The Mechanism Of PCSK9/IDOL In Curcumin Trinicotinate Increasing LDL Uptakes Of HepG2 Cells

Low-density lipoprotein cholesterol(LDL-C) is a major risk factor for the atherosclerotic cardiovascular disease(ASCVD). Over 70% of circulating LDL-C is metabolized by binding hepatic LDL receptor(LDLR). Moreover, genetic LDLR mutations usually result in hypercholesterolemia in the patients. Therefore, elevation of LDLR levels on hepatic cells would improve dyslipidemia. LDLR expression is usually regulated by the SREBP2/PCSK9 pathways. Targeting SREBP2/PCSK9 pathways by statins and human monoclonal PCSK9 antibody reduce the progression of ASVCD. Inducible degrader of LDLR(IDOL) is a novel regulator of LDLR. IDOL is an E3-ubiquitin ligase regulated via liver X receptors(LXRs) by binding to the upstream of translation start site of IDOL responding to extracellular cholesterol levels. IDOL has been shown to modulate LDLR distribution via ubiquitination and degradation of LDLR in lysosomes. Genome-wide association studies(GWAS) have revealed that the nonsynonymous substitution rs9370867 of IDOL probably affects the variability of circulating LDL levels in different populations. Recently studies also demonstrated that IDOL may influence PCSK9 expression in a LDLR/SREBP2-dependent manner. Specifically, loss of IDOL reduces LDLR distribution in the hepatic cell, and lows levels of in serum LDL-C in dyslipidemic patients. Taken together, IDOL might be a potential therapeutic target for the treatment of ASCVD.Curcumin trinicotinate(CurTn) is a kind of ester derivatived with multiple functional groups. Our previous studies showed that CurTn reduced the levels of plasma LDL-C and the deposition of lipid artery intima, but the mechanism is unknown. Experimental results showed that CurTn decreased IDOL and PCSK9 protein expression, and increased LDLR protein expression in vitro. In view of these, the applicant proposes that CurTn may promote hepatic clearance of plasma LDL-C via the PCSK9/IDOL axis. This project wants to elucidate that the molecular mechanism of the effect of CurTn on reducing plasma LDL-C through LV-IDOL/PCSK9, RNAi-IDOL/PCSK9 treated with HepG2 cells as experimental object. The implementation of the project is expected to develop CurTn as a clinical drug on improvement of dyslipidemia and to provide scientific basis for IDOL and PCSK9 as the targets to reduce ASCVD risk. PartⅠ: The effect of CurTn on LDL-C uptake in HepG2 cellsObjective: To make clear the mechanism of CurTn in reducing serum LDL-C levels involved in LDLR protein expression increased and LDL-C uptakes promoted.Methods: Co-incubated HepG2 cells with CurTn and LDL for 24 h, Cellular lipid was detected by oil red O staining. Cholesterol content was detected by cholesterol quantitative fluorometric kit. LDL uptakes were visualized by Di I-LDL and Hoechst33342. The m RNA and protein expression of INSIG1, SREBP2, LDLR, HMGCR, PCSK9 were analyzed by quantitative Real-time PCR(RT-Q-PCR) and Western blotting respectively.Results: Oil Red O staining showed that lipid droplets increased significantly in 10, 15 μmol/L CurTn groups. The content of Cholesterol and Di I-LDL uptake were higher in 10, 15 μmol/L CurTn groups than in control group. RT-Q-PCR and Western blotting showed that CurTn increased LDLR protein expression and decreased PCSK9 protein expression, although CurTn also increased SREBP2 m RNA expression. CurTn had no effect on LDLR m RNA expression in HepG2 cells, and also no effect on SREBP2, INSIG1, and HMGCR protein expression.Conclusion: The increased content of Cholesterol in HepG2 cells and higher LDLR protein expression treated with CurTn were associated with lower PCSK9 protein expression than control group’s.Part Ⅱ: The study of relationship between LDL-C uptake in HepG2 cells and PCSK9/IDOL expression on cell surface LDLR distribution treated with CurTnObjective: To make sure the relationship of proteins expression of PCSK9, IDOL and LDLR with LDL-C uptakes.Methods: Co-incubated HepG2 cells with CurTn and LDL for 24 h, LDL uptakes were visualized by Di I-LDL and Hoechst33342. The surface LDLR levels were detected by immunofluorescence flow cytometry. The protein expression of IDOL, K48, K63, and USP2 were analyzed by Western blotting respectively. LDLR ubiquitination were detected by Co immunoprecipitation.Results: Di I-LDL uptakes were higher in 10, 15μmol/L CurTn groups than that in control group. Western blotting results showed that CurTn decreased IDOL, K48, K63 protein expression and no effect on USP2 protein expression in HepG2 cells. Co immunoprecipitation testing showed that LDLR ubiquitination was lower in 15μM CurTn group than control group.Conclusion: LDL-C uptakes were associated with lower protein expression of IDOL and LDLR ubiquitination.Part Ⅲ: The effect of CurTn on the LDLR Levels of HepG2 cells treated with RNAi/LV-PCSK9/IDOLObjective: To study of the LDLR Levels in HepG2 cells treated with RNAi/LV-PCSK9/IDOL by CurTn.Methods: HepG2 cells infected with RNAi/LV-PCSK9/IDOL, and Q-RT-PCR and Western blotting were performed to test the m RNA and protein expression of PCSK9 and IDOL. Then co-incubated HepG2 cells with CurTn and LDL for another 24 h respectivelly, Western blotting were also performed to test the m RNA and protein expression of PCSK9 and IDOL. Finally LDLR protein was tested by western blotting to assure the effect of CurTn on LDLR levels of HepG2 cells infected by RNAi/LV-IDOL.Results: Firstly, Screen out the efficient RNAi-PCSK9/IDOL according to the results of Q-RT-PCR and Western blotting. Then, co-incubated HepG2 cells with CurTn and LDL for another 24 h respectivelly, the results showed that CurTn made IDOL/PCSK9 protein expression lower than their corresponding control groups, moreover also increased the LDLR protein expression.Conclusion: CurTn decreased the PCSK9/IDOL protein expression of HepG2 cells infected with RNAi/LV-PCSK9/IDOL and also promoted the LDLR level on HepG2 cells infected with RNAi/LV- IDOL..