关键词: 脑缺血再灌注, 一氧化氮合酶, 磷酸化, 应激活化蛋白激酶/c-Jun氨基末端激酶, p38 MAP激酶, 免疫组织化学, 蛋白免疫印迹, 大鼠

Abstract: 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(Thr183/Tyr185,Thr180/Tyr182)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 activa

Key words: Cerebral ischemiareperfusion, Nitric oxide synthase, Phosphorylation, Stressactivated protein kinase/cJun NH2terminal kinase, p38 Mitogenactivated protein kinase, Immunohistochemistry, Western blotting, Rat