通心络促进脂钙蛋白-2分泌而调节细胞自噬在缺血再灌注后心肌微血管保护中的机制研究

Myocardial ischemia/reperfusion injury is the primary cause of remained acute adverse events after reperfusion therapy for acute myocardial infarction. There are still no quite efficient treatments and no obvious progress in research on myocardial reperfusion injury. We considered the reperfusion injury of cardiac microvasculature as the trigger of myocardial injury, and demonstrated the protection of cardiac microvasculature was a core mechanism in myocardial preservation after reperfusion. We previously found that induction of autophagy by Tongxinluo protected cardiac microvascular endothelial cells from ischemia/reperfusion injury, and the results from protein microdot arrays indicated lipocalin-2 was greatly up-regulated by Tongxinluo in our additional study. On the basis of above, we raise a hypothesis that secretion of lipocalin-2 was promoted by Tongxinluo through the MEK/ERK positive feedback loop, which induced autophagy in cardiac microvascular endothelial cells, then protected microvasculature and preserved myocardium. We will apply models in reperfusion injury including the hypoxia/ reoxygenation of cardiac microvascular endothelial cells and the Ligation/loosening of Left anterior descending coronary artery in rat heart with Tongxinluo treatment to carry out the study. Some pathological and molecular biological methods will be used to test the regulatory mechanisms of autophagy or apoptosis by lipocalin-2, and the relationship between the MEK/ERK and lipocalin-2. We will demonstrate the hypothesis from three aspects including endothelial barrier function, microcirculation maintenance, and cardiac structure and function. Hopefully the study would provide new perspective and evidence for the treatment of cardiac microvascular dysfunction and myocardial reperfusion injury with Tongxinluo.

心肌缺血再灌注损伤是急性心梗血运重建后仍存在急性不良事件的主要原因，目前临床治疗欠佳。我们既往研究表明保护心肌微血管为防治心肌缺血再灌注损伤的重要靶点。我们前期工作发现了通心络可促进内皮细胞自噬而保护心肌微血管的机制，并且通过蛋白芯片筛选出通络干预后发生明显变化的靶点分子脂钙蛋白-2；结合前期基础，我们提出了通心络可激活MEK/ERK通路正反馈地促进心肌微血管内皮分泌脂钙蛋白-2而激活自噬，进而防治心肌缺血再灌注损伤的科学假设。我们将采用心肌微血管内皮缺氧/复氧模型以及大鼠前降支结扎/松弛缺血再灌注损伤模型，运用病理及分子生物学技术，紧密围绕脂钙蛋白2这一靶点及MEK/ERK通路，研究其对微血管内皮细胞凋亡及自噬的调控机制，并从微血管屏障功能、微循环血流灌注、再灌注心肌结构及功能三方面证明上述假设，从而为通络方药在心肌微血管缺血再灌注损伤的治疗及心肌保护提供新的理论及实验依据。

心肌缺血再灌注损伤是急性心梗血运重建后仍存在急性不良事件的主要原因，目前临床治疗欠佳。我们既往研究表明保护心肌微血管为防治心肌缺血再灌注损伤的重要靶点。在本研究中，我们采用心肌微血管内皮缺氧/复氧模型以及大鼠前降支结扎/松弛缺血再灌注损伤模型，证实了通心络可激活MEK/ERK通路正反馈地促进心肌微血管内皮分泌脂钙蛋白-2而激活自噬，进而防治心肌缺血再灌注损伤，改善心功能；我们收取了缺氧/复氧模型组、正常对照组及通心络处理组的心脏微血管内皮细胞培养基上清液，通过采用蛋白芯片以及生物信息学手段，证实了在缺血再灌注损伤的条件下通心络可调节心脏微血管内皮细胞分泌的包括LCN2在内的多种细胞因子，从而参与到细胞凋亡及自噬、生长及分化等过程；收集了相关处理后的细胞匀浆，对细胞内的蛋白变化进行蛋白组学质谱分析及生物信息学分析，发现相关的靶点蛋白及调节机制；此外，我们深入研究了自噬保护心脏微血管内皮细胞损伤的不同的调节机制，在体外实验中证实了IKKβ, IκBα and p65的磷酸化，NF-KB通路的激活与自噬的抑制相关，而通过药物抑制上述通路可以保护微血管内皮；体内实验中我们采用大鼠模型，证实了通心络显著改善了射血分数、减少梗死面积，促进了梗死周围区域的血管生成，其机制与促进AMPK/mTOR磷酸化激活、上调了自噬相关蛋白LC3的表达，进而下调了梗死区域凋亡相关蛋白Bax的表达有关。这些研究结果为不仅为通络方药作为一种微血管内皮保护剂在临床上的合理应用提供理论和实验基础，而且为自噬调节作为治疗缺血再灌注损伤干预靶点的机制方面提供可能的依据。