mTOR介导的细胞自噬在“痰瘀互结”致心肌无复流的作用及活血化痰中药干预机制研究

Although percutaneous coronary intervention has become the predominantly therapy for acute myocardial infarction, the no reflow or slow flow phenomenon during the procedure is prone to induce aggravating cardiac dysfunction. Our previous studies have confirmed that patients with Phlegm and blood stasis are vulnerable to no reflow during the procedure. Importantly, Danlou Tablet can reduce no reflow area via reducing myocardial ischemia/reperfusion injury and endothelial damage, but its exact mechanism has not been elucidated. During the process of ischemic reperfusion injury, autophagy can be induced and play important role in endothelial protection. Therefore, we speculate that "promoting blood circulation and eliminating phlegm" may reduce no reflow area by inducing autophagy. To confirm this hypothesis, we will construct myocardial ischemia/reperfusion injury model of phlegm and blood stasis, detecting autophagosome, marker protein and no reflow myocardium area, further to monitor the effect of Danlou Tablet on autophagy. Finally, in vitro cultured coronary artery endothelial cells, to observe the application of Danlou tablets on the influence of the autophagic process and its signal pahtway in hypoxia/reoxygenation condition. This research will further provide experimental evidence to clarify the mechanism underlying the treating both phlegm and blood stasis to protect coronary endothelial damage caused by ischemia/reperfusion, providing the new targets and new methods for no reflow .

经皮冠状动脉介入治疗是心肌梗死治疗的里程碑，但术中易于发生无复流或慢血流现象，加重心功能障碍。前期研究发现“痰瘀互结”证患者更易于发生无复流现象，而活血化痰中药丹蒌片能够减少无复流的发生，且实验研究显示丹蒌片具有内皮保护，并减轻小鼠心肌缺血/再灌注（I/R）损伤、缩小梗死面积的作用，但其机制尚不明确。缺血再灌注损伤过程中可发生自噬，而适度自噬可抑制I/R引起的内皮损伤。因此，我们推测“痰瘀同治”法可能是通过诱导自噬而发挥其内皮保护和减少无复流的作用。为了证实这一假说，我们将在整体和细胞两个水平复制心肌I/R损伤模型，观察应用丹蒌片对再灌注无复流小鼠模型自噬体的形成、自噬标志蛋白的表达以及无复流心肌面积的影响；并观察应用丹蒌片对缺氧/复氧冠状动脉内皮细胞功能、自噬过程及其信号通路的影响。本课题将从新的视角阐明“痰瘀同治”法保护冠脉内皮、抗I/R的机制，为防治无复流提供新靶点和新方法。

经皮冠状动脉介入治疗是心肌梗死治疗的里程碑，但术中易于发生“无复流”现象，加重心功能障碍。前期研究发现“痰瘀互结”证患者更易于发生“无复流”现象，而活血化痰中药丹蒌片能够减少“无复流”的发生，其机制不明。本研究构建了“痰瘀互结”型再灌注心肌无复流动物模型和缺氧／复氧细胞模型，从器官、细胞、分子水平探讨内皮细胞自噬在心肌缺血再灌注损伤发生中的作用，并从调节细胞自噬角度观察活血化瘀中药丹蒌片防治心肌无复流的作用。结果显示，丹蒌片减少了I/R损伤小鼠模型心肌梗死面积，降低缺血面积，改善心功能，提高左室射血分数；增加LC3Ⅱ、Bclin1、Atg5等自噬相关通路蛋白的表达；增加心肌自噬体的数量；降低心肌细胞凋亡蛋白Caspase-1的表达。细胞水平显示，丹蒌片减少了细胞凋亡，上调内皮细胞功能蛋白酶eNOS的表达，提高了LC3Ⅱ、Beclin1、 Atg5等自噬相关蛋白的表达；增加了GSK-3β,PI3KC3和mTOR的磷酸化水平。本研究显示，丹蒌片可能通过GSK-3β/mTOR信号通路，调节细胞自噬，抑制内皮细胞凋亡，从而发挥减缓心肌I/R损伤，改善“无复流”作用。目前研究成果已经发表论文6篇，SCI收录4篇，编写专著1部，申报专利3项，培养博士生1名、硕士生3名，研究成果被广泛应用于冠心病的防治。