转化生长因子β激活激酶1介导的细胞自噬在小鼠脑缺血-再灌注损伤中的作用

Role of TAK1 mediated cell autophagy after global cerebral ischemia-reperfusion in rats

目的评价转化生长因子β激活激酶1(TGFβ-activated kinase-1,TAK1)介导的细胞自噬在小鼠脑缺血-再灌注(ischemia-reperfusion,IR)损伤中的作用。方法雄性昆明小鼠72只,8周龄,体重(25.0±3.5)g,采用随机数字表法分为六组:空白对照组(C组)、假手术组(S组)、IR组、TAK1短发夹RNA(shRNA)慢病毒组(T组)、阴性对照慢病毒组(Y组)和生理盐水组(NS组),每组12只。T组、Y组和NS组分别以1μl/min侧脑室注射TAK1shRNA慢病毒、阴性对照慢病毒和等量生理盐水10μl。2周后IR组、T组、Y组和NS组制备脑IR损伤模型,缺血2h后恢复灌注;S组分离颈总动脉但不夹闭,其余手术步骤同IR组。再灌注24h后进行神经功能缺陷评分(neurological severity scores,NSS);NSS评分完成后处死小鼠,取脑组织测定脑梗死体积;Western blot法检测小鼠脑组织TAK1、LC3Ⅱ/LC3Ⅰ、Beclin1和p62蛋白含量。结果 IR组、T组、Y组和NS组NSS评分和脑梗死体积百分比明显高于C组(P<0.05);T组NSS评分和脑梗死体积百分比明显低于IR组(P<0.05)。IR组、Y组和NS组脑组织TAK1蛋白含量明显高于C组(P<0.05);T组脑组织TAK1蛋白含量明显低于IR组(P<0.05)。IR组、T组、Y组和NS组LC3Ⅱ/LC3Ⅰ比值、Beclin1蛋白含量明显高于C组(P<0.05),p62蛋白含量明显低于C组(P<0.05);T组脑组织LC3Ⅱ/LC3Ⅰ比值、Beclin1蛋白含量明显低于,p62蛋白含量明显高于IR组(P<0.05)。结论 TAK1介导的细胞自噬参与小鼠脑缺血-再灌注损伤机制。

Objective To study the effects of TGFβ-activated kinase-1（TAK1）mediated cell autophagy after global cerebral ischemia-reperfusion（IR）in rats.Methods Seventy-two male Kunming rats were randomly divided into six groups：control group（group C）,sham operation group（group S）,ischemia-reperfusion group（group IR）,TAK1 shRNA lentivirus group（group T）,negative lentivirus group（group Y）and normal saline group（group NS）（n=12each）.The rats in groups T,Y and NS received cerebral ventricles injection of TAK1 shRNA lentivirus,negative lentivirus and normal saline 10μl two weeks before preparing animal model.Using thread embolism of the right middle cerebral artery occlusion（MCAO）to cause focal ischemia for 2hand released for 24 h for reperfusion in groups IR,T,Y and NS.The common carotid arteries were separated but not ligated in group S,the rest of the procedure as the same as group IR.The rats of each group were evaluated by neurological severity scores（NSS）24hafter reperfusion,the cerebral infarct volume was measured with the method of TTC and the expression of TAK1,LC3Ⅱ/LC3Ⅰ,Beclin1 and p62protein in rat hippocampus were determined by using Western blot.Results The infarct volume and NSS in groups IR,T,Y and NS were significantly higher than those in group C（P〈0.05）.The infarct volume and NSS in group T were significantly lower than those in group IR（P〈0.05）.TAK1 protein of hippocampus in groups IR,Y and NS was significantly higher than that in group C（P〈0.05）.TAK1 protein of hippocampus in group T were significantly lower than that in group IR（P〈0.05）.LC3Ⅱ/LC3Ⅰand Beclin1 protein of hippocampus in groups IR,T,Y and NS were significantly higher than those in group C,and the p62 protein of hippocampus in groups IR,T,Y and NS was significantly lower than that in group C（P〈0.05）.The LC3Ⅱ/LC3Ⅰand Beclin1 in group T were significantly lower than those in group IR,and the p62 protein of hippocampus in group T was significantly higher than that in group C（P〈0.05）.Conclusion TAK1 mediated cell autophagy takes part in the mechanism of brain ischemia-reperfusion injury in rats.