脑缺血预处理诱导海马CA1区神经元Bad磷酸化及其蛋白表达的变化

Levels of Bad phosphorylation and protein expression in the hippocampal CA1 region of rat following cerebral ischemic preconditioning

目的： 观察大鼠脑缺血预处理（CIP）后不同时间点海马CA1区胞质及线粒体中Bcl-xl/Bcl-2相关死亡促进因子（Bad）和p-Bad蛋白水平变化,探讨其在缺血耐受机制中的作用.方法： 采用SD大鼠4动脉结扎全脑缺血模型,利用焦油紫染色、免疫印迹法检测神经元的损伤及蛋白表达.结果： 焦油紫染色显示,脑缺血再灌注（I/R）3d,CA1区神经元大量损伤,与I/R组相比,CIP组CA1区存活的神经元增加;免疫印迹结果显示,CIP组胞质中Bad磷酸化水平于再灌注3h和3d出现高峰,而其蛋白表达无明显变化;与I/R对应时间点相比,CIP后再灌注3h和3d p-Bad升高.CIP后线粒体中p-Bad在再灌注不同时间点无明显变化,而其蛋白表达在再灌注后期（6h～3d）逐渐下降.结论： 脑缺血预处理可有效减轻海马CA1区神经元损伤;同时诱导神经元胞质中p-Bad蛋白水平升高,线粒体Bad蛋白表达降低.脑缺血预处理后Bad线粒体转位的降低可能是缺血耐受的重要机制.

Objective： To investigate the levels of phospho-Bad and Bad in both cytoplasm and mitochondria, and to further clarify the possible role responsing to ischemic tolerance in the hippocampal CA1 region of rats. Methods：Four-vessel occlusion model （4-VO） of Sprague-Dawley rats was indeued and neuron injury was investigated using cresyl violet staining. Bad phosphorylation and its protein expression were detected by Western blotting analysis following cerebral is chemic preconditioning （CIP） or cerebral is chemia/reperfusion （I/R）. Results： 1. Cresyl violet staining results showed that I/R clearly reduced the number of surviving neurons at 3 d of reperfusion in the CA1 regioru Compared with I/R groups, CIP markedly induced the increase of survival pyramidal cells. 2. The results of immunoblot showed that Bad protein expression in the cytoplasm had no significant change following CIP, but the level of p-Bad significantly increased with two peaks occurring at ：3 h and 3 d of reperfusion respectively. The levels of p-Bad at 3 h and 3 d after CIP markedly increased compared with the same time point of I/R. On the other hand, in the mitochondria Bad phosphorylation had no significant change, while its protein expression gradually decreased from 6 h to 3 d of reperfusion. Conclusion： CIP significantly prevented neuronal injury from isehemia insult in the hippocampal CA1 region of rats. CIP induced an increase of p-Bad in the cytoplasm and a decrease of Bad expression in the mitochondria induced by CIP may be an important mechanism of cerebral ischemic tolerance.