从胶质细胞TAK1-NF-κB通路调控炎症反应失衡切入研究清脑滴丸对急性脑缺血的干预效应机制

Inflammatory response plays a critical factor in the process of acute cerebral ischemic damage. Gliacytes are the major effect cells in the cerebral inflammatory response. TAK1 is an important signaling protein of upstream of NF-κB, and the central link of NF-κB mediating inflammatory response. This subject is based on previous work and propose: One of the acute cerebral ischemical etiology and pathogenesis is hot (poison) and blood stasis blocking brain, its mechanism maybe the associated with imbulance and distribution of TAK1-NF-κB pathway mediating inflammtory response in gliacytes, The method of clearing heat-toxin and promoting blood circulation for removing obstruction in collaterals is an effective therapeutic approach.Culuring c6 glial cells lacked of glucose and oxygen induced cell damged model, focal cerebral ischemia made by suture occlusion technique induced middle cerebral artery occlusion in rats. Selecting Qingnao Dropping Pill intervention. Dynamic observing changes of the key facors in down stream of NF-κB signaling pathway, Further studying time and distribution of the NF-κB in different types of gliacytes. Clarifying the correlation between TAK1-NF-κB signal pathway mediated inflammatory response in gliacytes and acute cerebral ischemia, Defining the role of NF-κB target cells. Expliciting the effect target and integration mechanisms of Qingnao dripping pills, revealing the role link of method-syndrome. Providing a scientific basis for the clinical pharmacology on Qingnao Dropping Pill.

炎症反应应激过度是急性脑缺血损害的关键因素，胶质细胞是炎症反应主要的效应细胞，TAK1作为NF-κB上游重要的信号蛋白，是NF-κB调节炎症反应的关键调控枢纽，本课题结合前期工作提出：急性脑缺血主要病因病机之一是火热（毒）互结血瘀，阻滞脑络，其机制可能与胶质细胞TAK1-NF-κB通路调控炎症反应失衡及分布变化相关，清热解毒，活血宣窍法是其有效的治则。体外培养c6胶质细胞缺糖缺氧损伤模型和整体线栓法致急性脑缺血大鼠模型，选用清脑滴丸进行干预，动态观察TAK1-NF-κB通路上下游关键因子表达的变化，以及在不同类型胶质细胞中时空变化，阐述急性脑缺血胶质细胞与TAK1-NF-κB信号通路介导炎症反应的相关性，确定NF-κB作用的靶细胞，明确清脑滴丸的作用靶点和调节整合效应机制，阐明方证效应的作用环节，为清脑滴丸临床药理学提供科学依据。

炎症反应应激过度是急性脑缺血损害的关键因素，胶质细胞是炎症反应的主要的效应细胞，TAK1作为NF-κB上游重要的信号蛋白，是NF-κB调节炎症反应的关键调控枢纽。实验分为两部分，实验一线栓法制备大鼠大脑中动脉缺血再灌注模型（MCAO），随机分为正常组，假手术组，模型缺血1.5h分别再灌注6h、24h、48h、72h、7d，清脑滴丸各相应时间点治疗组。脑组织TTC染色显示模型各组均出现明显梗死灶，清脑滴丸治疗后，脑梗死面积明显缩减，再灌24h~72 h梗死面积减小最为明显（P<0.01）；降低神经细胞凋亡率，以再灌48h~72 h降低最为明显（P<0.01），降低TAK1、IκBα和p-IκBα、NF-κBp65的蛋白表达；在再灌注6h开始出现降低（P<0.01），在再灌注72h仍有下降（P<0.01），明显下调IL-1β和TNF-a（P<0.01）；且上调IL-10（P<0.05~0.01）。实验二培养C6胶质细胞，连二亚硫酸钠模拟细胞缺氧复氧损伤，清脑滴丸给予干预。结果发现清脑滴丸 5g/L 对C6细胞活力改善明显，可明显降低胶质细胞凋亡率（P<0.01），明显降低TAK1、IκBα、NF-κBp65蛋白表达（P<0.01）。结论：清脑滴丸能够明显降低急性脑梗死大鼠皮层与缺氧/复氧损伤的胶质细胞TAK1、IκBα和p-IκBα、NF-κBp65的蛋白表达，显著下调脑缺血再灌注后TNF-α、IL-1β，上调IL-10，缩小脑梗死面积，降低细胞凋亡率，减轻脑损伤，提示清脑滴丸治疗急性脑梗死作用机制可能与调节脑组织与胶质细胞的TAK1- NF-κBp65信号转导通路，调节促炎因子与抗炎因子达到动态平衡，以及降低胶质细胞凋亡率密切相关。