自噬相关基因Atg12和LC3-Ⅱ在脑缺血再灌注大脑表达的实验研究

Expression of Atg12 and LC3-Ⅱ in the cerebral cortex during focal cerebral ischemia-reperfusion injury

摘要:

目的：探讨局灶性脑缺血再灌注损伤（cerebral ischemia reperfusion injury，CIR）时大脑皮质自噬相关蛋白-12（autophagy related protein-12，Atg12）和自噬微管相关蛋白轻链3抗体Ⅱ（autophagy microtubule-associated protein light chain 3 antibodyⅡ，LC3-Ⅱ）的激活规律，在此基础上探讨其对脑组织自噬的影响。方法：实验分为假手术组（sham组）和缺血再灌注组（CIR组），且CIR组下设CIR 0、6、12、24、48、72h共6个亚组。除sham组和CIR 24h组为每组20只外（其中10只用于模型鉴定），其余为每组10只。采用大脑中动脉栓塞术制备局灶性脑缺血再灌注模型，再灌注24 h后采用神经功能评分、2，3，5-三苯基氯化四氮唑（2，3，5-Triphenyltetrazolium chloride，TTC）染色、脑组织含水量来鉴定脑缺血模型是否成功。采用免疫组化和免疫印迹方法检测大脑皮质Atg12和LC3-Ⅱ的表达规律。结果：与sham组比较，CIR 24h组可见明显的神经功能缺损、脑梗死和明显的脑水肿。免疫组化结果显示，Atg12和LC3-Ⅱ在大鼠脑缺血再灌注6 h开始表达[阳性率分别为（15.49±4.18）%、（18.54±3.62）%]，在24~48 h达高峰[（33.63±3.26）%、（29.62±1.73）%]，72 h之后逐渐减弱[（24.90±3.96）%、（20.36±3.51）%]。免疫印迹结果显示，Atg12和LC3-Ⅱ在大鼠脑缺血再灌注6 h开始表达[表达量为（0.372 3±0.076 5）、（0.148 4±0.011 5）]，在24~48 h达高峰[（0.741 7±0.071 8）、（0.451 3±0.019 0）]，72 h之后逐渐减弱[（0.365 0±0.042 0）、（0.245 1±0.030 3）]。结论：脑缺血时Atg12和LC3-Ⅱ在调节脑缺血的脑组织自噬方式中具有重要的作用。

Abstract:

Objective：To investigate the activation law of autophagy related protein-12（Atg12） and autophagy microtubule-associated protein light chain 3 antibodyⅡ（LC3-Ⅱ） in the cerebral cortex during focal cerebral ischemia-reperfusion injury（CIR），and to fur－ther explore its influence on brain autophagy on this basis. Methods：The rats were divided into sham operation group（sham group） and ischemia reperfusion group（CIR group），and the CIR group was subdivided into six subgroups：CIR 0，6，12，24，48h and 72 h. Ex－cept that sham group and CIR 24h group had 20 rats in each group（10 were used for model identification），there were 10 rats in other groups，respectively. Focal CIR model was established by middle cerebral artery occlusion. CIR model was identified by neurological score，2，3，5-Triphenyltetrazolium chloride（TTC） staining，and brain water content 24 h later. The expression pattern of Atg12 and LC3-Ⅱ in cerebral cortex was detected by immunohistochemistry methods and Western blot techniques. Results：Compared with sham group，there were obvious neuro－logical deficit symptoms，cerebral infarction and cerebral edema in CIR 24h group. The results of immunohistochemistry showed that the positive rates of Atg12 and LC3-Ⅱ appeared in rat CIR 6 h[（15.49±4.18）%，（18.54±3.62）%]，and peaked at 24 to 48 h[（33.63±3.26）%，（29.62±1.73）%]，then the expression gradually decreased 72 h later[（24.90±3.96）%，（20.36±3.51）%]. The results of Western blot showed that the expression of Atg12 and LC3-Ⅱ appeared in rat CIR 6 h[（0.372 3±0.076 5），（0.148 4±0.011 5）]，and peaked at 24 to 48 h [（0.741 7±0.071 8），（0.451 3±0.019 0）]，then the expression gradually decreased 72 h later[（0.365 0±0.042 0），（0.245 1±0.030 3）]. Conclusion：The expression of Atg12 and LC3-Ⅱ plays an important role in the regulation of brain autophagy during the CIR.