Metabonomic Study On The Prevention And Treatment Of Non-alcoholic Fatty Liver Disease By Astragalus Polysaccharide

Objective: In previous study,we have found and confirmed that Astragalus polysaccharides(APS)significantly palliated the obesity and non-alcoholic fatty liver disease(NAFLD)induced by high-fat diet.This study used a non-targeted metabolomics approach based on GC/TOFMS to research the serum and liver metabolites of APS-intervention mice,to screen and identify differential metabolites associated with APS intervention effects.On this basis,we looked for key differential metabolites that may be related to the prevention and treatment of NAFLD by APS,and further studied the effects of key differential metabolites on hepatocyte lipid accumulation and animal metabolism through in vitro and in vivo experiments.It was further clarified whether it might be involved in the prevention and treatment of NAFLD by APS.Methods:(1)According to body weight,epididymal fat weight and HE staining results,liver triglyceride(TG)level,liver HE staining results from grow for eight weeks feeding with different diet mice(control group feeding normal diet: Con group;High-fat diet group feeding high-fat diet: HFD group;APS group fed high-fat diet added 2%,4%,8% APS: APS-L group,APS-M group,APS-H group),eight mice were selected from each group for serum and liver metabolomics analysis.The results were screened to select for differential metabolites in the serum and liver that were significantly altered by APS.Functional annotation of differential metabolites was further screened to select potential functional metabolites.(2)In vitro functional experiments on the selected potential functional metabolites were performed to obtain2-hydroxybutyric acid(2-HB),a functional metabolite that regulates lipid accumulation in hepatocytes.The content of functional metabolite 2-HB in blood and liver was quantified by HPLC-MS/MS.(3)In vitro induction of adipocyte model on3T3-L1 cells,establishment of lipid increase model on primary hepatocytes,establishment of inflammation model on RAW264.7 cells,establishment of insulin resistance model on Hep G2 cells to explore whether 2-HB plays a regulatory role on cell lipid decomposition,inflammatory factor levels and insulin signaling.(4)50C57BL/6J mice were randomly divided into five groups,10 in the Con group,10 in the HFD group,and 30 in the 2-HB group(the 2-HB group contained three doses,the dose was 25 mg/ml,50 mg/ml and 100 mg/ml).The drug was administered to the mice simultaneously with the high-fat diet.The mice were sacrificed 2 weeks later to measure the blood and liver 2-HB content,serum and liver TG levels,liver and white lipid synthesis and decomposition genes and protein expression.(5)26 C57BL/6J mice were randomly divided into two groups,8 in the normal group and 18 in the high-fat group.In the fifth week,high-fat group added 30% sucrose water to intervene in drinking water,and in the eighth week,they were randomly divided into two groups,glycolipid group and 2-HB group.The 2-HB group was intraperitoneally injected with 2-HB 25 mg/ml on the basis of a combined diet of glycolipids.Glucose tolerance(GTT)was measured after two weeks and tissue was then collected for subsequent assays.Results:(1)APS can significantly regulate the weight gain and liver lipid accumulation in high fat diet mice.By screening the metabolites in serum and liver,three potential functional metabolites,3-Indolepropionic acid,2-HB and β-alanine,which are significantly altered by APS.(2)The functional metabolite 2-HB was screened in vitro study.The results of HPLC-MS/MS showed that the expression of2-HB in blood and liver was the same as non-targeted assay result,that is,APS could significantly reverse the 2-HB levels which were decreased by high fat diet.(3)2-HB shows that it can increase cell viability,promote lipid cell breakdown of fat cells,reduce the level of inflammatory factors,and improve insulin signaling.(4)In animal models of different modes of administration(gavage and intraperitoneal injection),2-HB plays a role in reducing liver and white lipid synthesis proteins and promoting lipolytic proteins.However,there was no significant regulation of mouse phenotype.Short-term intraperitoneal injection and intragastric administration of 2-HB are not suitable for exploring the mode of administration of 2-HB in vivo function.Conclusion:(1)APS significantly changed the serum and liver metabolite profiles of mice fed with high-fat diet;(2)APS intervention significantly increased the level of metabolite 2-HB in serum and liver of mice with high-fat diet;(3)2-HB can significantly palliate lipid accumulation in hepatocytes and significantly improved the expression of lipid-degrading proteins ATGL,HSL and CE1;(4)2-HB significantlydecreased the expression of lipid-synthesis protein FAS and ACC in liver and white lipids of mice fed with glycolipids.It also increases the expression of lipid-degrading protein CE1 in the liver and the expression of lipid-degrading proteins ATGL and HSL in white lipid.