The Mechanism Of Gypenosides Inhibiting PCSK9-LDLR Degradation Was Studied Through The Lipid Phagocytosis Pathway

Objective:Hyperlipidemia is an important risk factor for cardiovascular disease,especially dyslipidemia with elevated LDL cholesterol.One of the important means to improve arteriosclerotic diseases is to reduce the level of blood lipids.Lipid autophagy,also known as lipohagy,is an important way of lipid metabolism.Our research group has previously found that gypenosides can down-regulate PCSK9 in both cells and animals models.The aim of this study was to further explore whether the lipidlowering effect of gypenosides is related to the inhibition of PCSK9-LDLR degradation by regulating the lipid phagocytosis pathway.Research methods:1.Clinical research:A total of 52 highly high-risk patients with ASCVD treated in Jiangsu Provincial Hospital of Integrated Traditional and Western Medicine from January 2022 to January 2023 were selected and divided into control group(Western medicine standard treatment group)and treatment group(Western medicine standard treatment+Gypstock granules group).A total of 4 weeks were followed up.The levels of blood lipid,liver and kidney function and PCSK9 expression were compared before and after treatment.2.Experimental research(1)Animal experiments:A total of 36 golden hamster rats with 6 in each group were selected and divided into blank group,model group,Gypenoside low-dose group,gypenoside high-dose group,lipid phagocytic inhibitor group,lipid phagocytic inhibitor group+Gypenoside high-dose group.Except the blank group,the other groups were fed high-fat diet for 14 days to establish hyperlipidemia animal model.After 4 weeks of drug intervention in each group,weight of golden hamsters was weighed,serum and liver tissues were collected,and related indexes were detected.①TG,TC,LDL-C,HDL-C were detected;②Pathological sections of liver were made,HE staining and microscopic examination were performed.③Lipid degeneration of liver sections was evaluated by oil red O staining;④ Immunohistochemistry of P62 and LDLR were detected in liver sections.Westernblot was used to detect the protein expression of LC3、p62、PCSK9 and LDLR.(2)Cell experiment 1:HepG2 cells were induced by 100μg/ml LDL to establish a cell hyperlipemia model,and the non-toxic dose range of gypenosides was screened by MTT method.HepG2 cells were divided into blank group,model group,Gypenoside low-dose group,gypenoside high-dose group,liphage inhibitor group,liphage inhibitor+gypenoside high-dose group.① The levels of TC,TG and LDL were determined.② Oil red O staining method was used to stain intracellular lipid droplets,observe the effect of gypenosides on the number of intracellular lipid droplets,and determine the accumulation of lipid droplets.③ Autophagosome formation was observed by MDC staining.④The expression of LDLR on cell membrane was detected by immunofluorescence.The protein expressions of LC3,p62,PCSK9 and LDLR were detected by Westernblot.The distribution of lipid droplets,lipid vesicles and autophagosomes were observed by transmission electron microscopy.(3)Cell experiment 2:HepG2 cells were divided into blank group,normal control group,model group,Gypenoside low-dose group,gypenoside high-dose group,AMPK inhibitor group(AMPKi),AMPK inhibitor+gypenoside high-dose group.①The formation of autophagosomes was observed by MDC staining.The protein expressions of AMPK,p-AMPK,ULK1,p-ULK1,LC3 and p62 were detected by Westernblot.The research results:1.Clinical researchAfter treatment,both TC and LDL-C decreased significantly compared with those before treatment.Compared with the control group,TC and LDL-C in the treatment group were decreased.After treatment,PCSK9 in control group was significantly higher than before.The PCSK9 level of treatment group was lower than that of control group.After treatment,LDL-C compliance rate in the treatment group was significantly higher than other group.2.Experimental research(1)Animal experiments:Animal experiments found that gypenosides could reduce the contents of TC and LDLC in hamsters in a dose-dependent manner.The oil red O and HE staining of liver showed that gypenosides could reduce lipid deposition in hyperlipidemia golden hamster,and the higher the dose,the more obvious the effect.It was found that the total saponins of gypenosides could reduce the deposition of p62 downstream of LC3 and increase the expression of LDLR by immunohistochemical experiments in liver.Combined with Western-blot,it was found that the total saponins of gypenosides increased the expression level of LC3Ⅱ/Ⅰ decreased p62 level,increased LDLR level,and decreased PCSK9 level,and the dose was positively correlated.When autophagy level was reduced by autophagy inhibitor,gypenosides could increase the expression level of LDLR protein and decrease the PCSK9 level by increasing the amount of autophagy.(2)Cell experiment 1:Through the oil red O staining of cells,it was found that the total saponins of gypenosides could reduce the lipid deposition of hyperlipidemia golden hamster,and the higher the total saponins of gypenosides,the more obvious the effect.The MDC staining showed that gypenosides could further induce autophagy,and the higher the dose,the stronger the effect.When the autophagy inhibitor CQ decreased the autophagy level,the total saponins of gypenosides could enhance the decreased autophagy level.By cell immunofluorescence assay,it was found that total saponins of gypenosides could increase LDLR expression level which was reduced by LDL induction.Combined with Western-blot,it was found that the total saponins of gypenosides increased the expression level of autophagy associated LC3Ⅱ/Ⅰ protein,decreased p62 level,increased LDLR protein level,and decreased PCSK9 level,and it was positively correlated with dose.When autophagy level was reduced by autophagy inhibitor CQ,total saponins of gypenosides could increase the expression level of LDLR protein and decrease PCSK9 protein level by increasing the amount of autophagy.It was found that gypenosides could induce autophagosome lysosome fusion in OX-LDL-treated HepG2 cells,and gypenosides could induce further fusion and degradation of inhibited autophagosomes.(3)Cell experiment 2:This experiment found that through MDC staining experiment,total saponins of gypenosides could further induce autophagy,and the higher the dose,the stronger the effect;When AMPK inhibitor(COMPOUND C)decreased autophagy level,gypenosides enhanced the decreased autophagy level.Combined with Western blot,it was found that the total saponins of gypenosides increased the expression level of autophagy associated LC3 Ⅱ/Ⅰ protein,decreased the expression level of p62 protein,and increased the expression levels of p-AMPK and p-ULK1 protein,and were positively correlated with dose.When AMPK inhibitor(COMPOUND C)was used to reduce autophagy level,gypenosides increased autophagy and increased the decreased p-AMPK and p-ULK1 protein expression levels.Conclusion:1.The total saponins of gypenosides can further reduce blood lipid levels and improve the clinical compliance rate of LDL-C.2.The total saponins of gypenosides inhibit the degradation of PCSK9-LDLR through the lipid phagocytosis pathway and exert lipid lowering effect.3.The total saponins of gypenosides activate AMPK/ULK1 pathway by regulating lipid phagocytosis,thereby regulating lipid metabolism.