functions,but the mechanism of action of L.plantarum on alleviating hyperlipidemia,obesity,fatty liver and other diseases is not clear,and there are also strains specific.Secondly,little is known about the potential regulatory mechanism of intestinal flora homeostasis to host metabolism,as well as the relationship among intestinal flora,intestinal flora metabolites and liver metabolites.To study the regulation and mechanism of hepatic lipid regulation in mice by L.plantarum,the following six experiments were studied.In experiment one,11 strains of L.plantarum were evaluated for their tolerance to gastric acid and bile salt,cholesterol lowering and adhesion in vitro.Two strains of L.plantarum FRT4 and FRT10 with good lipid lowering and adhesion were selected comprehensively.In experiment two,the effects of FRT4 and FRT10 on lipid lowering were verified and the mechanism was studied at the cellular level.The lipid-degeneration model of Hep G2 cells was constructed with oleic acid.The results showed both FRT4 and FRT10 intervention significantly reduced the triglyceride content in hepatocytes,and down-regulated the expression of lipid synthesis genes SREBP-1,ACC and FAS,as well as inflammatory factors TNF-? and IL-6 at the m RNA level.In experiment three,it is not clear whether FRT4 and FRT10 can alleviate lipid accumulation in vivo.Therefore,FRT4 and FTR10 were used as the starting strains to conduct further studies on model animals.Nonalcoholic fatty liver disease(NAFLD)was modeled in mice by a high-fat diet for 8 weeks.The experiment was divided into normal control group(CT),high-fat diet control group(HF),FRT4 high dose treatment group(HF4H,5×109 CFU/m L),FRT4 lower dose treatment group(HF4L,5×108 CFU/m L),FRT10 high dose treatment group(HF10H,5×109 CFU/m L),and FRT10 lower dose treatment group(HF10L,5×108 CFU/m L).The mice were gavaged the corresponding dose of L.plantarum daily for 8 weeks.The results showed that FRT4 and FRT10 intervention significantly reduced the body weight,body weight gain,liver weight,fat weight,serum cholesterol,triglyceride,ALT level in liver(p < 0.05)and liver histopathology was improved.Further mechanism showed that,compared with the HF group,FRT4 and FRT10 could effectively up-regulate CPT1? involved in ?-oxidation and down-regulate SREBP-1,DGAT1 involved in lipid synthesis on the m RNA expression level.Besides,FRT4 and FRT10 reduced the m RNA expression level of endotoxin receptor TLR4 and inflammatory factor IL-6 in the liver,and increased the tight junction protein gene ZO-1,Occludin,and Claudin 1 in colon.In addition,FRT10 increased the expression of the bile acid synthesis rate-limiting enzyme CYP7A1,which could alleviate obesity by activating the PPAR?/CPT1? pathway in mice.These results suggest that FRT4 and FRT10 can be used as a single probiotic agent for dietary intervention to alleviate obesity.In experiment four,for the changes of hepatic lipid metabolites,the liver tissues were analyzed by using metabolomics.The results showed that compared with the normal diet group,the high-fat diet resulted in significantly higher liver levels of the intermediate metabolites of glycerophosphocholine,choline,glycerophosphocholine,phosphocholine,CDP-choline,sn-glycerol 3-phosphoethanolamine and ophosphoethanolamine(p < 0.05).After FRT4 intervention,the levels of choline,glycerophosphocholine,phosphocholine and o-phosphoethanolamine were significantly reduced(p < 0.05).Metabolic pathway analysis showed that glycerophospholipid metabolism is a potential target pathway closely involved in the FRT4 alleviating the NAFLD.FRT10 has been shown to play a major role in glycerophospholipid metabolism,galactose metabolism,and protein digestion and absorption pathways in alleviating obesity.In experiment five,in order to explore whether FRT4 and FRT10 alleviated obeisity are related to the regulation of intestinal microbes,16 S r RNA gene high-throughput sequencing was performed on cecal contents in this study.The results showed that,compared with the normal control group,high-fat diet caused significant changes in intestinal microbes in Firmicutes and Bacteroides(p < 0.05).Both FRT4 and FRT10 can significantly change the composition of intestinal microorganisms(p < 0.05).FRT4 induced different changes in intestinal flora structure and key phylotypes.Compared with high-fat diet group,FRT4 intervention significantly increased the relative abundance of Alistipes,Intestimonas,Butyicicoccus,and Butyricimonas(p < 0.05).The relative abundance of Oscillibacter and Lachnoclostridium decreased significantly(p < 0.05).Spearman's correlation analysis of intestinal microorganisms and liver metabolites showed that some specific genera were significantly correlated with glycerolphospholipids metabolites(p < 0.05).FRT10 significantly regulated intestinal flora dysregulation induced by high-fat diet,significantly increased Butyricicoccus,Butyricimonas,Odoribacter and Alistipes,and significantly lowered Desulfovibrionaceae,Roseburia and Lachnoclostridium.These results suggest that both FRT4 and FRT10 can ameliorate high-fat diet-induced intestinal flora,thereby alleviating obesity and lipid abnormalities in the liver.In experiment six,intestinal metabolites play a key role in the “gut-liver” axis.To explore changes in intestinal microbial metabolites by intestinal flora,Cecal metabolome was performed and showed that the metabolic substances mainly involved in the glycerophospholipid metabolism,fatty acid metabolism,primary bile acid synthesis and secretion of bile,among them,the high-fat diet induced lipid,small peptide increased significantly(p < 0.05).After FRT4 and FRT10 intervention,some lipid and small peptide significantly reduced(p < 0.05).The correlation analysis of intestinal differential metabolites and bacteria flora showed that Lyso PC(18:1(9Z))was positively correlated with Dorea and Enterorhabdus(p < 0.05),and negatively correlated with Bacteroides,Bilophila,Butyricimonas,and Intestinimonas(p < 0.05).These results showed that the intestinal flora structure and diversity were significantly changed,resulting in changes in intestinal contents.In addition,the analysis of gut metabolites and hepatic metabolites related pathways showed that L.plantarum played a major role in lipid lowering in glycerophospholipid metabolism pathway.In conclusion,this research comprehensively selected two L.plantarum FRT4 and FRT10 through the cholesterol degration,adhesion and the evaluation experiments on lipid degeneration in Hep G2 cells.Hepatic steatosis model in mice was constructed by high-fat diet.Both FRT4 and FRT10 could alleviate obesity and lipid abnormalities caused by high-fat diet.From the growth performance,physiological and biochemical indexes,liver histology,lipid-related genes,liver metabolite composition,gut microbes structure and the composition of gut microbes metabolites analysis,the results showed that L.plantarum intervention caused significant changes in the diversity and structure of gut microbiota,thereby caused significant changes in the intestinal contents,and then affected the lipid metabolism in the liver.From the study,we can have a more comprehensive understanding of the lipid-lowering mechanism of L.plantarum.It is of great significance to prevent and treat fatty liver.