电针预处理抑制脑缺血后甘露聚糖结合凝集素激活TLR4的机制研究

Complement activation have been suggested in the pathogenesis of stroke, mannose-binding lectin (MBL) were found to plays an important role during the process. Our studies demonstrate that the serum MBL levels were significantly higher in acutely ischemic stroke patients as compared to normal controls.There was a correlation between serum levels of MBL and NIHSS score. Our most recent pilot studies demonstrate that electroacupuncture (EA) preconditioning inhibites MBL after cerebral ischemia. The expression of TLR4 in MBL knockout mice decreased. We therefore put forward the central hypothesis of this project: EA preconditioning-induced decrease of MBL and consequent activation of TLR4 signaling, inhibites TLR4-MyD88-NF-kB signaling and consequently decrease inflammatory and apoptosis, induce neuronal tolerance neuroprotection. We will test this hypothesis with ischemic model on MBL knockout mice and TLR4 knockout mice, using Immunohistochemical and Immunofluorescense,Western blot,Real time PCR,to detect the mechanism of EA preconditioning inducing the inhibition of MBL and consequent activation of TLR4 signaling after cerebral ischemia. These novel studies will provide a novel solid base on stroke treatment.

甘露聚糖结合凝集素（MBL）补体活化途径在脑缺血损伤中发挥重要作用。申请人研究发现：急性缺血性脑卒中患者MBL水平显著性增高，并且与患者NIHSS得分呈正相关；预实验结果显示：电针预处理可下调缺血再灌注小鼠MBL水平，MBL基因敲除小鼠脑缺血后TLR4表达下调。有研究发现：MBL对TLR信号转导通路具有调节作用。综上，我们提出本课题假说：电针预处理引起MBL表达下调，抑制脑缺血后MBL激活TLR4，进而通过干预TLR4-MyD88-NF-kB信号传导途径，减少脑缺血再灌注后促炎因子的表达和细胞凋亡，激活内源性保护机制，诱导脑保护效应。本课题拟以MBL基因敲除小鼠和TLR4基因敲除小鼠为平台，采用免疫组化、免疫荧光、Western blot、实时定量PCR等方法，深入研究电针预处理抑制脑缺血后MBL激活TLR4的机制，为脑缺血损伤防治提供新的理论依据，体现针灸治未病理念。

脑缺血耐受可以对缺血后神经细胞起到一定的保护作用，电针预处理、缺血预处理、缺血后处理是目前比较公认的诱导脑血耐受手段。脑缺血耐受的具体机制十分复杂，甘露聚糖结合凝集素（MBL）补体活化途径介导的神经炎症损伤在脑缺血后发挥重要作用，本课题利用野生小鼠、MBL基因敲出小鼠、TLR4基因敲出小鼠大脑中动脉栓塞模型研究针灸预处理诱导脑缺血耐受的机制，并与缺血预处理、缺血后处理进行对照。本课题研究证实脑缺血后MBL基因敲出小鼠、TLR4基因敲出小鼠神经损伤明显低于野生小鼠；电针预处理引起MBL表达下调，抑制脑缺血后MBL激活TLR4，进而通过干预TLR4-MyD88-NF-κB信号传导途径，减少脑缺血再灌注后促炎因子的表达和细胞凋亡，激活内源性保护机制；电针预处理诱导产生的脑缺血耐受与缺血预处理、缺血后处理治疗对脑缺血小鼠的神经保护作用相当。目前急性脑卒中的治疗手段多告以失败，卒中后康复对于各种功能障碍也无确实有效的干预措施，80%患者以上的患者遗留各种程度不同的功能障碍，因而脑卒中的预防应该必须引起重视。脑卒中防治研究中应该将方向集中于探索能够充分调动脑内源性保护机制的措施方法，以取得理想的神经保护效应。本课题的科学意义在于从脑缺血后补体激活引起神经炎症机制角度证实了电针预处理对脑缺血后神经损伤的可能机制，从与缺血预处理、缺血后处理进行对照角度证实了电针预处理诱导脑缺血耐受的作用，从补体激活途径为脑缺血损伤防治提供了的临床策略，体现针灸治未病理念。