内源性硫化氢通过PCSK9/LDLR途径抑制大鼠高胆固醇血症

Endogenous hydrogen sulfide (H2S) is a cardiovascular regulatory gasotransmitter. Our previous studies showed that H2S inhibited the development of atherosclerosis possibly through controlling the cholesterol metabolism in hepatocyte. To investigate the effect of H2S on the hypercholesterolemia and its mechanisms, the present project will target on proprotein convertase subtilisin/kexin type 9 (PCSK9) and examine the significance of regulatory effect of H2S on the PCSK9/low density lipoprotein receptor (LDLR) pathway in the pathogenesis of hypercholesterolemia in rat. Firstly, the applicants shall detect the change of hepatic H2S pathway and PCSK9/LDLR pathway in high-fat diet rats. Secondly, the applicants shall explore the effect of endogenous H2S on hypercholesterolemia and hepatic PCSK9/LDLR pathway by upregulating or downregulating the expression of endogenous H2S generating enzymes cystathionine γ lyase (CSE) or cysthathionine β synthase (CBS) in rat liver. Thirdly, the researchers plan to investigate if PCSK9 is a target, on which H2S regulates hepatic LDL metabolism via overexpression or knockdown of PCSK9. Mechanistically, the applicants plan to use the mass spectrometry, the specific probe for protein sulfhydration and site-directed mutation to clarify if the sulfhydration modification of PCSK9 by H2S might be the molecular mechanism by which H2S inhibits hypercholesterolemia. Based on the abovementioned studies, it is expected to discover a novel regulatory mechanism by which H2S inhibits hypercholesterolemia and provide new idea for exploring the prevention and treatment strategy for atherosclerosis.

内源性硫化氢(H2S)是心血管调节的气体信号分子，本课题组前期研究发现H2S可抑制动脉粥样硬化，其作用可能与其调节肝细胞胆固醇代谢有关。为阐明H2S对高胆固醇血症的调节作用及机制，本项目将以前蛋白转化酶枯草溶菌素9(PCSK9)为靶点，研究内源性H2S对PCSK9/低密度脂蛋白受体(LDLR)通路的调控在大鼠高胆固醇血症发病中的意义。首先，在整体水平研究高脂饮食大鼠肝脏H2S及PCSK9/LDLR通路的变化；进一步通过过表达或敲低肝脏H2S生成酶CSE及CBS，揭示内源性H2S对高胆固醇血症及PCSK9/LDLR通路的调节作用；通过过表达或敲低PCSK9，阐明PCSK9是H2S调节肝细胞LDL代谢的分子靶点；采用质谱分析结合硫氢化修饰探针及定点突变，揭示H2S对PCSK9的硫氢化修饰是其抑制高胆固醇血症的分子机制。以期发现H2S调节高胆固醇血症的新机制，为动脉粥样硬化的临床防治提供新思路。

以低密度脂蛋白胆固醇（LDL-c）升高为主要改变的高脂血症与高血压、动脉粥样硬化、冠心病、脑卒中等心脑血管病发病密切相关，揭示血脂调控的分子机制，探索防治高脂血症的新靶点是心血管领域的重要科学问题。PCSK9是近年来降脂治疗药物的研发热点。虽然各种PCSK9抑制剂如单克隆抗体、封闭肽、小RNA及反义寡核苷酸等新型药物的临床试验已经广泛开展并取得了一定的临床疗效，但是仍然存在价格昂贵、副作用多等缺点，亟待研发新的PCSK9抑制分子。本项目组在既往关于内源性H2S与心血管调节的研究基础上，研究发现高脂饮食大鼠肝脏内源性H2S生成体系显著下调，H2S可抑制高脂饮食大鼠肝脏PCSK9表达，增加LDLR含量，降低血浆LDL水平；PCSK9过表达及PCSK9蛋白可阻断H2S抑制肝细胞LDL摄取的效应；H2S可抑制HNF1A活性，继而在转录水平抑制PCSK9表达；揭示H2S抑制HNF1A转录活性的分子机制，阐明H2S通过HNF1A/PCSK9/LDLR通路促进肝细胞LDL摄取的关键分子靶点。综上，本研究结果提示维持肝细胞内源性H2S稳定可能是控制PCSK9，进而抑制高LDL-c血症的新型治疗策略，内源性H2S有望成为PCSK9抑制剂研发的新靶点。围绕上述研究成果，共发表SCI论文8篇，在全国学术会议特邀讲座3次，项目负责人入选教育部青年长江学者，获得茅以升北京青年科技奖，培养博士研究生2人，硕士研究生2人。