益气活血中药调控线粒体自噬保护缺血心肌的机制研究

Mitochondrial autophagy （mitophagy）are closely related to the ischemic heart disease. Previous studies have showed that Chinese medicine Recipe, Shuangshen Tongguan with the effects of reinforcing QI and activating blood circulation, can improve myocardial energy metabolism, regulate autophagy and apoptosis, reduce the opening degree of the mitochondrial membrane permeability transition pore (mPTP), protect myocardial mitochondria, which can effectively protect the ischemic myocardium, reduce myocardial necrosis and protect heart function. So, we believe that the cardio-protective effect of Chinese medicine with the effects of reinforcing QI and activating blood circulation is closely related to its regulation of mitochondrial autophagy pathway PINK1 / Parkin and NIX / Bnip3 / FUNDC1. This study will take the chronic ischemic heart disease and the nutrient deficiency in miniature pig to induce cardiomyocyte autophagy model as experimental object. The cardiac magnetic resonance (CMR), transmission electron microscopy, immunohistochemistry, flow cytometry , Western blot method are applied to reveal the mechanism of regulating autophagy to protect myocardial mitochondria by Chinese medicine with the effects of reinforcing QI and activating blood circulation through the observation of the myocardial cell ultrastructure, cytolysosome, mitochondria morphological structure and though testing the expression of mitochondria autophagy key protein PINK1, Parkin, Bnip3, NIX, FUNDC1.

线粒体自噬（mitophagy）与缺血性心脏病的发生发展有密切关系。既往研究证明，益气活血中药双参通冠方可改善心肌能量代谢，调控自噬、凋亡，降低心肌细胞线粒体膜通透性转换孔（mPTP）开放程度，保护心肌线粒体，从而有效保护缺血心肌，减少心肌坏死，保护心功能。基于此，我们认为益气活血中药的心肌保护作用与其调控线粒体自噬通路PINK1/Parkin和 NIX/Bnip3/FUNDC1密切相关。本研究将以小型猪慢性缺血性心脏病模型和营养缺乏诱导心肌细胞自噬模型为实验对象，应用心脏磁共振（CMR）、透射电子显微镜、免疫组化、流式细胞、Western blot等技术方法，通过观测心肌细胞超微结构、自噬小体、线粒体形态结构，检测调控心肌细胞线粒体自噬关键性蛋白PINK1、Parkin、Bnip3、NIX、FUNDC1、LC3、ULK1等的表达情况，揭示益气活血中药调控线粒体自噬保护心肌的作用机制。

心肌缺血其本质是缺血造成心肌细胞氧供和能量不足所致使心肌细胞处于“饥饿”状态。线粒体自噬与缺血性心脏病的发生发展有密切关系。心肌细胞能量代谢需求高，线粒体含量多。心肌缺血后多种原因造成线粒体损伤，线粒体自噬可清除受损的线粒体，保护心肌。课题组既往研究证明，益气活血中药双参宁心胶囊可改善心肌能量代谢，调控自噬、凋亡，降低心肌细胞线粒体膜通透性转换孔开放程度，保护心肌线粒体，从而有效保护缺血心肌，减少心肌坏死，保护心功能。课题组提出益气活血中药的心肌保护作用可能与其调控线粒体自噬通路PINK1/Parkin和NIX/Bnip3/FUNDC1密切相关。本研究以大鼠和小型猪心肌缺血再灌损伤模型、心肌细胞缺氧复氧损伤模型为实验对象，应用心脏磁共振、透射电子显微镜、免疫组化、Westernblot等技术方法，观测心肌细胞超微结构、自噬小体、线粒体形态结构，检测调控心肌细胞线粒体自噬关键性蛋白PINK1、Parkin、NIX、FUNDC1、LC3等表达情况。结果发现，SSNX通过可通过开放线粒体ATP敏感性钾通道使缺血心肌线粒体膜电位（△Ψm）增高，减少大鼠心肌细胞线粒体自噬相关蛋白LC3B、PINK1、Parkin、FUNDC1的表达，减轻心肌细胞线粒体自噬，保护大鼠MIRI。双参宁心胶囊对心肌缺血再灌注损伤小型猪心肌组织也有同样的保护作用，可减轻心肌组织结构损伤及维持线粒体功能相对完整，其机制可能是通过抑制PINK1/Parkin 通路介导的线粒体自噬过度激活，从而保护心肌缺血再灌注损伤后小型猪心脏功能。其有效成分人参皂苷Rg1、延胡索乙素、丹酚酸B可抑制FUNDC1介导的线粒体自噬保护H9c2心肌细胞H/R损伤。推测人参皂苷Rg1、延胡索乙素、丹酚酸B是SSNX调控FUNDC1介导的线粒体自噬保护MIRI的物质基础。中药SSNX有望成为调控线粒体自噬对抗MIRI的有效治疗药物。