一氧化氮激活应激活化蛋白激酶/c-Jun氨基末端激酶及p38MAP激酶诱导脑缺血再灌注后神经元凋亡

目的研究脑缺血区周边组织一氧化氮合酶（NOS）的表达与应激活化蛋白激酶／c—Jun氨基末端激酶（SAPK／JNK）及p38MAP激酶（p38MAPK）激活的关系；探讨一氧化氮（NO）诱导脑缺血再灌注后神经元凋亡的可能机制。方法采用TUNEL染色法观察脑缺血再灌注不同时段模型鼠缺血区周边组织凋亡的阳性神经元数量；免疫组织化学、蛋白免疫印迹方法检测活化型Caspase-3、神经元型一氧化氮合酶（nNOS）、诱导型一氧化氮合酶（iNOS）和SAPK／JNK，p38MAPK及其磷酸化组分的表达。结果再灌注后1h、2h缺血区周边组织nNOS表达明显增强；自1h起iNOS开始表达，12h达到高峰。1hp-SAPK／JNK表达较强，以后逐渐减弱；p38MAPK各时段表达均明显增强，以6h为著，p-p38MAPK表达高峰亦在6h。6h活化型Caspase．3开始表达，12h达到高峰；12h开始出现TUNEL阳性神经元，24h达到高峰。结论缺血区周边组织NOS表达的增强可能通过激活SAPK／JNK及p38MAPK诱导脑缺血再灌注后神经元凋亡。

THE ACTIVATION OF SAPK/JNK AND p38MAPK MAY BE THE POSSIBLE MECHANISM OF NEURONAL APOPTOSIS INDUCED BY NO AFTER CEREBRAL ISCHEMIA-REPERFUSION

Objective To establish transient focal cerebral ischemia rats model,explore the relationship between the expression of NOS,the activation of SAPK/JNK and p38 MAPK in the boundary zone of the infarcts area after reperfusion,and uncover the possible mechanism of NO inducing neuronal apoptosis after cerebral ischemia-reperfusion. Methods Transient focal ischemia models of middle cerebral artery occlusion were induced by inserting a filament through left internal carotid artery for 2h.Rats were grouped as following: control,sham operation,model.Coronal brain sections were collected after 1h,2h,4h,6h,12h,24h of reperfusion,Neuronal injury in the boundary zone of the infarcts area was evaluated by TUNEL staining;The expression of activated Caspase-3,nNOS and iNOS,total SAPK/JNK,p38MAPK and their phosphorylation(Thr~(183)/Tyr~(185),Thr~(180)/Tyr~(182))was investigated by immunohistochemistry and Western blotting with corresponding antibodies. Results After reperfusion,nNOS immunoreactivity increased markedly at lh and 2h time point in the boundary zone of the infarcts area(P<0.01 it was compared with control,sham and the other time points);The expression of iNOS protein appeared at 1 h and enhanced gradually,peaked at 12h and 24h(P<0.01 compared with the other time points).SAPK/JNK immunoreactivity did not increase at each time point in the boundary zone of the infarcts area after reperfusion,p-SAPK/JNK immunoreactivity increased significantly at 1h(P<0.01 compared with the other time points) and decreased gradually;p38MAPK immunoreactivity was enhanced at each time point(P<0.01 compared with normal,sham),peaked at 6h(P<0.01 compared with the other time points),p-p38MAPK was induced after reperfusion and the activation peaked also at 6h(P<0.01 compared with the other time points).Activated Caspase-3 immunoreactivity appeared at 6h in the boundary zone of the infarcts area and peaked at 12h(P<0.01 compared with 12 h,24 h);TUNEL positive neurons appeared at 12h and became more abundant at 24h(P<0.01 compared with 12 h).Conclusion The increased expression of NOS in the boundary zone of the infarcts area may induce neuronal apoptosis by activation of SAPK/JNK and p38 MAPK.