NOS、p38MAPK、Caspase-3介导大鼠脑缺血神经细胞凋亡可能通路的实验研究

To investigate the potential pathway of neuronal apoptosis resulting from NOS,p38MAPK and Caspase-3 after ischemic injury in rat MACO model

目的探讨脑缺血后细胞凋亡发生的可能机制以及神经元型一氧化氮合酶（neuronal nitric oxide synthase，nNOS）、诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS），p38丝裂原活化蛋白激酶（mitogen activated proteinkinase p38．p38MAPK）和半光氨酸蛋白酶-3（caspase-3）在脑缺血后神经细胞凋亡中的共同作用机制。方法采用线栓法闭塞大鼠大脑中动脉（middle cerebral artery occlusion，MACO）建立脑缺血SD大鼠模型，应用透射电镜观察脑缺血对脑组织超微结构的影响，流式细胞仪方法（FCM）分别定量检测细胞凋亡率，半定量RT—PCR检测nNOS、iNOS，p38MAPK和Caspase-3 mRNA表达水平。结果透视电镜下脑缺血6h出现核固缩，缺血12h出现细胞核分裂，缺血24h出现凋亡小体；FCM检测细胞凋亡百分率随着缺血时间延长而增加，缺血72h达到高峰，约70．37％；RT—PCR产物的琼脂糖凝胶电泳显示nNOS、iNOS、p38MAPK和Caspase-3 mRNA的特异性片段大小分别为501、342、250和342bp，但mRNA。表达量不一致，nNOSmRNA主要在缺血早期表达，iNOS，p38MAPK和Caspase-3 mRNA在缺血中晚期表达，并在缺血3～5d，后三种基因的表达量达到高峰。结论脑缺血区域发生典型的神经细胞凋亡现象，nNOS来源的NOS在缺血早期发挥神经毒性作用，INOS来源的NOS在缺血晚期发挥神经毒性作用；NOS，p38MAPK和Caspase-3三种基因的相互关系可能构成介导缺血神经细胞凋亡的通路之一。

Objective To explore the mechanisms of neuronal apoptosis after ischemic injury and the potential pathway of neuronal apoptosis resulting from NOS,p38MAPK and Caspase-3. Methods Focal cerebral ischemia model was established via MCAO with the intraluminal filament technique. The ultrastructure of brain tissue after ischemic injury was observed by transmission electromicroscope. The apoptosis rate, nNOS, iNOS, p38 MAPK and Caspase-3 in positive cells were measured with FCM. The expression of mRNA of iNOS, p38 MAPK and Caspase-3 was semi-quantified by RT-PCR. Results Under the observation of transmission electromicroscope, cells showed pyknosis after 6h ischemia, karyorrhexis after 12h and apoptotic bodies after 24h. Agarose gel electrophoresis showed that mRNA express of nNOS, iNOS, p38MAPK and Caspase-3 differed, nNOS mRNA was expressed largely in the early phase whereas mRNA of iNOS, p38MAPK and Caspase-3 dominated in the late injury peaking at 3 -5 day after MCAO. Conclusions Typical apoptosis was detected in the focal cerebral ischemia, could be early affected by NO produced by nNOS lated by iNOS. The relation among NOS, p38MAPK and Caspaseo3 might constitute a pathway for ischemia-induced apoptosis.