脑衰蛋白调节蛋白2对新生大鼠缺氧缺血后轴突及树突损伤的调控作用

The Involvement of CRMP-2 on Axonal and Dendrictic Injury after Hypoxic-Ischemic Brain Injury in Neonatal Rat

目的探讨在体内缺氧缺血（hypoxia-ischemia,HI）条件下,脑衰蛋白调节蛋白2（collapsin response mediator protein 2,CRMP-2）与轴突及树突损伤的关系。方法以10日龄新生SD大鼠为研究对象,行右侧颈总动脉结扎手术,8%氧氮混合气缺氧2.5h制作HI模型。Western blot法检测HI后大鼠皮层及海马脑组织CRMP-2总蛋白和磷酸化蛋白、淀粉样前蛋白（amyloid precursor protein,APP）表达变化;使用蛋白激酶B（Akt）抑制剂渥曼青霉素（wortmannin）和LY294002后,Western blot和免疫组化染色法检测CRMP-2总蛋白和磷酸化蛋白在HI后4h、24h改变;HI后72h,Western blot和免疫组化检测渥曼青霉素对APP表达的影响,电镜检测轴突及树突损伤。结果 HI后CRMP-2总蛋白表达无明显变化;磷酸化CRMP-2（p-CRMP-2）蛋白表达在0.5h短暂上升,此后一直处于低水平;HI后APP蛋白表达在2h开始上升,此后2-24h一直处于稳定增高水平,48-72h表达量再次增高。HI后4h和24h,渥曼青霉素或LY294002干预未改变CRMP-2总蛋白表达量,但p-CRMP-2的表达量增加。HI后72h,渥曼青霉素预处理增加APP的表达量,加重轴突及树突损伤。结论 CRMP-2作为Akt信号通路下游蛋白,参与调控新生大鼠HI后神经元轴突和树突损伤。

Objective To investigate the activity of collapsin response mediator protein 2（CRMP-2）and its possible regulation for axonal and dendrictic injury under hypoxia-ischemia（HI）.Methods Postnatal day 10 SD rats were suffered the right common carotid artery ligation and 8% mixture of oxygen and nitrogen hypoxia 2.5hto produce HI model.The expression of total and phosphorylated CRMP-2and amyloid precursor protein（APP）were detected by Western blot after HI.After pretreatment of protein kinase B（Akt）inhibitor,wortmannin or LY294002,western blot and immunohistochemistry were applied to detect the expression of total and phosphorylated of CRMP-2at 4hand 24 hafter HI.At 72 hafter HI,APP was detected by Western blot and immunohistochemistry,axonal and dendrictic injury was determined by electron microscope.Results Total CRMP-2was not obviously changed after HI,compared with that of sham controls.However,the phosphorylated CRMP-2（p-CRMP-2）protein transiently increased at 0.5h,started to decrease at 2h,remained at a low level at the rest of the time points,compared with that of sham controls.APP protein started to increase at 2h,remained at a high level at 4,8,24 h,and then progressively increased at 48 and 72h.In wortmannin and LY294002 group,CRMP-2protein was not obviously changed at 4hand 24 hafter HI.However,p-CRMP-2increased at 4hand 24 h.At 72 h,wortmannin pretreatment increased APP expression,leading to more severe ultrastructure failure of axon and dendrite.Conclusion As a downstream effector of Akt pathway,CRMP-2is involved in axonal and dendritic injury regulation after HI in neonatal rat.