益气养阴活血中药抑制线粒体mPTP减轻缺血再灌注心肌损伤的分子机制

Coronary heart disease is the leading cause of death globally. Following an acute coronary artery occlusion, timely myocardial reperfusion using either primary percutaneous coronary intervention or thrombolytic therapy remains the most effective treatment strategy. However, the full benefits of myocardial reperfusion are not realised, given that myocardial ischemia reperfusion injury will be a major clinical problem obstructing the development. Yiqi Yangyin Huoxue Chinese herb preparation （Shenmai Injection and salvia miltiorrhiza injection）have definite effect on MIRI due to multiple components and multiple targets to treat disease. The mechanisms have not been reported yet. The objective of research are to clarify that Yiqi Yangyin Huoxue Chinese herb preparation exert cardioprotective effects against MIRI in isolated perfused rat hearts and cardiomyocytes and the mechanisms involve activation of specific survival signals reperfusion injury salvage kinase (PI3K-Akt pathway and ERK1/2 pathway), hence inhibiting mitochondrial permeability transition pore opening(mPTP) directly. What is called "tonifying qi and yin to help strengthen healthy energy"； The mechanisms also involve reducing ROS generation and inhibition of calcium overload. Hence inhibiting mitochondrial permeability transition pore opening indirectly. What is called "promoting blood circulation to help eliminate pathogens". The research techniques, such as Western bloting, electron microscope technique and laser scanning confocal microscope, some reagents, such as opener of mPTP and specific inhibitor of PI3K-Akt and ERK1/2 pathways are used in the research. We expect to clarify the basic theory of healthy qi and pathogenic factors in traditional Chinese medical science at mitochondrial level. This project help us research Chinese medicine against MIRI better and promote the development of Chinese Medicine Theory.

冠心病是全球范围造成死亡的最主要原因，经皮冠脉介入、溶栓等心肌再灌注成为急性心梗的最有效的治疗方法，但缺血再灌注心肌损伤（MIRI）成为面临的主要难题，益气养阴活血中药（参麦注射液与丹参注射液合用）发挥中药多成分多靶点的协同作用，治疗MIRI疗效确切，但作用机制尚未见报道，本课题拟应用大鼠离体心脏、乳鼠心肌细胞，采用Western、电镜、激光共聚焦显微镜等方法，借助mPTP开放剂、PI3K及ERK1/2特异性抑制剂，研究益气养阴活血中药通过激活再灌注损伤营救激酶途径中PI3K-Akt和ERK1/2信号通路，直接抑制mPTP开放，提高线粒体的耐受性，"益气养阴以扶正"；减少活性氧产生、抑制钙超载，降低诱导mPTP开放的因素，"活血通络以祛邪"。阐明益气养阴活血中药抗MIRI的分子机制，从线粒体水平阐释中医正邪理论的科学内涵，有助于抗心肌损伤中药的研究及中医理论水平的提高。

随着经皮冠脉介入、溶栓等治疗方法的迅速发展，如何减轻心肌缺血再灌注损伤(MIRI)成为心血管研究的热点之一，MIRI属于中医“胸痹”、“心悸”、“怔忡”，气阴两虚、瘀血内阻是临床常见中医证型，治以益气养阴、活血通络，益气养阴以“扶正”，常用参麦类方剂；活血通络以“祛邪”，常用丹参类方剂，益气养阴活血中药(参麦注射液与丹参注射液联合应用，YYH)是临床常用的治疗MIRI的合用药物。线粒体透性转换孔(mPTP)是存在于线粒体内膜的一种非特异性通道，mPTP 开放是导致线粒体功能障碍、细胞凋亡和坏死的主要因素，抑制其开放成为心肌保护作用的最终效应器。.本课题应用Langendorff离体心脏缺血再灌注(I/R)损伤模型、原代培养乳鼠心肌细胞缺氧复氧(H/R)及过氧化氢(H2O2)损伤模型，采用Western blotting、透射电镜、mPTP开放的检测等技术或方法，借助PI3K及ERK1/2特异性抑制剂LY294002 、PD98059，研究YYH抗MIRI的分子机制。结果表明，YYH通过通过激活RISK途径中PI3K-Akt 和ERK1/2 信号通路，直接抑制mPTP 开放，提高线粒体的耐受性，“益气养阴以扶正”；减少ROS产生、抑制钙超载，降低诱导mPTP 开放的因素，“活血通络以祛邪”，减轻了MIRI，发挥了心肌保护作用。YYH主要通过减少诱发mPTP开放的因素-ROS的产生和线粒体钙超载，“间接”抑制mPTP开放。参麦注射液可“直接”抑制mPTP开放，提高线粒体耐受性，发挥了“扶正”作用，主要有效成分包含人参皂苷Rb1。.在本项目的支持下，超额研究了“扶正”药物参麦注射液能“直接”抑制mPTP 开放，而活血药物丹红注射液无此作用，为以后的工作奠定了基础。利用本项目建立的模型及方法，进行了相关的益气温阳药物参附注射液及活血药物丹红注射液的相关研究，取得了一定成果。发表论文6篇，培养已毕业硕士生各1名，在读硕士生1名。