从小胶质细胞TLR4/NF-κB通路探讨针刺治疗缺血性中风的抗炎机制

Immunoinflammatory response of cerebral ischemia can promote secondary cerebral injury. While the activation of microglia cells which are the main immune cells of central nervous system is the key initiating factor of immunoinflammatory response of cerebral ischemia. Our previous study indicated acupuncture could reduce the activation of microglia cells after cerebral ischemia. To investigate the mechanism, we will construct MCAO model rats and observe the effect of acupuncture on MCAO rats and the effect of acupuncture on MCAO rats after TLR4 is blocked. The brain function of MCAO rats will be detected by neurobehavioral score. Necrosis and apoptosis of nerve cells will be detected by HE and TUNEL staining. The area of infarct focus will be detected by TTC staining. The content of inflammatory factor in brain tissue will be detected by ELISA. The activation of microglia cells, the expressing of key molecular of TLR4/NF-κB signaling pathway on microglia cells will be detected by immune fluorescence labeling、laser scanning confocal microscope and RT-PCR. We will analyze the relation of the above factors, then discuss the presumption that "acupuncture can regulate the activation of microglia cells by influencing TLR4/NF-κB signaling path, finally restrain the immunoinflammatory damage of cerebral ischemia". This project will be not only a further study on our previous research, but also provide new research thought and method for clarifying protective effect of acupuncture on apoplexy from ischemia.

脑缺血免疫炎症反应促进了继发性脑损害，而中枢神经系统最主要的免疫效应细胞- - 小胶质细胞的活化则是脑缺血免疫炎症反应的关键始动因素。课题组以往的研究提示针刺可以减少脑缺血后小胶质细胞的活化，为深入探讨其机制，本项目拟建立MCAO大鼠模型，观察针刺对MCAO大鼠及TLR4阻断后MCAO大鼠的影响，采用神经行为学评分检测大鼠脑功能，HE及TUNEL染色检测神经元的坏死及凋亡，TTC染色检测梗塞灶面积，ELISA检测脑组织炎症因子含量，免疫荧光标记、激光共聚焦显微镜及原位RT-PCR检测小胶质细胞的活化、TLR4/NF-κB信号通路关键分子在小胶质细胞的表达。分析上述各因素之间的关系，论证"针刺可能通过影响小胶质细胞TLR4/NF-κB信号通路而调控小胶质细胞活化，抑制脑缺血免疫炎症损伤"的假说，本项目既是对以往研究的深入，也为阐明针刺治疗缺血性中风的脑保护机制提供新的研究思路和手段。

针刺治疗中风的机制一直是针刺基础研究领域的重点及热点，但其机制尚未完全阐明。目前许多研究均支持免疫性炎症反应参与了缺血性中风后的脑损伤，脑缺血时炎症反应促进了继发性脑损害，是脑缺血损伤的主要原因之一，而脑组织最主要的免疫效应细胞——小胶质细胞(microglia，MG)的活化是脑缺血免疫炎症反应的关键始动因素。因此，如何抑制小胶质细胞的过度活化，将炎症反应控制在适当水平，将为脑缺血的治疗提供新的途径。小胶质细胞的活化依赖于受体与配体的结合，其中TLR4/NF-κB通路是脑缺血炎症损伤的重要信号通路，因此，小胶质细胞的活化的调控是脑缺血后免疫炎症反应的关键，而TLR分子无疑就是"信号调控的开关"。本项目建立MCAO大鼠模型，研究针刺对脑功能的恢复，脑组织炎症介质TNF-α、IL-1β 和 IL-6的含量以及TLR4/NF-κB通路在小胶质细胞表达的影响。研究结果显示，针刺内关、曲池能显著改善MCAO大鼠的神经体征评分、脑组织梗塞率、脑海马神经元死亡率，并且能明显抑制脑缺血后脑组织TLR4/NF-κB通路关键分子的异常表达，抑制小胶质细胞的活化，降低脑缺血后脑组织TNF-α，IL-1β，IL-6含量的异常升高。提示针刺能够通过影响小胶质细胞TLR4/NF-κB信号通路的活化从而调控脑缺血继发的炎症反应。本研究为阐明针刺治疗缺血性中风的脑保护机制提供新的研究思路和手段。