基于凝血酶诱导自噬探讨急性缺血性中风“瘀毒致风”的分子机制

Previous studies have confirmed that thrombin-inflammatory reaction is an important biological basis for pathogenesis of blood stasis & toxin of acute ischemic stroke. Recently, it is also found that thrombin can induce autophagy. This project suggests that thrombin-induced autophagy is involved in the development of acute ischemic brain damage and plays an important role in the formation of stroke pathogenesis by blood stasis & toxin. The middle cerebral artery occlusion rat model and thrombin-stimulated glucose-oxygen deprived rat brain microvascular endothelial cells, neurons and glial cells are selected as subjects. Electron microscopy, immunohistochemical technique, WB, confocal laser scanning microscope, mRFP-GFP-LC3 double fluorescent indicator technique are used to observe the autophagosomes, phagosome autolysosome, autophagy flow, the ratio of LC3-II/I, PI3K/Akt/mTOR signaling pathway, PPAR-gamma and AMPK activation of the above related cells and the intervention effect of detoxification and removing stasis method; The research results will elucidate the modern biological basis of the pathogenesis of acute ischemic stroke caused by blood stasis and toxin from the perspective of cerebral collateral relating cells’ autophagy and the regulation of cellular signaling pathways based on syndrome detecting from recipe used . It will also provide an important evidence for understanding the pathogenesis of acute ischemic stroke caused by blood stasis and toxin, and new ideas and strategies for promoting the clinical practice of preventing and treating ischemic stroke by the method of detoxification and removing stasis.

课题组前期研究证实，凝血酶-炎症反应是急性缺血性中风瘀毒病机的重要生物学基础。新近发现凝血酶可以诱导细胞自噬。本项目提出凝血酶诱导自噬参与了急性缺血性脑损伤的发生发展过程，在“瘀毒致风”病机形成中具有重要作用。以MCAO大鼠模型和凝血酶刺激的糖氧剥夺大鼠脑微血管内皮细胞、神经细胞和神经胶质细胞为受试对象，运用透射电镜、免疫组化、WB、激光共聚焦、mRFP-GFP-LC3 双荧光自噬指示等技术观察相应细胞吞噬泡、自噬小体、自噬溶酶体、自噬流、LC3-II/I比值、PI3K/Akt/mTOR信号通路、AMPK活化以及化瘀解毒法的干预作用。研究结果将从脑络相关细胞自噬及细胞信号通路调控的角度、通过以方测证的方法阐明急性缺血性中风“瘀毒互结”病机的现代生物学基础，为深入理解急性缺血性中风“瘀毒致风”的病机实质提供重要依据，对促进化瘀解毒法防治缺血性中风的临床实践提供新的的思路与策略。

课题组前期研究证实凝血酶-炎症反应是急性缺血性中风瘀毒病机的重要生物学基础，现有研究发现凝血酶可诱导自噬，过度自噬会导致脑损伤。本项目提出凝血酶诱导自噬参与了急性缺血性脑损伤的发生发展过程，在“瘀毒致风”病机形成中具有重要作用。在整体动物部分建立了缺血性中风瘀毒互结证大鼠模型，使用该大鼠模型观察到造模后 12 h和24 h脑组织凝血酶与自噬表达上升最明显，且具有相关性，化瘀解毒方干预后，两个时间点的凝血酶及自噬表达皆明显下降。结合TTC染色、病理切片、透射电镜等检测方法得出化瘀解毒方可通过抑制凝血酶的过表达下调自噬的发生，从而发挥脑保护作用。另于造模后24h观察脑组织自噬及脑血管内皮结构和功能的变化及化瘀解毒方的干预作用，化瘀解毒方对模型大鼠脑组织过度自噬导致的脑血管内皮结构和功能损伤具有保护作用。在细胞水平上观察了PI3K/Akt/mTOR信号通路激活程度，该通路的抑制诱导自噬，结果显示化瘀解毒方含药血清对OGD或OGD加凝血酶共同作用模型的脑微血管内皮细胞、星形胶质细胞、PC12细胞都具有保护作用，化瘀解毒方能抑制细胞过度自噬，与该通路抑制剂LY294002的作用相反，化瘀解毒方能抵消部分LY294002的作用。结合体内、体外部分实验结果我们得出结论：瘀毒互结是急性脑缺血损伤的重要病机。凝血酶表达升高并诱导自噬过度激活从而引起脑血管和神经细胞损伤，这可能是瘀血阻滞、毒损脑络病机的现代科学内涵。基于瘀毒病机理论组建的化瘀解毒方能减轻脑梗死体积、抑制凝血酶及其诱导的过度自噬，并且通过抑制过度自噬保护血管内皮结构和功能，从而减少脑缺血损伤，且化瘀解毒方对PI3K/Akt/mTOR信号通路的干预是其中重要的调节机制。本研究丰富和完善了急性缺血性中风瘀毒病机理论的现代科学内涵，为中医药治疗急性缺血性中风临床实践提供新的思路与策略。