mTORC1 信号在天麻素治疗心肌缺血再灌注损伤中的作用及其调节机制

According to the World Health Organization, acute coronary occlusion is the leading cause of morbidity and mortality in the world and will continue to be the major cause of death in the world by the year 2020. Reperfusion therapy is beneficial to prevent cardiomyocyte death and contractile dysfunction after myocardial infarction (MI). However, numerous studies have shown that reperfusion itself may enhance the injury, resulting in extension of infarct size after ischemia, and termed myocardial ischemia/reperfusion injury (MIRI). Currently, it has been widely accepted that cardiac ischemic preconditioning (IPC) and ischemic postconditioning (IPost) can attenuate both contractile dysfunction and development of myocardial infarction following an MIRI insult. However, IPC and IPost have their limitations in that pre- and post-conditioning mimetic must be applied before and after the index ischemic event, which are unpredictable and impractical in the clinical setting of acute myocardial infarction. Accordingly, pharmacological pre- and post-conditioning were developed. Therefore, the identification and characterisation of agents that can protect the heart from the damaging effects of MIRI are of considerable importance..Gastrodin might play an important role in the protection against cardiovascular diseases, but its role and signal transduction pathway in anti-pathogenesis of myocardial ischemia/reperfusion injury (MIRI) have remained unexplored. Our preliminary results suggested that gastrodin regulated phosphoinositide 3-kinase (PI3-K) / protein kinase B (PKB) signaling and AMP-activated protein kinase (AMPK) / a mammalian ortholog of Sir2 (SIRT1) signaling-related mTOR signaling in the pathogenesis of MIRI. The current project aims to identify the roles and molecular and cellular mechanims of gastrodin on mammalian target of rapamycin complex 1 (mTORCI) signaling which plays a crucial role in determinating others signal transduction pathway in the anti-pathogenesis of MIRI. The inducible and cell-specific knockout mice and cellular models will be used, in which the mTORCI in cardiomyocytes, vascular endothelial cells and vascular smooth muscle cells in mice could be specifically activated or inhibited. The effects of mTORCI on proliferation, autophagy and apoptosis of these cells, secretion of cytokines and inflammatory factors and signaling pathways in the gastrodin-treated mice and cellular models will be investigated..Taking together, the research further our knowledge regarding the pathways used by gastrodin in exerting its protective effects and support our belief that gastrodin may hold future clinical promise in the development of novel therapeutic strategies for the treatment of MIRI. Furthermore, the research will provide experimental evidence for the novel application of natural products of Yunnan Province.

前期研究表明：在心肌缺血再灌注损伤(MIRI)病理过程中，天麻素通过调控哺乳动物雷帕霉素靶蛋白(mTOR)相关通路磷脂酰肌醇3－激酶/蛋白激酶B(PI3-K/Akt)信号和AMP活化蛋白激酶/Sir2哺乳动物同源物(AMPK/SIRT1)信号发挥心肌保护的作用，但天麻素调控 mTOR 的中心靶蛋白的信号网络机制不清楚。本项目拟采用可诱导、细胞特异基因敲除的小鼠与细胞模型，使小鼠心肌细胞、血管内皮细胞和血管平滑肌细胞的 mTOR 复合物-1(mTORC1)被特异性地激活或抑制，深入研究 mTORC1 在天麻素治疗MIRI中对调控上述细胞的增殖、炎性反应、自噬与凋亡的信号网络机制。从分子、细胞和整体层面探讨 mTORC1 通路在天麻素治疗 MIRI 中的关键性作用，为临床应用天麻素治疗 MIRI 提供新的靶点与新的分子机制。同时，为进一步拓展云南优势特色天然药物新用途提供坚实的理论依据。

随着心脑血管疾病发病率迅猛增高的势头，其防治已成为我国医学研究的重点和热点领域之一。在心肌缺血再灌注损伤病理过程中，天麻素通过调控哺乳动物雷帕霉素靶蛋白(mTOR)相关通路磷脂酰肌醇3－激酶/蛋白激酶B(PI3-K/Akt)信号和AMP活化蛋白激酶/Sir2哺乳动物同源(AMPK/SIRT1)信号发挥心肌保护的作用，但天麻素调控mTOR的中心靶蛋白的信号网络机制不清楚。本研究针对天麻素在心血管疾病中的保护作用，从广度和深度方面展开系统性探索。从分子、细胞及整体层面探讨天麻素抗心肌缺血再灌注损伤病理发生过程中的作用及其信号网络分子机制，取得了较好的研究成果，主要研究发现如下：通过体内和体外相结合的方式研究了心肌缺血再灌注过程中天麻素对心脏的保护作用，发现天麻素能有效地减少大鼠心肌梗死面积，抑制心肌细胞的死亡。机制学上，天麻素能激活mTOR，抑制自噬小体的累积，恢复自噬流，从而抑制缺血再灌注诱导的心肌细胞凋亡以及炎症反应。研究证实RBM3和UBE2D3调控的自噬在心肌缺血再灌注损伤中的作用。发现在缺血再灌注过程中，RBM3的表达升高，随后与Raptor相互作用，增加自噬活性，抑制缺血复灌诱导的心肌细胞凋亡。另外，在缺血再灌注过程中，上调的UBE2D3促进p62的泛素化进而加重自噬流受损，增加心肌细胞的凋亡，抑制UBE2D3的表达可显著降低缺血复灌诱导的心肌细胞凋亡。在此基础上，拓展了心肌缺血再灌注损伤的发生机理，研究了心肌缺血再灌注过程中心肌微血管内皮功能障碍的病理发生机制。发现缺血再灌注导致微血管内皮细胞和心肌组织中NLRP3与caspase-4炎性小体活化和焦亡；抑制NLRP3与caspase-4后可对缺血再灌注诱导的微血管损伤起到保护作用。进一步深入研究发现心肌缺血再灌注导致微血管内皮细胞和心肌组织中TNFAIP3的表达被抑制，促进了NLRP3的活化；过表达beclin1可上调TNFAIP3的表达，抑制微血管内皮细胞和心肌组织中NLRP3的活性，从而缓解缺血再灌注诱导的微血管损伤。本研究为天麻素发挥心肌保护作用提供新的细胞分子生物学依据。进一步拓展云南优势特色天然药物新用途提供坚实的理论依据。