| Objective: 1.To establish the cell model of non-alcoholic fatty liver in vitro and to find the best model conditions;2.To explore the lipid-lowering effect of terpenoids of Alismatis rhizoma on fatty liver and the effect on lipid-lowering gene expression in cells,so as to further clarify the lipid-lowering mechanism of Alismatis rhizoma;3.To establish a chromatographic method for the determination of four monomers in Alismatis rhizoma;4.To explore the influence of Lactobacillus,the dominant intestinal strain,on the content changes of components of Alismatis rhizoma after fermentation,and to find a method to improve the effective terpenoids of Alismatis rhizoma.Methods: 1.In vitro cell culture,sodium palmitate(PA)and sodium oleate(OA)were used to simulate human lipid supersaturation phenomenon,and lipid deposition was observed by detecting intracellular triglyceride content and total cholesterol content and oil red staining.Hep G2 fatty liver cell model was established.2.To study the lipidogenic effects of the main terpenoid monomers 24-acetazenol A,23-acetazenol C and epoxisenol on NAFLD cells,and to detect the effects of the monomers on the expression of Insig1 and Nr1D1 in cells by Western blotting(WB).3.Chromatography was used to determine the content change of Alismatis rhizoma.4.The effects of fermentation time,the addition amount of fermentation strains and the ratio of fermentation material to liquid on the content of terpenoids in Alismatis rhizoma were investigated by single factors.Results: 1.The optimal conditions for the establishment of fatty liver model were as follows:1 m M PAOA(1:2)mixture culture for 24 h with low inhibition rate of cell growth and ideal lipid accumulation;2.Compared with the model group and the normal control group,the contents of intracellular TG,TC,ALT and AST were decreased under the treatment of23-acetyliseol C and epoxiseol.The lipid-lowering effect of 24-acetyliseol A was not obvious within the dose range used in the experiment.3.The results of WB development and gray value analysis showed that under the same modeling conditions,compared with the model group and the normal control group,the expression of Insig1 and the expression of Nr1D1 were increased by 23-acetyliseol C and epoxiseol,thus playing a lipid-lowering role.4.Influence of single factor on fermentation: when the fermentation day was 7 d,the content of24-acetylalismatol A increased significantly,and the ratio of solid to liquid 1: Under the condition of 10,the content of 24-acetazenol A increased significantly,and the content of24-acetazenol A and 24-acetazenol A increased significantly when the strain added amount was 5 m L.The dried fermented powder of Alismatis rhizoma was detected by chromatography,and it was found that 24-acetazenol A and epoxidazenol A increased the most.Conclusion: 1.Fatty liver cell model can be successfully established by 1 m M PAOA(1:2)mixture culture for 24 h,in which OA plays a protective role;2.Alisma monomer 23-acetyl aliseol C and epoxy aliseol can play a lipid-lowering effect on cells and prevent the infiltration and invasion of cells by lipid supersaturation;3.Alismatis rhizoma monomer can increase the expression of Insig1,an important gene in the lipid-lowering pathway,and decrease the expression of Nr1D1,which further proves that the lipid-lowering effect of Alisma alisma is closely related to these two gene targets.4.The most obvious effects of Lactobacillus fermentation experiment on terpenoids in Alismatis rhizoma were 24-acetyl Alismatol A and epoxide Alismatol.Lactobacillus,as the dominant intestinal strain,had A certain effect on terpenoids in Alismatis rhizoma. |
