从miR-210介导VEGF/PI3K/Akt通路研究清热解毒通络治疗急性脑梗死血管新生的作用机制

Fire-toxin, transformed from the accumulation of wind, fire, phlegm and blood stasis during acute cerebral infarction, is the turning point causing apostasis and exacerbation of the disease. Previous study (No. 81173233) has confirmed that this process corresponds to (inosculates with) ischemic cascade damage, which is based on inflammatory reaction. It is currently a hot research area that ischemic cascade reaction damages both the structure and function of neurovascular unit (NVU) and inhibits angiogenesis. Clearing heat, detoxicating and dredging collaterals protects the injured NVU, during acute cerebral infarction, with the key process adjusting miR-210 which mediates VEGF/PI3K/Akt pathway and promoting angiogenesis. Taking MCAO rat model and vitro cultured neuron, astrocyte, endothelial cells as objects, our research takes advantage of miR-210 agonist thereby to specifically observe the different effects of Kudiezi Injection on VEGF/PI3K/Akt pathway mediated by miR-201 at different times of cerebral ischemia, and explicit its molecular mechanism of promoting angiogenesis. The key process and target of clearing heat, detoxicating and dredging collaterals protecting injured NVU is also illuminated. Therefore, eliminating pathogen and strengthening healthy qi, which represents the holism of Chinese medicine, can be further enriched from the individual, cell and molecularlevel.

脑梗死急性期风火痰瘀蕴化形成火毒是病情骤变、加重恶化的转折点，前期工作（No.81173233）证实此过程与以炎性反应为中心环节的缺血级联损伤相吻合，当前缺血级联损伤导致神经血管单元结构及功能破坏、抑制血管新生是研究热点。清热解毒通络对急性脑梗死神经血管单元损伤具有保护作用，其调控miR-210介导VEGF-PI3K/Akt通路，促进血管新生是重要环节。本课题以大鼠MCAO模型和体外培养神经元、星形胶质细胞、微血管内皮细胞为研究对象，利用miR-210激动剂特异性观察苦碟子注射液对脑缺血不同时程miR-210介导VEGF-PI3K/Akt通路的作用，明确其促进血管新生的分子调控机制，阐明清热解毒通络对急性脑梗死神经血管单元保护作用的关键环节和靶点，从整体、细胞、分子层面进一步丰富祛邪扶正中医治病的整体观。

本研究分为动物实验和细胞实验两部分。分别采用雄性SD大鼠和各自原代培养神经元、大鼠星形胶质细胞，大鼠脑微血管内皮细胞，线栓法阻断左侧大脑中动脉（MCAO）模型，缺氧/复氧建立氧糖剥夺（OGD）损伤模型，构建miR-210质粒用于大鼠，构建miR-210重组慢病毒载体用于三种细胞，分为假手术组/空白组、MCAO模型组/OGD模型组，苦碟子注射液组，miR-210激动剂组，苦碟子注射液+miR-210激动剂组。Garcia 评分法和TTC染色法观察苦碟子注射液也对急性脑梗死大鼠疗效作用，MTT和LDH法以及细胞凋亡率观察苦碟子注射液对三种细胞损伤的保护作用，免疫荧光染色法观察大鼠缺血侧皮层微血管密度vWF表达以及脑微血管内皮细胞的VEGF2表达，Matrigel管腔生成实验检测细胞管腔形成情况。RT-PCR法观察大鼠缺血侧皮层以及三种细胞的miR-210表达，western blotting法观察VEGF/VEGF2/PI3K/Akt信号通路的蛋白表达。结果：与MCAO模型组比较， 苦碟子注射液组大鼠明显增加 Garcia JH神经功能缺损评分，减轻脑梗死体积，增加缺血侧皮层miR-210表达，显著增加VEGF、VEGFR2、PI3K、AKT蛋白表达。与OGD模型组相比，苦碟子注射液明显增加三种细胞的活性，降低LDH漏出率，降低细胞凋亡率，增加细胞miR-210表达，增加VEGF、VEGFR2、p-PI3K/PI3K、p-AKT/AKT蛋白表达，增加管腔生成数量。结论：苦碟子注射液可能通过调控miR-210表达诱导VEGF/VEGFR2/PI3K/Ak信号通路蛋白表达增加，促进血管新生，增加神经元、星形胶质细胞、脑微信内皮细胞活性，降低细胞凋亡率，减少脑梗死体积，减轻脑损伤，发挥脑保护作用。