基于自噬信号通路研究参芎注射液抗MIR及心律失常的分子机制

Myocardial ischemia reperfusion (MIR) and induced arrhythmia, which symptoms are similar to ‘chest impediment’, ‘heart pain’, ‘palpitations’ in traditional Chinese medicine, are the most lethal pathophysiological processes in cardiovascular diseases.It causes a heavily family and social economic burden. To date, the commonly prescribed drugs against myocardial ischemia reperfusion and induced arrhythmia merely bring about unsatisfactory effects, and/or possessing obvious side effects. The anti myocardial ischemia reperfusion and induced arrhythmias effects of Shenxiong Glucose Injection(SGI) had been firmly demonstrated except its mechanisms. Currently, it believed that the autophagy is excessively promoted under myocardial ischemia reperfusion by activating of energy receptor AMPK and nutrition receptor mTOR leading to endogenous injury. Therefore, it´s a new strategy to prevent excessive autophagy injury to against MIR. Based on our previous studies, the efficacy of SGI on myocardial ischemia reperfusion and induced arrhythmia are planned to be evaluated in this study target on PI3K-Akt-mTOR-Atg1、JNK1-Bcl-2-Beclin1、Ca2+/CaMKK-AMPK signaling pathways with myocardial ischemia/reoxygenation, langendorff and rat MIR models, and to investigate SGI effects on ion channels, calcium overload, mPTP and mitochondrial membrane potential with patch clamp and molecular biology technology to identify scientific hypothesis. This study can elucidate the mechanisms and scientific connotation of blood-activating, qi-moving, stasis resolving medicine SGI on ‘chest impediment’, ‘heart pain’ and ‘palpitations’ with different models in vitro and in vivo.

心肌缺血再灌注损伤(MIR)及其所致心律失常(与中医学“胸痹”、“心痛”、“心悸”等病症相似)是目前心血管疾病的主要致死原因，家庭与社会不堪重负。常用药物疗效不甚理想，和/或副作用明显。参芎注射液对MIR及心律失常等病症疗效确切、安全但机制不清。MIR损伤时机体通过调节能量感受器AMPK与营养感受器mTOR信号分子促进自噬的过度激活反而带来损害，因此对其进行干预是抗MIR的新策略。本项目在预实验基础上通过心肌细胞缺氧/复氧、离体心脏灌流和大鼠MIR模型，提出参芎注射液可能通过干预自噬PI3K-Akt-mTOR-Atg1、JNK1-Bcl-2-Beclin1、Ca2+/CaMKK-AMPK通路的多个关键蛋白，从多个层面调控自噬及心肌细胞离子通道，抑制过度自噬、钙超载、mPTP开放的科学假说，以期阐明活血、行气、化瘀中药防治“胸痹”、“心痛”、“心悸”的分子机制及现代科学内涵。

缺血性疾病已成为中国乃至全球范围内的头号致死原因。心肌缺血再灌注 (Myocardial Ischemia Reperfusion, MIR)引起的并发症，特别是心律失常是造成缺血性心脏病致死的主要原因。虽然丹参素、川芎嗪防治MIR 有一定研究基础，但两者配伍及以之为主要药效成分的复方制剂防治MIR 心律失常的分子机制尚不明确，本项目从体内外验证参芎葡萄糖注射液(SGI) 可能通过干预自噬通路的多个关键蛋白，从多个层面调控自噬及心肌细胞离子通道，抑制过度自噬、钙超载、mPTP 开放的科学假说。.首先成功建立H9c2心肌细胞缺氧/复氧模型、筛选SGI及其药效成分对H9c2心肌细胞的给药剂量，通过存活率和生化指标检测，明确了SGI及其药效成分对缺氧/复氧H9c2心肌细胞的药效。随后建立大鼠离体心脏灌流模型，观察 SGI 及其药效成分对 MIR 损伤模型心功能及所致心律失常的影响。接下来通过结扎大鼠左冠状动脉前降支的方法，以手术造模缺血30min、再灌注24h成功建立大鼠心肌缺血再灌注损伤模型，深入探讨SGI抗MIR及心律失常的药效和分子机制。此外还建立了斑马鱼缺氧/复氧模型和异丙肾上腺素诱导斑马鱼心衰模型，结果表明SGI能显著改善缺氧/复氧和异丙肾上腺素处理后斑马鱼心率，对物理诱导和化学诱导的心肌损伤均有保护作用。.综上，本研究证实SGI可通过减轻氧化应激反应，减少心肌酶的过度释放保护心肌细胞，同时通过Bcl-2-p-JNK1-Beclin1以p-AKT-mTOR/p-mTOR-Beclin1信号通路下调Beclin1、ATG5、ATG7、LC3B等自噬蛋白的表达改善过度自噬造成的心肌细胞损伤，发挥抗大鼠心肌缺血再灌注损伤效应，对拓宽SGI这种中药复方制剂的临床应用，对降低缺血性心脏病的病死率、致残率，恢复患者生活自理能力，减轻家庭及社会沉重的负担有着重要的现实意义，阐明了中药防治“胸痹”、“心痛”“心悸”“怔仲”的科学内涵。