从TLR2∕MyD88信号通路探讨补阳还五汤促慢性脑缺血神经发生机制研究

Ischemic stroke is one of the leading causes of death and disability in the world. Neurological sequela caused by cerebral ischemia is the fundamental reason for neuronal loss, thus promoting the formation of new neurons (neurogenesis) after ischemia is expected to become the target of treatment. Neurogenesis depends on neural stem cell proliferation, differentiation and survival. Because the Toll decided to proliferation, differentiation and survival of embryonic stem cell, as Toll analogs of Toll like receptor 2 (TLR2) signal transduction pathway triggered by which may affect the proliferation, differentiation and survival of neural stem cells. Buyang Huanwu Decoction is a classical prescription in treating ischemic apoplexy sequela, which has significant clinical efficacyt. Experiments in vivo and in vitro show that it can significantly promote the neurogenesis, yet its underlying molecular mechanisms are not completely understood. In this study, we will employ hypoxia/reoxygenation conditions for primary hippocampal neural stem cell cultures as an in vitro model of stroke as well as the in vivo model of rat focal middle cerebral artery occlusion (MCAO), and which will be be treated with Buyang Huanwu Decoction. We use the antibody blocking technique, BrdU technique, immunofluorescence technique, molecular biology and bioinformatics methods from the in vitro and in vivo experiment, to explore possible mechanisms of Buyang Huanwu Decotion promoting nerve regeneration after cerebral ischemia from the TLR2/MyD88 Signal transduction pathway. The research results will provide a basis for revealing adjustment mechanism of neurogenesis after cerebral ischemia and for the treatment of cerebral ischemia. And the results of this study provide a method and a new experimental evidence for Chinese medicine treatment of cerebral injury disease.

缺血性中风所致后遗症根本原因是神经元减少，因此促进缺血后新的神经元的形成（神经发生）有望成为其治疗的的靶点。神经发生依赖于神经干细胞增殖与分化。由于Toll决定果蝇胚胎干细胞的增殖与分化方向，作为Toll同源类似物Toll样受体2（TLR2）所引发的信号转导通路可能影响神经干细胞的增殖与分化。补阳还五汤是治疗缺血性中风后遗症的经典名方，临床疗效确切。体内外实验表明其有显著促神经发生作用，但其机制未明。本研究拟制备动物与细胞两种脑缺血模型，给药补阳还五汤干预；并采用抗体阻断技术、BrdU标记技术、免疫荧光技术、分子生物学技术及生物信息学方法拟从动物体内实验与细胞培养实验两水平通过TLR2/MyD88信号通路探讨补阳还五汤促脑缺血后神经发生的可能机制。该研究结果为揭示脑缺血后神经发生的调节机制和脑缺血的治疗提供基础，为中医药治疗脑损伤性疾病提供新的方法和实验依据。

目的: 本课题运用特异性阻断、realtime- PCR、western blot、免疫荧光等技术探讨神经发生影响的可能分子机制及补阳还五汤促神经发生的机理，揭示脑缺血后神经发生的调节机制和新的作用靶点，为中医药治疗脑损伤性疾病提供新的方法和实验依据。 . 方法:采用大脑中动脉线栓法建立左侧局灶性脑缺血大鼠模型，将动物随机分为模型+补阳组、模型组、假手术组、模型+侧脑室PBS组、模型+侧脑室TLR2与TLR4的阻断剂OxPAPC组、模型+侧脑室TLR2与TLR4的阻断剂OxPAPC +补阳还五汤组、模型+侧脑室侧脑室TLR4的阻断剂CLI-095 +补阳组。各大组内分三个时间点即于14d和21d处死动物，每组动物每个时间点取5只动物腹腔注射Brdu检测海马神经干细胞，取10只神经功能评分，取6只进行免疫印迹、Realtime-PCR指标检测。采用阻断来观察TLR信号通路对脑缺血后神经干细胞增殖的影响，采用免疫荧光双标法观察BrdU标记干细胞增生情况，采用免疫单标法检测各组样本TLR2的表达，采用WB、Realtime -PCR法检测补阳还五汤对大鼠脑缺血后TLR2信号通路分子表达的影响。.结果：.(1)与相同时间相模型组比较补阳还五汤可减轻大鼠局灶性脑缺血后神经功能缺损（P＜0.05）。.(2)补阳还五汤组与相同时间模型组相比BrdU阳性细胞数显著增多（P＜0.05）。.(3) 在慢性脑缺血大鼠海马，与相同时间相模型组比较补阳还五汤组TLR2信号通路分子表达水平高（P＜0.05）。.(4) TLR2阻断后在慢性脑缺血大鼠，TLR2信号通路分子有下调的趋势（P＜0.05）。. (5) TLR2阻断后大鼠脑缺血后海马神经干细胞的增殖减少（P＜0.05）。.结论:.（1）补阳还五汤能促进大鼠脑缺血后 （慢性期）神经功能恢复。.（2）补阳还五汤对大鼠脑缺血病理损伤具有神经保护作用，并促进缺血后神经干细胞增殖，后者可能在其促进缺血后神经功能恢复中发挥作用。.（3）TLR2介导免疫炎症通路对脑缺血后（恢复期与慢性期）神经干细胞增殖有促进作用。.（4）通过TLR2介导免疫炎症通路，可能是补阳还五汤实现脑保护作用与促进脑缺血后神经再生机理之一。