姜黄素烟酸酯调节IDOL泛素化降低血浆LDL-C的作用机制

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 expression would improve dyslipidemia. LDLR expression is regulated by the levels of transcription and post-translation. Inducible degrader of LDLR (IDOL) is a novel regulator of LDLR. IDOL is an E3-ubiquitin ligase regulated via liver X receptors (LXRs) binding to the upstream of translation start site of IDOL by 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. Specifically, loss of IDOL reduces LDLR distribution in the hepatic cell, and lows levels of 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 derivatives 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 protein expression, and increased LDLR protein expression in vitro. In view of these, the applicant proposes that CurTn may through the IDOL/LDLR axis to promote hepatic clearance of plasma LDL-C. This project wants to elucidate that the molecular mechanism of the effect of CurTn on reducing plasma LDL-C through LV-IDOL, RNAi-IDOL and MG132 treated with HepG2 cells as experimental object. At the same time, the rabbits as the experimental object, the further study of the molecular target of CurTn on promoting hepatic clearance of plasma LDL-C by IDOL/LDLR. 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 as the target to reduce ASCVD risk.

IDOL是改善血脂异常的新靶点，可诱导LDLR泛素化在溶酶体降解，升高血浆LDL-C。LDLR经内吞循环负责着血浆LDL-C的清除，降低LDL-C是防治血脂异常的首要目标。前期研究发现姜黄素烟酸酯(CurTn)可降低血浆LDL-C，减少动脉内膜下脂质沉积，其机制不明。我们预实验发现CurTn可升高LDLR蛋白表达，并在翻译后水平降低IDOL蛋白含量，因IDOL主要经泛素化在蛋白酶体降解而减少，为此，申请者提出CurTn促进IDOL泛素化降低血浆LDL-C的工作设想。本课题将在肝细胞分析CurTn对IDOL泛素化的影响，并利用慢病毒稳转系统及RNA干扰技术探讨CurTn调节IDOL泛素化降低血浆LDL-C的分子机制，As模型动物实验证实CurTn通过促进IDOL泛素化降低血浆LDL-C的作用靶点。本项目的实施，将有望为CurTn改善血脂异常及防治动脉硬化性心血管疾病提供理论依据。

寻求降低以LDL-C为主的心血管疾病高风险因素是临床降低心血管事件的主要目标。该项目以此为目标，利用课题组合成的姜黄素烟酸酯可降低小鼠血浆LDL-C，机制不明为契机。进一步作用于高脂饲喂的Wistar大鼠8周，检测肝组织的脂质及血浆脂质的水平，发现姜黄素烟酸酯可显著降低TC及LDL-C的水平，而对HDL-C及TG的含量无影响；利用Western boltting及QPCR检测到肝组织LDLR的蛋白表达水平升高，而IDOL的蛋白表达降低，同时意外的是发现药物处理后肝脏的脂质含量并没有比模型组高。体外利用HepG2和Lo2肝细胞为研究对象，油红O及荧光没法检测结果显示：姜黄素烟酸酯作用24小时促进了肝细胞脂质含量的增加，且胆固醇含量也显著增多；同时也发现阳性药物瑞舒伐他汀也出现了相似的效应，结果提示姜黄素烟酸酯促进了肝细胞胆固醇的摄取。我们进一步利用流式细胞术检测了细胞膜表面LDLR的分布及DiI-LDL的摄取，结果显示姜黄素烟酸酯可使肝细胞膜表面LDLR分布增加，并促进了肝细胞摄取LDL-C；利用免疫共沉淀检测到姜黄素烟酸酯还使LDLR泛素化水平降低，却升高了IDOL的泛素化水平，以上研究结果提示姜黄素烟酸酯促进LDLR在细胞膜表面的分布与升高IDOL泛素化水平有关。我们构建的过表达及干扰IDOL慢病毒载体转染HepG2及Lo2细胞，姜黄素烟酸酯作用24小时后，油红O染色及胆固醇含量检测发现姜黄素烟酸酯可使干扰IDOL慢病毒感染的HepG2及Lo2细胞TC和FC含量进一步增多，以上研究结果提示姜黄素烟酸酯升高肝细胞膜表面LDLR分布，促进肝细胞清除血浆LDL-C与促进IDOL泛素化密切相关。