Studies On The Hypolipidemic Effects Of Ginsenoside Compound K By Enhancing Lipophagy And Lipase ATGL

Imbalances between lipid synthesis and lipolysis can lead to lipid accumulation and dyslipidemia,contributes to the development of obesity,diabetes and other metabolism-related diseases.Lipid-lowering drugs have been widely used to treat hyperlipidemia,but these drugs are all single targeted drugs,also have ther limitations.Therefore,the development of safe and efficient lipid-lowering drugs had become the focus of the global biopharmaceutical industry.Ginsenosides are the major active compounds of ginseng.To date,more than 60 kinds of ginsenoside monomers have been discovered structures.In our previous study,we identified ginsenoside Rg2 as a novel autophagy inducer that could regulate HFD-induced metabolic diseases.Here,we tested the influence of sugar moiety in ginsenosides on their activities,hoping to identify the structure-activity relationship of ginsenosides in regulation of lipid metabolism.Next,we explored the mechansim of ginsenosides CK in reducing body weight and blood lipid.The detail contents and results are as follows:In this thesis,we explored the roles of sugar moiety in ginsenosides on lipid metabolism.We used ginsenosides Rb1,GXVII,GLXXV,CK and PPD,which containing the same aglycon and different sugar moiety.We treated hepatocytes with high concentrations of fatty acid to evaluate its ability in improving insulin resistance,and we also subjected obese mice to compare its regulation in lipid metabolism.Taken together,these results indicated that the smaller the number of sugar moiety,the stronger the ginsenosides affected the hypolipidemic ability,but sugar moiety was essential.Ginsenoside CK had the most significantly effect in reducing body weight and blood lipid.The hypolipidemic activity and molecular mechanism of ginsenoside CK in vivo were explored.We analyzed the effect of CK on body weight,fat compostion and the metabolic activity in obese mice.Compared with orlistat,CK significantly reduced the body weight and blood lipid levels,affected fat deposition.CK treatment increased energy metabolism,whereas it did not promote carbohydrate consumption.LC-MS mass spectrometry,proteomics,western blotting and qPCR were used to screen differential proteins in CK-treated liver.Data showed that CK downregulated fatty acid synthesis-related factors,upregulated fatty acid oxidation-related factors,stimulated cAMP/PKA/HSL signaling pathway and enhanced lipases(ATGL and HSL)expressions to promote lipolysis.Meanwhile,CK activated AMPK rather than mTOR signaling pathways.Furthered studied on the mechanism of CK-stimulated autophagy.CK induced autophagosomes appeared in the early stage and continued to the autophagosome degradation in the late stage.We observed CK produced LC3 puncta co-localize with FIP200,WIPI2 and Eva1 A by confocal microscopy,which initiated autophagy from phagophore formation,autophagosomes maturation to autophagolysosomes formation.Further western blotting assay showed that CK activated autophagy in an AMPK/ULK1-dependent and mTOR-independent manner.We treated hepatocytes with high concentrations of FA to mimic the conditions observed in the obese liver mice.CK increased the co-localization of lipid dropets and autophagosomes(LC3)as well as lysosomes(LAMP1).The results showed that ginsenoside CK drived specific lipophagy,reduced the number of fat droplets.However,we observed that CK enhanced the expression of lipolytic factors,while the ATG7 siRNA did not affect the expression of lipase induced by CK.Therefore,CK could enhance lipolysis by two independent processes,including the induction of lipophagy and enhancement of lipase activity.Finally,we needed to reveal the target of CK on activating lipophagy and enhancing lipases expressions.Quantitative analysis with UPLC-MS to analyze the transport of CK in hepatocytes.The content of CK entered the cells increased with the treatment time extension.The pull-down,fluorescently-labeled dexamethasone competitive and immunofluorescence assays were used to explore the target molecule of ginsenoside CK.We confirmed that CK could strongly bind to GR and increase the nuclear translocation of the GR.The promoter-luciferase dual reporter vector and ChIP-pcr were used to explore the active domain of lipase ATGL.CK promoted binding of GR at the-430 ～-310 bp region of ATGL promoter upstream.Furthermore,treatment with GR siRNA inhibited the activation of lipophagy lipase activity.In summary,we found that CK promoted binding of GR at the lipase ATGL promoter,enhanced lipophagy and ATGL activity,promoted lipolysis,reduced fat accumulation.Therefore,ginsenoside CK serve as natural autophagy regulators for reducing fat content and weight.Our research data will provide a candidate molecule for improving obesity,diabetes and other lipid metabolism related diseases therapy.