姜黄素对肝细胞LDLR表达作用的分子机理研究

【作者】 窦晓兵；

【导师】 沃兴德；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2007， 博士

【摘要】 血液中胆固醇特别是LDL-C浓度的升高是引起动脉粥样硬化和冠心病的最主要危险因素，降低血液胆固醇浓度可明显减少冠心病的发病率和死亡率。体内大部分胆固醇结合于LDL，经肝细胞表面的LDLR代谢和清除。LDLR是位于细胞表面的跨膜受体，其表达受到胆固醇和其它许多物质在转录水平或转录后水平的调控，上调肝脏LDLR的表达是降低血液胆固醇浓度的最主要手段。目前临床应用最广泛的能上调肝脏LDLR表达的药物是他汀类，他汀类药物能竞争性抑制HMG-CoA还原酶的活性，使细胞内胆固醇合成减少。总体来讲他汀类具有出色的降胆固醇疗效，但是也有部分患者因不良反应(如肝功能损害等)不能耐受其治疗。因此，筛选新型降胆固醇药物一直是调血脂药物的研究重点。目的：通过对传统中草药物的筛选，我们发现从活血化瘀药姜黄中提取的姜黄素能显著降低血清胆固醇、LDL-C含量的作用，从而起到降血脂和抗动脉粥样硬化的作用。为了研究姜黄素降胆固醇作用的分子机制，我们设计了一系列实验。本课题就是在以往研究的基础上，拟从基因表达水平和细胞信号调控角度探寻姜黄素与信号调控途径中关键蛋白分子以及基因调控元件间的相互作用关系，从而阐明姜黄素对LDLR表达影响的机理。方法：第一部分姜黄素诱导LDLR表达的药效研究首先利用人肝HepG2细胞作为LDLR基因表达检测系统，经姜黄素干预培养后，利用Western-Blot、Rt-PCR、荧光显微镜和流式细胞仪等技术分析肝细胞内源性LDLR蛋白的表达量及其表达活性，确定姜黄素对LDLR基因表达的影响，从受体蛋白水平阐明姜黄素降胆固醇作用的机理。第二部分姜黄素诱导LDLR表达的机理研究：①将绿色荧光蛋白GFP报告基因构建在调控低密度脂蛋白受体基因表达的固醇调控元件SRE-1的下游，经脂质体介导法导入HepG2细胞，建立LDLR基因表达的固醇调控报告系统及其稳定细胞株HepG2／SRE-GFP，通过姜黄素的干预培养，从基因表达调控水平研究姜黄素对启动子SRE-1的影响，探寻姜黄素的作用机理。②利用脂质体介导法将pCMV-Insig2导入HepG2细胞，建立稳定的细胞株HepG2／Insig2，通过姜黄素的干预培养，研究姜黄素对Insig-2的影响，从信号调控通路水平探寻姜黄素降胆固醇作用可能的分子或基因靶点。结果：第一部分研究表明，①姜黄素能够增强HepG2 LDLR对LDL的摄取，具有明显的时间依赖性和剂量依赖性。并且在25μmol／L和作用24小时的时候LDLR的活性最强。②姜黄素能够显著促进HepG2 LDLR的表达量，具有明显的时间依赖性和剂量依赖性。③姜黄素可能通过促进HepG2 LDLR mRNA的转录上调LDLR的表达。第二部分研究结果表明，①姜黄素能够激活启动子调控元件SRE-1上调LDLR表达。②一定浓度的姜黄素能够通过逆转受Insig2抑制的SREBP通路，上调LDLR的表达。结论：综上所述，我们从受体水平、基因表达水平到信号调控通路水平对姜黄素降胆固醇的机理进行了一系列研究。所有证据都证明姜黄素很可能是通过作用于SREBP通路中的某个调节因子，促使LDLR表达起到降胆固醇作用的。但是究竟作用于SCAP还是Insig2或者其它蛋白仍有待进一步研究。更多还原

