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目的:观察脑缺血后两种抗氧化剂对NF-κB蛋白表达及活性变化和IκB磷酸化水平变化的调节机制。方法:采用四动脉结扎全脑缺血模型,应用p65,p50抗体进行免疫印迹分析,凝胶电泳迁移改变分析法测定NF-κB的DNA结合活性,尼氏染色观察神经元的死亡。结果:NF-κB的蛋白表达和结合活性在脑缺血复灌不同时间呈明显的时间依赖性;NF-κB的活性在缺血/复灌6小时后达到最高;其活性变化均能被吡咯烷二硫氨基甲酸盐(PDTC)和N-乙酰半胱氨酸(NAC)所抑制;组织学观察也证明了两种抗氧化剂对脑缺血的保护作用。结论:抗氧化剂对脑缺血/复灌引起的损伤的保护作用是通过降低NF-κB的活性而实现的,而NF-κB的活化和失活主要受抑制蛋白IκBα磷酸化降解调控。
OBJECTIVE: To observe the regulatory mechanism of the changes of NF-κB protein expression, activity and IκB phosphorylation after two kinds of cerebral ischemia. Methods: The model of global cerebral ischemia was established by ligating the four arteries. The p65 and p50 antibodies were used for Western blotting. The DNA binding activity of NF-κB was determined by gel electrophoretic mobility shift assay. The neuron death was observed by Nissl staining. RESULTS: The protein expression and binding activity of NF-κB were significantly time-dependent at different time points after cerebral ischemia / reperfusion. The activity of NF-κB reached the peak at 6 hours after ischemia / reperfusion. The changes of NF- Alkane dithiocarbamate (PDTC) and N-acetylcysteine (NAC). Histological observations also demonstrated the protective effect of the two antioxidants on cerebral ischemia. CONCLUSION: The protective effect of antioxidants on the injury induced by cerebral ischemia / reperfusion is achieved by decreasing the activity of NF-κB. The activation and inactivation of NF-κB are mainly regulated by the phosphorylation and degradation of IκBα.