A New Mechanism For Negative Feedback Regulation Of LDL/LDLR:The Lipophagy Mediated PCSK9-LDLR Degradation Pathway

Atherosclerosis(AS)is a chronic inflammatory disease characterized by lipid accumulation in the wall of large and medium-sized arteries.Dysregulation of lipid metabolism leads to the accumulation of lipids in the vascular intima,especially the accumulation of low-density lipoprotein(LDL),which is the initiating factor for the development of atherosclerotic lesions.LDL in plasma is mainly metabolized by low density lipoprotein receptor(LDLR)uptake into cells.The LDL/LDLR pathway is a negative feedback regulatory system that plays an important role in maintaining plasma and intracellular lipid homeostasis.Lipophagy is a new type of selective autophagy,which can regulate lipid metabolism by autophagosomes selectively recognizing lipids and transporting them to lysosomal for degradation.Lipophagy plays an important role in removing excess lipids,maintaining cell homeostasis,and preventing the progression of AS.Proprotein convertase subtilisin kexin type 9(PCSK9)is a gene closely related to the regulation of lipid metabolism,which participates in lipid metabolism by mediating the degradation of LDLR on the cell surface.So,is it possible for lipophagy to influence the expression of LDLR by regulating the expression of PCSK9,thereby regulating lipid metabolism.Therefore,the purpose of this study intends to assess the effects of lipophagy on the degradation pathway of PCSK9-LDLR in Hep G2 cells,and to provide a new theoretical basis for the negative feedback regulation mechanism of LDL/LDLR.Part I: Effect of lipid load on lipophagy and the expression of PCSK9 and LDLR in Hep G2 cellsObjectives: To investigate the effect of lipid load on lipophagy and the expression of PCSK9 and LDLR in Hep G2 cellsMethods: Hep G2 cells were treated with 100μg/ml LDL for 24 hours,oil red O staining and Dil-LDL was observed of lipid uptake in Hep G2 cells;Immunofluorescence was used to detect the expression of LDLR on the cell membrane surface;Western blot was used to detect LC3,P62,LAMP1,PCSK9,LDLR protein expression levels;Immunofluorescence co-localization to observe the co-localization of LC3 and Bodipy,LAMP1 and Bodipy,LC3 and LAMP1.Results: Compared with the Control group,LDL treatment increased the lipid uptake of Hep G2 cells and reduced the expression of LDLR on the cell membrane surface;the protein expression of LC3-II,LAMP1 and PCSK9 increased,and the protein expression of P62 and LDLR decreased;LC3 and Bodipy,LAMP1 and Bodipy,LC3 and LAMP1 have a lot of colocalization.Summary: LDL treatment increased the lipid uptake of HepG2 cells,caused negative feedback effect of LDLR,and reduced the expression of LDLR on the cell membrane surface;increased the protein expression of LC3-II and LAMP1,decreased the protein expression of P62,increase the co-localization of LC3 and Bodipy,LAMP1 and Bodipy,LC3 and LAMP1,and induced the occurrence of lipophagy;increased the protein expression of PCSK9 and decreased the protein expression of LDLR.Part Ⅱ : Effects of lipophagy inhibitor Chloroquine(CQ)on the expression of PCSK9 and LDLR,and lipid accumulation in LDL-treated Hep G2 cellsObjectives: To investigate the effect of inhibition of lipophagy on the expression of PCSK9 and LDLR,and lipid accumulation in LDL-treated Hep G2 cellsMethods: Hep G2 cells were pretreated with the lipophagy inhibitor CQ,and then Hep G2 cells were treated with 100μg/ml LDL for 24 hours.Western blot was used to detect the protein expression of LC3,P62,PCSK9,and LDLR.Dil-LDL to observe lipid accumulation in Hep G2 cells.Results: Compared with the LDL group,CQ and LDL treatment increased the protein expression of LC3-II,P62,and LDLR,and decreased the protein expression of PCSK9.The expression of LDLR on the cell membrane was restored,thereby increasing lipid accumulation in Hep G2 cells.Summary: CQ and LDL treatment increased the proteins expression of LC3-II and P62,and inhibited the occurrence of lipophagy;CQ and LDL treatment decreased the protein expression of PCSK9,which in turn increased the protein expression of LDLR and increased lipid accumulation in Hep G2 cells.Conclusion: LDL load induces the occurrence of lipophagy,which affects the negative feedback effect of LDLR by regulating the expression of PCSK9,which provides a new theoretical basis for the regulation mechanism of LDL / LDLR negative feedback.