天麻素在心肌缺血再灌注损伤治疗中的作用及其信号机制研究

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. By using the MIRI models of the distal segment of left anterior descending artery will be blocked with balloon angioplasty in Xishuangbanna mini pigs, the isolated rat hearts which were perfused in the Langendorff mode, cardiomyocytes, monocyte-derived macrophages, vascular endothelial cells and vascular smooth muscle cells will be harvested for investigation of the therapeutic role of gastrodin in MIRI. Effects of gastrodin on reperfusion injury salvage kinase (RISK.) pathways which play a crucial role in determinating the pathogenesis of MIRI such as phosphoinositide 3-kinase (PI3-K) / protein kinase B (PKB) / mammalian target of rapamycin (mTOR) signal pathway, phospholipase C-γ1 (PLC-γ1) / protein kinase C (PKC) signal pathway, mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signal pathway, AMP-activated protein kinase (AMPK) /a mammalian ortholog of Sir2 (SIRT1) signal pathway and the expression of apoptosis-regulatory proteins and autophagy-regulatory proteins will be examined. 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：天麻素对心肌损伤有一定的保护作用，但天麻素在调控心肌缺血再灌注损伤(MIRI)病理过程中的信号网络机制不完全清楚。本课题采用近交系西双版纳小耳猪通过介入球囊堵闭冠脉的方法与Langendorff离体SD大鼠心脏灌注构建心脏MIRI模型；心肌细胞、单核巨噬细胞、血管内皮细胞和血管平滑肌细胞等构建体外MIRI细胞模型，系统研究天麻素在MIRI过程中保护作用及对关键信号通路、中心环节靶蛋白（如：磷脂酰肌醇3－激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白、磷脂酶C-γ1/蛋白激酶C、有丝分裂原活化蛋白激酶与核因子-κB、AMP活化蛋白激酶/Sir2哺乳动物同源物和凋亡与自噬蛋白等）信号网络系统的调控作用。从分子、细胞和整体层面探讨天麻素在MIRI治疗中的信号网络调控机制，为临床应用天麻素治疗MIRI提供新的靶点与新的机制。同时，为进一步拓展云南优势特色天然药物新用途提供理论依据。

本研究阐明天麻素在心肌缺血再灌注损伤病理过程中的保护作用及其信号网络机制，同时探索了云南特色药物三七的有效成分人参皂苷 Rg1 和 Rb1在心肌缺血再灌注损伤病理过程中的保护作用及其信号网络机制，从研究的广度和深度开展了拓展性系统探索。在研究体系的构建中，引入天麻素缓释生物可降解材料在心血管系统修复中的作用等交叉学科研究体系。完成了以下方面的研究工作：（1）初步阐明天麻素在SD大鼠离体心脏、西双版纳小耳猪在体心脏和C57BL/6J小鼠在体心脏MIRI模型等三种动物模型中的保护作用。（2）天麻素通过AMPK信号通路抑制血清剥夺诱导的H9C2心肌细胞的自噬发挥抗凋亡的保护作用。（3）天麻素通过下调凋亡蛋白Beclin-1介导自噬流从而减轻H9C2心肌细胞缺血再灌注损伤中炎症反应。（4）探索了天麻素通过抑制溶酶体酸化改善心肌缺血再灌注损伤介导的心肌细胞自噬流，天麻素改善自噬通量与mTOR的激活无关，而是通过介导Beclin 1的下调抑制溶酶体酸化实现。（5）天麻素通过抑制 NLRP3/caspase-1信号通路抑制心肌缺血再灌注损伤诱导的心肌微血管内皮细胞和心肌组织的焦亡。（6）天麻素通过激活蛋白激酶B/p38MAPKs信号通路，抑制血清剥夺/复灌诱导的细胞凋亡，从而发挥抗心肌缺血再灌注损伤的保护作用。（7）天麻素通过AMPK/FoxO1/TFEB信号途径，抑制了FoxO1的活性，以此促进TFEB入核，增加泡沫细胞的溶酶体生物发生和自噬及抑制泡沫细胞的形成。（8）人参皂苷 Rg1改善心肌缺血再灌注损伤后的心肌生理功能，减少流出液中心肌酶的释放和心肌梗死面积，从而发挥抗心肌缺血再灌注损伤的保护作用。（9）人参皂苷Rb1抑制心肌缺血再灌注损伤诱导的心肌细胞凋亡与mTOR信号通路的激活有关。（10）深入开展了基于柠檬酸的荧光和光声双模式成像的电活性生物可降解材料的研究，开发了一种具有成像和治疗的多功能柠檬酸盐基生物材料平台。（11）通过构建具不同天麻素缓释量聚氨酯弹性体，探讨其对炎症反应和凝血的调控作用，最终促进血管新生，为小径血管支架的设计提供依据。研究阐明了天麻素在心肌缺血再灌注损伤中的保护作用以及信号转导机制，为天麻素在心血管保护的作用及其机制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。