细胞凋亡信号调节激酶1在脊髓缺血再灌注损伤中的作用

The role of apoptosis signal-regulating kinase 1 in reperfusional injury of ischemic spinal cord

目的:探讨细胞凋亡信号调节激酶1(apoptosis signal-regulating kinase1,ASK1)在脊髓缺血再灌注损伤中的作用及机制。方法:20只新西兰大白兔随机分成4组,每组5只,分别为对照组(A组)、缺血30min/再灌注15min组(B组)、缺血30min/再灌注1h组(C组)、缺血30min/再灌注24h组(D组),脊髓缺血再灌注损伤模型应用腹主动脉阻断法制作。光镜及电镜观察各组脊髓组织病理变化,Westernblot检测脊髓组织中ASK1的蛋白表达及活化情况,免疫共沉淀分析ASK1与14-3-3蛋白间相互作用,免疫组织化学染色观察活化ASK1(pASK1)及14-3-3蛋白在细胞内的定位表达。结果:光镜检查B组的脊髓组织形态学与A组比较无明显改变,C组及D组脊髓间质明显出血,神经细胞肿胀;电镜检查C组及D组神经细胞出现细胞核浓缩、染色质聚边、脱髓鞘改变等早期凋亡征象;Westernblot蛋白电泳显示B组ASK1无明显活化,C组及D组ASK1显著活化;免疫共沉淀分析提示ASK1与14-3-3蛋白在C组及D组发生蛋白分离;免疫组织化学染色显示缺血再灌注时pASK1与14-3-3蛋白均在细胞浆内表达。结论:ASK1介导的凋亡信号转导途径参与脊髓缺血再灌注的损伤过程,14-3-3蛋白与ASK1蛋白的分离可能是导致ASK1活化的机制之一。

Objective：To investigate the role and mechanism of apoptosis signal-regulating kinase 1（ASK1） in reperfusional injury of ischemic spinal cord.Method：20 New Zealand white rabbits were divided into four groups randomly,including control group （group A,n=5）,ischemia 30 minutes/reperfusion 15 minutes group （group B,n=5）,ischemia 30 minutes/reperfusion 1 hour group（group C,n=5） and ischemia 30 minutes/reperfusion 24 hours group （group D,n=5）.The model of spinal cord followed by ischemia and reperfusional injury was produced by occluding the abdominal aorta with an arterial clamp.Changes in spinal cord morphology were observed by hematoxylin and eosin （HE） stain and electron microscopy.The activation levels of ASK1 （pASK1） were assessed by immunoblotting.The interaction between 14-3-3 protein and ASK1 were analyzed by immunoprecipitation.The localization of pASK1 and 14-3-3 protein were analyzed by immunohistochemistry. Result：For HE stain,hemorrhagic focuses were found and the neuronal cells were swollen in group C and group D compared with group A and B.Electron microscopic examination of spinal cord revealed demyelination,coarse chromatin condensation,and breakdown of the nucleus into discrete fragments in group C and group D.For immunoblotting,the expression of pASK1 did not increase in group B but significantly increase in group C and group D compared with group A.The dissociation of 14-3-3 protein from pASK1 was found in group C and group D.14-3-3 protein and pASK1 were both expressed in the site of cytoplasm.Conclusion： ASK1-mediated transmission of apoptosis signals contribute to spinal cord ischemia/reperfusional injuries,and the activation mechanism of ASK1 may be caused by dissociation of 14-3-3 protein from ASK1.