氧化应激对脑缺血再灌注损伤后自噬调控机制  

The regulation effect of oxidative stress on autophagy after cerebral ischemia-reperfusion injury

作　　者：王文静[1] 孙昱皓[1] 戴敏超[1] 汤耀辉[2] 孙青芳[1] 卞留贯[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院神经外科,200025 [2]上海交通大学MED-X研究院神经科学与神经工程中心

出　　处：《中国微侵袭神经外科杂志》2013年第6期275-279,共5页Chinese Journal of Minimally Invasive Neurosurgery

基　　金：上海市科委重点课题资助项目(编号:09JC1409700)(10JC1410700);上海市科委生物医药重点课题资助项目(编号:10411954200)

摘　　要：目的研究小鼠局灶性脑缺血再灌注损伤后早期自噬的变化和活性氧调控自噬的作用机制。方法线栓法制作小鼠短暂性大脑中动脉闭塞(tMCAO)模型56只,随机分成假手术组,对照组(仅建立tMCAO损伤)、tMCAO+N-乙酰-L-半胱氨酸(NAC)组和tMCAO+雷帕霉素组。Western blotting检测皮质和纹状体再灌注后不同时间点(再灌注后1、3、6、12、24 h)自噬相关蛋白LC3Ⅰ、LC3Ⅱ和Beclin-1的表达。双氢溴乙啶染色检测细胞内活性氧水平及对自噬活性的影响,免疫荧光染色观察NAC或雷帕霉素诱导下的自噬变化。2,3,5-氯化三苯四氮唑染色观察脑梗死面积的变化。结果与假手术组比较,缺血再灌注损伤后1 h Beclin-1开始升高,6 h LC3Ⅱ/LC3Ⅰ表达上调。与对照组对比,NAC条件下LC3Ⅱ/LC3Ⅰ和Beclin-1表达趋势明显升高。缺血再灌注损伤后1 h活性氧开始升高,给予NAC后表达下降。在NAC和雷帕霉素干预下,梗死面积缩小。结论小鼠局灶性脑缺血再灌注损伤后自噬表达上调,活性氧水平升高,抗氧化剂可能通过激活细胞的自噬途径保护神经细胞,减小脑梗死面积。Objective To study the changes of early autophagy after focal cerebral ischemia-reperfusion injury resulting from temporary middle cerebral artery occlusion（tMCAO） and regulatory mechanism of reactive oxygen species on the autophagy in mice.Methods The focal cerebral ischemia was induced by tMCAO by applying a modified intraluminal filament technique in 56 mice.The mice were randomly divided into sham group,control group（tMCAO alone）,tMCAO ＋ N-acetyl-L-cysteine（NAC） group and tMCAO ＋ rapamycin group.The reperfusion was performed at 1,3,6,12 and 24 h,the expressions of autophagy-related proteins LC3Ⅰ,LC3Ⅱ and Beclin-1 in the cortex and striatum after ischemia-reperfusion were detected by Western blotting at each time point.The level of reactive oxygen species was detected by staining technique with dihydroethidium（DHE）.Effects of antioxidant NAC or rapamycin on autophagy were evaluated using fluorescence microscope.The change of infarct volume was determined by 2,3,5-triphenyltetrazolium chloride（TTC） staining.Results Compared with the sham group,the expression of Beclin-1 was increased 1 h after focal ischemiareperfusion, while LC3Ⅱ/ LC3Ⅰexpression was upregulated at 6 h.Compared with the control group,LC3Ⅱ/LC3Ⅰ and Beclin-1 were upregulated under NAC administration.The level of reactive oxygen species started to elevate,and its expression reduced after NAC treatment.Therapeutic intervention with NAC and rapamycin decreased infarct volume.Conclusions The expression of autophagy is upregulated and level of reactive oxygen species elevates in the central nervous system after ischemia-reperfusion.Antioxidants can protect neural cells and decrease infarct volume possibly by activating autophagic pathway of cells.

关 键 词：脑缺血 再灌注损伤 自噬 氧化性应激 

分 类 号：R743[医药卫生—神经病学与精神病学]