脑缺血再灌注致小鼠神经元退行性变进程中自由基水平和SOD-1表达的变化

Level of free radicals and expression of SOD-1 in neuronal degeneration mice due to cerebral ischemia-reperfusion

目的探讨脑缺血再灌注(ischemia/reperfusion,I/R)致神经元退行性变自由基和SOD-1表达的时程变化。方法 NIH小鼠160只,分为I/R组和假手术组,每组80只。抽取并回输约40%总血量加双侧颈总动脉夹闭20 min建立I/R损伤模型。分别于术后5、15、30、60 d以光学显微镜观察神经元损伤;Morris水迷宫评价学习记忆能力;分光光度计法检测SOD-1活性和丙二醛(MDA)含量;RT-PCR和Western blot分别检测海马SOD-1 mRNA和蛋白表达。结果假手术组脑组织MDA含量、SOD-1活性及蛋白、mRNA表达分别为(0.549±0.049)nmol/mg、(55.134±4.076)U/mg、30.413±2.962和104.541±18.825。与假手术组比较,I/R组小鼠学习记忆能力显著降低,术后5、15、30、60 d脑组织MDA含量[(0.673±0.087)、(0.782±0.101)、(0.719±0.094)、(1.084±0.140)nmol/mg]进行性显著升高(P<0.05);术后5 d和15 d I/R组SOD-1活性[(42.522±3.701)、(37.011±4.843)U/mg]和蛋白表达(23.145±3.476、15.746±2.344)显著降低(P<0.05);术后30 d和60 d I/R组SOD-1活性[(51.801±6.706)、(50.202±6.488)U/mg]与假手术组间差异无显著性(P>0.05),但SOD-1蛋白表达(40.587±6.154、44.263±6.597)较假手术组显著升高(P<0.05);各组SOD-1 mRNA表达水平无显著差异(P>0.05);组织学检测显示I/R组海马出现进行性神经元核固缩加重和神经元丢失。结论 I/R致小鼠神经元退行性变与SOD-1表达和功能异常和氧化应激进行性增强有关。

Objective To study the changes of three radicals and expression of SOD-1 in neuronal degeneration due to cerebral ischemia/reperfusion（I/R） in mice.Methods One hundred and sixty NIH mice were divided into I/R group and sham operation group,80 mice in each group.A cerebral I/R model was established by drawing out and reperfusing 40% of the whole blood volume and clamping the bilateral carotid arteries for 20 min.On days 5,15,30,and 60 after cerebral I/R,neuronal injury was observed under a light microscope,learning and memory ability of the mice was assessed with Morris water maze,hippocampal SOD-1 activity and malonaldehyde（MDA） level were measured with a spectrophotometer and expressions of SOD-1 mRNA and protein in the hippocampus were detected by RT-PCR and Western blotting,respectively.Results The MDA level,SOD-1 activity,expression level of SOD-1 protein and mRNA in sham operation group were 0.549±0.049 nmol/mg,55.134±4.076 U/mg,30.413±2.962 and 104.541±18.825,respectively.The learning and memory ability of mice was significantly lower in I/R group than in sham operation group.The MDA level in brain tissue of I/R group was significantly higher on days 5,15,30,and 60 days after I/R（0.673±0.087,0.782±0.101,0.719±0.094,and 1.084±0.140 nmol/mg） than sham operation group（P0.05）.The activity of SOD-1（42.522±3.701 and 37.011±4.843 U/mg） and the expression level of SOD-1 protein（23.145±3.476 and 15.746±2.344） were significantly lower than sham operation group after I/R on days 5 and 15（P〈0.05）.No significant difference was observed in SOD-1 activity after I/R 30 and 60 d between the two groups（51.801±6.706 vs 50.202±6.488 U/mg,P〈0.05）,while the expression of SOD-1 protein was significantly higher in I/R group than in sham operation group（44.263±6.597 vs 40.587±6.154,P〈0.05）.However,no significant difference was found in expression level of SOD-1 mRNA（P〉0.05）.Histology showed the progressive loss and karyopyknosis of neurons in hippocampi of I/R group.Conclusion Neuronal degeneration due to cerebral I/R is related with the progressively increased oxidative stress and abnormal SOD-1 expression and activity.