益气活血药对心肌缺血线粒体能量代谢和信号转导影响的研究

The status of myocardial ischemia and coronary microcirculation disorder could be evaluated, relying on the classical model of myocardial infarction and cell ischemia model, using the techniques of cardiac magnetic resonance imaging, co-immunoprecipitation, RNA interference technology, microscopic image analysis and immune-histochemical technology. The mechanism of energy metabolism and regulation of hypoxic-ischemic injury could be clarified by observing the changes of function, structure, adenosine triphosphate, adenosine diphosphate and adenosine monophosphate of myocardial mitochondria. The status of mitochondrial fusion, fission, autophagy and the process of myocardial injury repair could revealed by observing the expression of proteins and genes like mitochondrial associated molecules Mfn2、Drp1、FUNDC1、LC3、Pink1. The regulatory pathways of Notch signal transduction in ischemic myocardium could be revealed by observing the functions of Notch1、RBP-J、Hes1、NF-κB in Notch signal transduction. Then, the mechanism of "cross talk" between Notch and mitochondrial metabolism could be clarified. It also based on the qi and blood theory of traditional Chinese medicine. The changes of energy metabolism, heart and microvascular structure and function and other related indicators were analyzed. Then, the theoretical basis of heart qi and / or heart qi deficiency could be explored based on the observations of tonifying Qi and activating blood traditional Chinese medicine promoting the role of blood supply and oxygen supply of myocardium, improving myocardial ischemia.

依托经典心梗模型和细胞缺血模型，借助心脏核磁、免疫共沉淀、RNA干扰技术、显微图像分析和免疫组化等技术，通过观察心肌梗死、心脏和微血管结构与功能的变化，评价心肌缺血及冠脉微循环障碍状态；观察心肌细胞线粒体功能、结构及ATP、ADP、AMP的变化，阐明能量代谢及调控缺血缺氧损伤的作用；观察线粒体相关分子Mfn2、Drp1、FUNDC1、LC3、Pink1蛋白和基因的表达，揭示线粒体融合、分裂、自噬状态及参与心肌损伤修复的过程；观察Notch信号Notch1、RBP-J、Hes1、NF-κB的作用，揭示Notch信号转导在缺血心肌中的调控途径，进而阐明Notch与线粒体代谢"交叉对话"机制；在观察益气活血中药作用上述调控机制促进心肌供氧供血、改善心肌缺血的基础上，以中医气血理论为指导，分析基于能量代谢、心脏和微血管功能结构与功能等相关指标的变化情况，探索阐释心气和/或心气虚的理论基础。

由多种原因引起的心肌梗死常出现心室重构、心功能不全、甚至心衰或死亡，如何快速恢复血流，挽救濒死心肌，保护心脏功能具有重要研究意义。心脏是一种动力器官，能量变化对其功能影响重大，本研究以线粒体能量代谢、线粒体动力学和线粒体自噬为切入点，在术后7天、28天两个时间节点动态观察相关指标变化情况，揭示心肌缺血缺氧的发生发展并探究益气活血方对心肌的保护作用。本项目以结扎大鼠冠状动脉左前降支方法建立在体心肌梗死模型，采用H9c2心肌细胞系作为实验对象，构建心肌缺血缺氧模型，发现在能量代谢方面，益气活血方能够增加ATP生成，适度增加葡萄糖氧化供能，防止脂肪酸过度氧化，保护心肌细胞线粒体，进而发挥心肌保护作用；在线粒体结构和功能上，不同时间点模型组心肌细胞线粒体生物合成能力降低，益气活血方组PGC-1α、NRF1、mtTFA mRNA和蛋白表达升高，模型组线粒体膜电位降低，而益气活血方则可逆转线粒体去极化现象；电镜下模型组形成典型的自噬体和自噬溶酶体的自噬现象，益气活血方组可见少量自噬泡存在，自噬相关分子LC3、Beclin 1的表达在心肌梗死初期降低，28天时增强。信号通路的相关变化方面，心肌梗死后Notch、LKB1、AMPK蛋白和mRNA表达明显降低，使用Notch抑制剂DAPT后，28天时与模型组相比，不仅Notch蛋白和mRNA表达下降，LKB1、AMPK蛋白和mRNA表达也明显降低，提示Notch与AMPK信号通路基于LKB1存在交叉对话。以上这些的研究从线粒体结构、功能变化和能量代谢角度入手，揭示了心肌缺血后益气活血方对心肌产生保护作用可能的机制。