SREBP-1c/PCSK9介导虎杖苷调节2型糖尿病糖脂代谢的机制研究

The disorder of glucose and lipid metabolism is the basis of microvascular complications in type 2 diabetes mellitus, which has high mortality and sounds the alarm to develop drugs with both hypoglycemic and lipid regulation effects. Nowadays researchers have found that proprotein convertase subtilisin/kexin type 9 (PCSK9) is highly correlated with glucose and lipid metabolism. In our previous study, we found that Polydatin could bind to the active pocket of PCSK9 crystal structure, besides, abnormal glucose and lipid metabolism in the liver of diabetic model animals was accompanied by increased SREBP-1c nucleoprotein level and PCSK9 expression, which could be reversed by Polydatin primely. Further research found that the nucleus translocation of SREBP-1c improved, with the increase of PCSK9 expression in insulin resistant HepG2 cells, and Polydatin can not only inhibit their expression but also reduce the combination of PCSK9-LDLR.Thus we propose a hypothesis: in addition to directly binding to PCSK9, Polydatin can also reduce PCSK9 transcription and translation by inhibiting the activity of SREBP-1c-PCSK9 promoter, thus synergistically exerting the hypoglycemic and lipid-regulating effects. This project intends to use the vitro and vivo models, screening the key protein of glucolipid metabolism, and explore PCSK9 correlated signaling pathway in glucolipid metabolic regulation under the condition of Polydatin treatment, which will provide a scientific basis for developing Polydatin as a new drug with a specific target in hypoglycemic and lipid regulation.

糖脂代谢紊乱是2型糖尿病微血管并发症的基础病变，致死率高，亟待开发兼有降糖调脂作用的药物。近年发现前蛋白转化酶枯草溶菌素9（PCSK9）与糖脂代谢存在极大关联，我们前期发现：虎杖苷可结合于PCSK9晶体活性口袋，且糖尿病模型动物肝脏糖脂代谢异常的同时，伴随SREBP-1c核蛋白水平和PCSK9表达的增加，而虎杖苷可有效逆转该现象。进一步研究发现：胰岛素抵抗HepG2细胞中，SREBP-1c核转位增多，伴随PCSK9表达的增加，虎杖苷抑制二者表达的同时还可减少PCSK9-LDLR的结合。由此我们提出假说：虎杖苷除直接结合PCSK9外，还可通过抑制SREBP-1c-PCSK9启动子活性来减少PCSK9转录翻译，协同发挥降糖调脂作用。本项目拟采用体内外模型，筛选出虎杖苷糖脂代谢调节的关键蛋白，并探寻PCSK9介导虎杖苷糖脂代谢调节相关通路，为将虎杖苷开发为具有特定靶点的新型降糖调脂药物提供依据。

糖脂代谢紊乱是2型糖尿病微血管并发症的基础病变，致死率高，亟待开发兼有降糖调脂作用的药物。虎杖苷作为中药虎杖成分中一种具有多种生物活性的天然物质,具有良好的药理活性，课题组早期研究发现：虎杖苷在糖尿病治疗中发挥抗炎、抗氧化、调节脂质代谢、改善胰岛素敏感性等多重作用，对虎杖苷的进一步深入研究将有利于寻找更有针对性的糖尿病药物靶点。近年发现前蛋白转化酶枯草溶菌素9（PCSK9）与糖脂代谢、炎症反应及免疫调节均存在极大关联，是心血管疾病发生发展的独立危险因素，基于PCSK9靶点的药物研发成为热点。本研究通过表面等离子共振技术(Surface plasmon resonance，SPR)证实：虎杖苷和人重组 PCSK9 蛋白之间存在相互作用，结合力随虎杖苷浓度增加而增强，但未呈现明显的剂量依赖性。进一步动物实验证实：虎杖苷可增加糖尿病模型鼠肝糖原生成，减少肝脏脂质沉积，降低血甘油三酯及胆固醇水平，有效改善糖脂代谢紊乱，这与虎杖苷抑制肝脏SREBP-1c表达、减少PCSK9及FAS 转录翻译，从而使得肝脏胆固醇合成减少，脂肪摄取增加密切相关，细胞膜LDLR水平的升高是逆转高脂血症的关键因素。细胞实验发现：虎杖苷可有效改善胰岛素抵抗状态下 HepG2 细胞糖摄取障碍，逆转脂肪过度沉积，该过程伴随SREBP-1c核转位的减少以及PCSK9蛋白表达的降低，细胞内脂肪合成减少是逆转HepG2 细胞脂肪沉积的重要因素，同时虎杖苷可提高IRS1的转录及磷酸化水平并通过增加GCK蛋白表达发挥糖代谢调节作用。总之，虎杖苷除直接抑制PCSK9-LDLR结合外，还抑制SREBP-1c-PCSK9分子轴从而减少PCSK9转录翻译，同时通过改善胰岛素敏感性协同发挥降糖调脂作用。本项目筛选出了虎杖苷调节糖脂代谢的关键蛋白PCSK9、LDLR、IRS1、 GCK和核因子SREBP-1c，并深入了解PCSK9介导虎杖苷糖脂代谢调节的具体分子机制，为将虎杖苷开发为具有特定靶点的新型降糖调脂药物提供了更多理论支撑,对虎杖苷的进一步深入研究将有利于寻找更有针对性的糖尿病药物靶点，同时也为传统中药学科发展，新药研发提供新策略。