失荅剌知丸对局灶性脑缺血再灌注模型大鼠MAPK-ERK通路的影响

"Shidalazhiwan" is the representative prescription of treating ischemic cerebrovascular disease recorded in Huihuiyaofang, Previous studies have demonstrated the mechanism of anti apoptosis and the improvement of neurological cells function in cerebral ischemia. In order to further study the mechanism of anti apoptosis, promoting the proliferation and differentiation of neural cells, On the fundation of PI3-K/AKT signaling pathway, this research start to explore the brain protection mechanism of shidalazhiwan by the MAPK-ERK pathway. this project make model of focal cerebral ischemia-reperfusion by the thread occlusion, 238 SD rats will be divided into 7 groups randomly with normal group, sham-operated group, model group, Nimodipine group,high-dose group of shidalazhiwan, middle-dose group of shidalazhiwan,low-dose group of shidalazhiwan, then we will evaluate the nervous system signs at various time-points. After death, we will observe pathological changes in brain issues, count apoptosis brain cells in ischemia-reperfusion ,the number of positive cells with propagating makers, detect the expression of ERK1、ERK2、ERK5 in each point, To explore the intervention mechanism of shidalazhiwan by MAPK-ERK pathway after ischemia reperfusion.The results will provide a experimental basis for further development and use of this prescription.

“失荅剌知丸”是《回回药方》中治疗缺血性脑血管疾病的经典方剂，前期开展的研究从机理方面验证了该方具有抗神经细胞凋亡、改善脑缺血后神经功能的作用。为进一步深化研究该方在抗凋亡、促进神经细胞增殖、分化机制，在前期PI3-K/AKT信号通路研究基础上，拟从与其紧密相关MAPK-ERK通路上，探讨该方发挥脑保护的机制。本研究采线栓法制备MCAO模型，将238只SD大鼠分为空白组、假手术组、模型组，尼莫地平组及失荅剌知丸高、中、低剂量组共17组，于造模后各时间点进行神经功能评分后处死，观察各时间点缺血区脑组织形态学改变、神经元细胞凋亡情况及增殖细胞标记物阳性计数；分别检测各组各时间点ERK1、ERK2、ERK5酶的表达水平。借以探讨失荅剌知丸对脑缺血再灌注后大鼠MAPK-ERK通路的干预机制，为进一步开发和运用该方提供实验依据。

脑卒中是临床常见病和多发病，其致残率、复发率和病死率均高，且发病率呈逐年增长趋势，发病年龄趋于年轻化，严重影响着患者的生活。失荅剌知丸是防治脑系疾病的经典方剂，前期基础研究表明失荅剌知丸可改善脑缺血再灌注大鼠神经功能，可能的作用机制是调控血清内炎性分子、脑细胞凋亡、修复血管蛋白及因子的表达。本课题在前期基础上采用线栓法制备脑缺血再灌注大鼠模型，开展“失荅剌知丸、失荅剌知丸拆方、巴豆霜及巴豆叶”对大鼠脑缺血再灌注模型MAPK-ERK通路的影响的实验。主要研究结果表明：（1）失荅剌知丸及各拆方组、巴豆霜、巴豆叶组均可改善大鼠脑缺血再灌注后的神经功能损伤，抑制神经细胞凋亡，促进细胞的增殖分化、修复受损的神经元，可能的作用机制是其调节MAPK-ERK通路中ERK1/2、ERK5、JNK相关蛋白的表达；（2）各拆方组中以巴豆、柴胡、黑诃子、芦荟四味药物的组方治疗效果最佳，考虑该组药物可能是失荅剌知丸能有效抑制局灶性脑缺血再灌注损伤的主要药物组成；巴豆可能是四味药中发挥脑保护作用的主要药物之一；（3）巴豆叶各浓度组均能有效改善大鼠脑缺血再灌注损伤后神经功能缺损、修复神经细胞损伤，其作用机制可能是抗海马CA1区、抗海马CA3/DG区神经元细胞凋亡及下调JNK通路的蛋白表达；其中巴豆叶高浓度7d组作用效果最好。