失荅剌知丸对大鼠局灶性脑缺血再灌注Notch通路的影响

"Shidalazhiwan " is a representative prescription of prevention and treatment of ischemic cerebrovascular disease in the classical medical of the Hui-medicine—Huihuiyaofang,about the preliminary series of experimental studies of this prescription,including the funds subject PI3-K/AKT pathway mediated by neuron apoptosis mechanism research,all tips show that shidalazhiwan has great development potential and good application prospect in the aspect of prevention and treatment of ischemic cerebrovascular disease. In order to further investigating the protection mechanism in the party of promote vascularin after ischemia-reperfusion injury. On the fundation of PI3-K/AKT signaling pathway, this research start to explore the mechanism of shidalazhiwan from the party upstream of Notch Signaling pathway. this project make model of focal cerebral ischemia-reperfusion by the thread occlusion, 204 SD rats will be divided into 17 groups randomly with normal group, sham-operated group, model 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 Notch1、Deltal、Jagged in each point by Western-Blot and RT-qPCR, To explore the intervention mechanism of shidalazhiwan by Notch Signaling pathway after ischemia reperfusion.The results will provide a experimental basis for further development and use of this prescription.

“失荅剌知丸”是回医药典籍—《回回药方》防治缺血性脑血管病之代表方剂。前期开展的该方系列实验研究，包括国基金课题在内的PI3-K/AKT通路介导下的抗神经元凋亡机制研究，均提示该方在防治缺血性脑血管病方面具有很大的开发潜力和良好的应用前景。为进一步探讨该方对脑缺血后损伤的促血管、神经元再生及保护机制，本研究在PI3-K/AKT信号通路研究基础上，拟从其上游Notch信号转导通路入手，探求该方之作用机理。采用线栓法制备MCAO模型，将204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，观察其缺血区脑组织形态学改变、神经元细胞凋亡及增殖细胞标记物计数；并分别检测其Notch1、Deltal、Jagged蛋白及基因的表达水平。以探讨失荅剌知丸对大鼠脑缺血再灌注后Notch通路的干预，为进一步开发和运用该方提供依据。

采用线栓法制备脑缺血再灌注大鼠模型，研究失荅剌知丸对Notch通路的影响。（1）失荅剌知丸能促进脑缺血再灌注后大鼠神经功能的恢复；（2）对Delta样配体4表达的影响：WB、PCR、免疫荧光示：各治疗组与模型组比较Delta样配体4蛋白表达增高(P＜0.01)；其中失荅剌知丸高剂量组各时间点Delta样配体4蛋白表达明显增高（P<0.01）；失荅剌知丸各组中，与低剂量组比较，中、高剂量组Delta样配体4 蛋白表达有上调，其中以高剂量组最显著（P<0.01）；与1d组比较，3d、7d组Delta样配体4蛋白的表达有增高，其中以7d组最为明显（P<0.01）。（3）对Notch1受体表达的影响：各治疗组与模型组比较Notch1表达增高(P＜0.01)，WB、PCR、免疫荧光示:失荅剌知丸低、中、高剂量组，随着每一个时间点的延长，Notch1活性表达依次呈上升趋势，其中尤以7d组表达最高。（4）对Hes1表达的影响：WB、PCR、免疫荧光示：各治疗组与模型组比较Hes1的表达明显升高(P＜0.01)，与尼莫地平组Hes1表达情况，以及失荅剌知丸组中的低剂量、中剂量组Hes1表达情况比较，失荅剌知丸高剂量组各对应时间点Hes1表达显著升高，提示失荅剌知丸高浓缩药物具有更好的促进该因子表达的作用；（5）对NSCs蛋白表达的影响：各治疗组与模型组比较NSCs蛋白表达增多（P ＜0.05），与尼莫地平组同期比较，失荅剌知丸低剂量组新生的NSCs个数少于尼莫地平组（P ＜0.05），失荅剌知丸中剂量组新生的NSCs个数与尼莫地平组相当（P ＞0.05），荅剌知丸高剂量组新生的NSCs个数多于尼莫地平组（P ＜0.05）；（6）对Nestin基因mRNA转录水平表达的影响：PCR检测结果示：各治疗组与模型组比较Nestin基因mRNA转录水平的相对量均增高（P ＜0.05），与尼莫地平组同期比较，荅剌知丸高剂量组Nestin基因mRNA转录水平的相对量高于尼莫地平组（P ＜0.05）；（7）对Nestin蛋白、Jagged1蛋白表达影响：WB检测结果示：各治疗组与模型组比较Nestin蛋白、Jagged1蛋白表达均明显增高（P ＜0.05），与尼莫地平组同期比较，荅剌知丸高剂量组Nestin蛋白表达高于尼莫地平组（P ＜0.05）。