【Abstract】 The elevation of serum cholesterol levels, especially LDL-C, is by far one of the mostrelative risk factors that cause atherosclerosis and CHD. Lowering LDL-C levels bymedication can reduce the mortality of CHD significantly. The majority of LDL-C are takenup and cleared by hepatic LDLR. LDLR is a trans-membrane receptor located on cell surface,the expression level of which is tightly regulated by sterols or non-sterols pathway both onthe transcription and post-transcription level. Up-regulation of hepatic LDLR is the mosteffective method to lower serum LDL-C levels. The most widely-used cholesterol-loweringdrug at present is statins. Statins can inhibit the enzymatic activity of HMG-CoA reductasecompetitively, and then inhibit the cellular cholesterol synthesis. In general, statins haveexcellent effect in hypercholesterolemia. However, a small part of patients can not toleratestatins because of adverse effects such as liver function damage. So, the efforts for screeningnew types of LDLR up-regulators have being done for a long time.Objective:Based on the traditional Chinese herbal medicine on the screen, we discovered HuoxueHuayu turmeric extract of curcumin could significantly reduce serum cholesterol, LDL-C andplayed the role of hypolipidemic and anti-atherosclerotic effect. In order to study themolecular mechanism of the curcumin effects on cholesterol-lowering, we designed a seriesof experiments. In the basis of previous research, from the level of gene expression and cellsignaling regulation, the issue is order to clarify the mechanism of curcumin on the LDLRexpression and search for the interaction between curcumin and the key protein moleculesand genes in the signal pathway.Methods:PartⅠis the effects of curcumin-induced expression of LDLR.Firstly, using human liver HepG2 cells as LDLR gene expression detection system, aftercurcumin treated, liver cells-derived LDLR protein expression and activity of endocytosis areanalysised by Western-Blot, Rt-PCR, Fluorescence microscopy, flow cytometry and othertechniques. Then the effects of curcumin on LDLR on the gene expression are identified, andthe role of cholesterol-reducing mechanism was clarified from the receptor protein levels.PartⅡis the mechanism of curcumin-induced expression of LDLR.①Green fluorescent protein (GFP) reporter gene is constructed in the downstream oflow-density lipoprotein receptor gene control components, SRE-1, and then is transductedinto HepG2 cells by liposome-mediated methods. Then LDLR gene expression regulationreporting system is conducted and the HepG2/SRE-GFP stable cell line is established. Aftercurcumin treated, the mechanism of curcumin on the promoter SRE-1 is explained from the level of gene expression regulation.②The CMV-Insig2 plasmid is also transducted into HepG2 cells by liposome-mediatedmethods and then constructs the stable cell lines, HepG2/Insig2. After curcumin treated, themechanism of curcumin on the Insig2 is revealed from the level of gene expression regulation.And the possible molecular or genetic target of curcumin in the role of cholesterol lowering isexplored.Results:PartⅠresults showed that,①curcumin can enhance HepG2 LDLR uptake of LDL,Obviously with the time-dependent and dose-dependent manner. And the 25μmol/L and the24-hour time LDLR have the strongest endocytosis activity.②Curcumin can significantlypromote the expression in HepG2 LDLR, with obvious time-dependent and dose-dependentmanner.③Curcumin can up-regulate the expression of LDLR mRNA to a significant extentin the human hepatoma cell line HepG2.PartⅡresults showed that,①Curcumin can activate the promoter element SRE-1 toincrease LDLR expression.②Certain concentration of curcumin can reverse and restore theSREBP pathway inhibited by the Insig2 and up-regulate the expression of LDLR.Conclusion:In summary, from the receptor level, gene expression levels and the signal regulationpathway level, we discussed the mechanism of the curcumin on the cholesterol-lowering by aseries of studies. All the evidence proved that curcumin, as a cholesterol-lowering drug, canpromote the LDLR expression by acting with some regulational molecular in the SREBPpathway. But which step the curcumin acts on remains to be further studied.更多还原