蛋白激酶B激活对脑缺血再灌注海马CA3区神经元的保护作用

目的 探讨丝氨酸-苏氨酸蛋白激酶B（Protein kinase B,Akt）对脑缺血再灌注损伤后海马CA3区神经元的保护作用.方法 采用四动脉结扎法建立大鼠全脑缺血模型,再灌注0 min、30 min、3h、6h、12h、1d和3d各时间点,运用免疫印迹技术检测海马CA3区Akt1的磷酸化水平,采用免疫组化和焦油紫染色分别检测LY294002（PI3K/Akt特异性抑制剂）对缺血再灌注后磷酸化Akt1（p-Akt1）的表达及神经元存活的影响.结果 缺血再灌注后30 min,与假手术（sham）组相比,海马CA3区p-Akt1的表达显著升高（P＜0.05）,再灌后3h至峰值,一直持续至3d仍维持在较高水平（P＜0.05）;与相应的再灌注组相比,给予LY294002后,p-Akt1的蛋白表达明显降低[R6 h组和LY294002＋ R6 h组的阳性细胞数（个/mm^2）分别为（134.6±7.8）和（54.8±5.1）],海马CA3区的神经元大量死亡[R5 d组和LY294002 ＋R5 d组神经元数量（个/mm^2）分别为（152.0±17.4）和（62.7±5.1）]（P＜0.05）.结论 脑缺血再灌注后蛋白激酶Akt的持续激活,是海马CA3区神经元存活的重要因素.

Neuroprotective effect of Akt activation on neurons of hippocampal CA3 regions after cerebral ischemiareperfusion

Objective To explore the neuroprotective effect of serine-threonine protein kinase B （Akt） on neurons of hippocampal CA3 regions after cerebral ischemia-reperfusion （I/R）.Methods Cerebral ischemia was induced by four-vessel occlusion in SD rats,then the rats were decapitated at 0,30 min,3 h,6 h,12 h,1 d and 3 d after ischemia,and the time courses of the expression and phosphorylation of Akt1 after I/R in hippocampal CA3 regions were investigated using Western blot.The effect of LY294002 （a specific inhibitor of the PI3K/Akt） on p-Akt1 expression and neuronal survival in hippocampal CA3 regions was examined by immunohistochemical staining and cresyl violet staining,respectively.Results Compared to sham group,p-Akt1 expression was significantly increased at 30 min（P〈0.05） and reached peak at 3 h of reperfusion,and remained a high level until 3 d in hippocampal CA3 regions （P〈0.05）.Pretreatment with LY294002,significantly decreased p-Akt1 immunoreactivity compared with the R6 h group （the positive cells：R6 h,134.6±7.8/mm^2 ;LY294002＋R6 h,54.8±5.1/mm^2） （P〈0.05）,and aggravated the injury of neurons in hippocampal CA3 regions,compared with the R5 d group （the neuronal density：R5 d,152.0±17.4/mm^2 ; LY294002＋R5 d,62.7±5.1/mm^2） （P〈0.05）.Conclusion The activation of Akt induced by ischemic stress plays an important role in neuronal survival of hippocampal CA3 region.