p-Akt、Bid与小鼠局灶性脑缺血后处理保护作用的关系

Relationship between Akt signal pathway and the protective effects of focal cerebral ischemic postconditioning in mice

目的探讨p-Akt、Bid在小鼠局灶性脑缺血再灌注(I/R)及缺血后处理(IP)后的启动规律,探讨p-Akt、Bid与IP保护作用的关系。方法将315只雄性昆明小鼠采用线栓法建立大脑中动脉栓塞模型后,随机分成假手术组(Sham组)、缺血再灌注组(I/R组)、缺血后处理组(IP组)、缺血后处理+PI-3K抑制剂LY294002组(IP+LY组)4组,于再灌注相应时间点取材,免疫组织化学方法检测p-Akt、Bid的表达及分布情况;免疫印迹法检测皮质p-Akt、Bid蛋白表达情况。氯化三苯基四氮唑(TTC)染色测定脑梗死体积。结果与Sham组比较,I/R组神经元胞质中可见p-Akt及Bid的表达明显上调,p-Akt表达于30min增加,1 h达高峰(P<0.05),24 h回落至基线水平,Bid表达于6 h增加,24 h达高峰(P<0.05),48～72 h有所回落;IP组各相应时间点较I/R组p-Akt表达显著增加(P<0.05),Bid表达明显降低(P<0.05)。应用PI-3K特异性阻断剂LY294002阻断PI-3K/Akt信号系统活化的同时,也抑制了IP的保护作用。p-Akt蛋白表达量与Bid免疫反应性呈负相关(r=-0.8265,P<0.05)。结论 p-Akt、Bid参与缺血后处理过程;缺血后处理可能通过PI-3K/Akt通路发挥脑保护作用;Akt可能通过死亡受体通路调控Bid的表达。

Objective To investigate the changes in p-Akt and Bid after ischemia reperfusion（I/R）injury and ischemic postconditioning（IP） ,and the relationship between p-Akt and IP. Methods Focal ischemia was generated by transient middle cerebral artery occlusion （MCAO）. Totally 315 male Kunming mice were randomly divided into four groups ： Sham group, I/R group,IP group, IP plus LY group. At the right time point, the brain tissue samples were prepaired for brain histology and determination of the experssion of p-Akt and Bid,by means of immunohistochemistry and Western blotting. The volume ratio of the cerebral infarction was estimated by 20 g/L TTC staining. Results In the cytoplasm of nerve cells,the levels of Akt and Bid were higher in I/R mice than those in Sham mice. The expression of p-Akt in the cytoplasm began to increase at the 0.5 hour, increased to the highest level at the 1 st hour （ P 〈 0.05 ） , and then gradually returned to the basic level. But the expression of Bid began to increase at the 6th hour, increased to the highest level at the 24th hour（P 〈 0. 05 ）, and then returned gradually; At the corresponding time, compared with I/R group, the expression of p-Akt increased remarkably in IP group（P 〈 0.05） ; meanwhile, the expression of Bid decreased significantly in IP group （P 〈0. 05 ）. LY294002, a specific blocker of PI-3K, notably blocked PI-3K/Akt signal pathway and inhibited the protective effects of IP in the brain. The experssion of p-Akt negatively correlated with the activated Bid （r = -0. 826 5, P 〈 0.05）. Conclusion The protective effects of IP in the mice brain is exerted through regulating Akt signal pathway. Perhaps,the up-regulation of the activation of P1-3K/Akt system is one of the mechanism through which the protective effects of IP can exert. Bid is involved in the process of IP. Akt can regulate the expression of Bid through the death receptor signal pathway.