通窍活血汤调控PI3K/Akt/mTOR自噬信号通路修复脑微血管内皮细胞损伤的作用机制研究

The impairment of brain microvascular endothelial cells（BMEC）is the the early lesions and fundamental reason of cerebral ischemia injury..Autophagy is closely related with the pathomechanism of ischemic cerebrovascular disease.Can TongQiao HuoXue Decoction regulate autophagy of BMEC to exert vascular protection so as to treat ischemic stroke? Is PI3K/Akt/mTOR signal transduction pathway involved in this process? Our previous study had showed TongQiao HuoXue Dection had proctective effect on cerebral ischemia-reperfusion injury in rats, significantly inhibited the damage of hippocampal neurons and reduced the opening of TJ on blood–brain-barrier by increasing the expression of tight junction proteins.Based on our previous research,this program will revolve around the theme of “Changes of cerebral blood flow in rats→Changes of vasomotor function→Structure and function of BMEC”to illustrate the regulation of autophagy to protect against BMEC damage during the Oxygen-Glucose Deprivation at the the levels of”Animal-Organ-Cell”, then confirm the cerebrovascular protection and investigate the mechanism of TongQiao HuoXue Dection on proctecting BMEC damaged by Oxygen-Glucose Deprivation/Reoxygenation by regulating autophagy via PI3K/Akt/mTOR.This work would provide the new train of thought for the research of Traditional Chinese Medicine on treating ischemic stroke.By using BMEC,which is the structural basis of “brain collateral”as the breakthrough point,the scientific connotation of “Quickenening the blood and dispersing stasis,freeing the collateral vessels and opening orifice”will be elucidated.

脑微血管内皮细胞损伤是脑缺血损害的早期病变和基本动因，细胞自噬和缺血性脑血管疾病病理机制密切相关。通窍活血汤是否可以通过调控脑微血管内皮细胞的自噬发挥血管保护作用抗缺血性中风？PI3K/Akt/mTOR信号是否参与该调控作用？我们前期研究显示通窍活血汤对大鼠脑缺血再灌注损伤模型有较好的脑保护作用，在此基础上，本项目以脑组织缺血缺氧损伤时“脑血流→脑血管舒缩→脑微血管内皮细胞结构与功能”的变化为主线，从“整体动物→器官→细胞”层次阐明缺糖缺氧损伤后细胞自噬调节和脑微血管内皮细胞损伤的关系，明确通窍活血汤调控自噬的脑血管保护作用，并围绕PI3K/Akt/mTOR信号，探讨其血管保护作用的自噬机制，为治疗缺血性中风的中药研究提供新思路。同时，以作为“脑络”结构组成基础的脑微血管内皮细胞为切入点,阐明传统经典方通窍活血汤在中风治疗中“活血祛瘀,通络开窍”的科学内涵。

脑微血管内皮细胞损伤是脑缺血损害的早期病变和基本动因，细胞自噬和缺血性脑血管疾病病理机制密切相关。本研究从整体动物实验和细胞实验两种水平，阐明缺血缺氧损伤后细胞自噬调节和BMEC损伤的关系，明确通窍活血汤调控自噬的脑血管保护作用，并围绕PI3K/Akt/mTOR信号，探讨其血管保护作用的自噬机制。动物实验的研究结果表明，大鼠脑缺血再灌注损伤后自噬被激活，透射电镜下可见双层膜结构的自噬体，随着再灌注时间的增加，自噬相关蛋白Beclin1和LC3的表达也逐渐增强，再灌注24h时达到峰值，此时通过尼氏染色说明神经损伤也最严重。免疫荧光双染法表明自噬在神经元、星形胶质细胞和脑微血管内皮细胞中均有表达。TQHXD能够激活PI3K/AKt/mTOR通路，抑制脑缺血损伤后的神经细胞自噬，继而发挥了神经保护的作用。细胞实验的研究结果表明，OGD/R损伤大鼠BMECs后自噬被激活，细胞活性降低，细胞内自噬体数量增多，细胞凋亡程度加重。通窍活血汤含药脑脊液可以增强受损BMECs的活力，减少自噬体数量，改善细胞凋亡情况，说明通窍活血汤含药脑脊液对BMECs具有一定的保护作用。这种调节作用与调节PI3K/Akt/mTOR自噬信号通路有关，即通窍活血汤含药脑脊液能够激活PI3K/Akt/mTOR自噬通路，对OGD/R损伤后的BMECs有所改善，进而发挥其保护作用。结合体内外实验，得出结论：TQHXD通过激活PI3K/AKt/mTOR通路，抑制脑缺血损伤后的神经细胞自噬，发挥了脑血管保护保护作用。本研究验证了科学设想，完成了预期计划。为治疗缺血性中风的中药研究提供新思路。一定程度上阐释了传统经典方通窍活血汤在中风治疗中“活血祛瘀,通络开窍”的科学内涵与调节自噬、保护损伤脑血管有关。