热休克蛋白A5介导的自噬在小鼠脑缺血/再灌注损伤中的作用

摘要

目的:探讨热休克蛋白A5(HSPA5)诱导的自噬在小鼠脑缺血/再灌注损伤中的作用.方法:将36只BALB/c小鼠随机分为sham、缺血再灌注(I/R)、vehicle+ I/R、3-甲基腺嘌呤(3-MA)+I/R、scramble siRNA+ L/R和HSPA5 siRNA+ I/R组(n=6).Sham组只进行手术操作,不插入线栓.L/R采用大脑中动脉阻塞(MCAO)60 min后再灌注24h.Vehicle+ I/R组和3-MA+ I/R将5μl0.9％NaCl或3-MA (30 mg/ml)在MCAO前30 min侧脑室注射.scramble siRNA+ I/R组和HSPA5 siRNA+ I/R组将5μl scramble siRNA或HSPA5 siRNA(2μg/μl)在MCAO前24h侧脑室注射.检测神经细胞内自噬体、缺血大脑皮层(LC3)-Ⅱ/LC3-Ⅰ表达、神经元损伤程度及神经功能缺损.结果:显微镜下sham组小鼠大脑皮层神经细胞形态正常;I/R组小鼠缺血大脑皮层神经元胞质中细胞器减少,自噬体形成.与sham组比较,I/R组缺血大脑皮层LC3-IⅡ/LC3-Ⅰ蛋白表达水平显著增高(P＜0.05);与I/R组相比,3-MA+I/R组或HSPA5 siRNA+ I/R组缺血大脑皮层LC3-Ⅱ/LC3-Ⅰ蛋白表达明显减少(P＜0.05);3-MA+ I/R组及HSPA5 siR-NA+ I/R组I/R后脑缺血性损伤及神经系统症状加重(P＜0.05).结论:HSPA5诱导自噬可能在小鼠局灶性I/R损伤中发挥保护作用.%Objective:To determine the role of heat shock protein A5 (HSPA5) induced autophagy on cerebral ischemia/reperfusion injury in mice.Methods:Thirty-six BALB/c mice were randomly divided into sham group,ischcmia/reperfusion (I/R) group,vehicle + I/R group,3-Methyladenine(3-MA) + I/R group,scramble siRNA group and HSPA5 siRNA + I/R group(n =6).In sham group,the operation was only performed,did not insert line switch.Focal cerebral ischemia was performed using the method of middle cerebral artery occlusion (MCAO) for 60 min and 24 h reperfusion.In vehicle + I/R group and 3-MA + I/R group,2 μl 0.9％ NaCl or 3-MA(30 mg/ml) was administered by intracerebroventricular injection 30 min before MCAO;In scramble siRNA + I/R group and HSPA5 siRNA + I/R group,5 μl scramble siRNA or HSPA5 siRNA(2 μg/μl) was administered by intracerebmventricular injection 24 h before MCAO.Autophagosome in neuron,the expression of microtubule-associated protein light chain 3 (LC3)-Ⅱ/LC3-Ⅰ in ischemic cortex,the degree of cerebral ischemic injury and neurological function score were detected.Results:Initial electron microscopy showed that neuronal morphology appeared to be normal in the sham group.At 24 h after I/R,cell shrinkage,loss of cellular organelles and formation of autophagosomes were observed in the ischemic cerebral cortex of I/R group.In addition,autophagosomes were less frequently observed than that in I/R group.The expressions of LC3-Ⅱ/LC3-Ⅰ and Beclin-1 protein were increased significantly in I/R group compared with that in sham group(P ＜ 0.05).Compare with I/R group,the LC3Ⅱ/LC3-Ⅰ protein levels induced by I/R in 3-MA + I/R group or HSPA5 siRNA + I/R group was decreased effectively (P ＜ 0.05).In addition,the cerebral ischemic injury and neurological symptoms after I/R in 3-MA + I/R group or HSPA5 siRNA + I/R group were exacerbated significantly (P ＜ 0.05).Conclusion:These results suggest that HSPA5 induced autophagy may play a protective role in focal I/R damage in mice.