全脑缺血再灌注诱导大鼠海马CA1区Trx巯基去亚硝基化机制的研究

Mechanism of Cerebral Ischemia/Reperfusion-induced Denitrosylation of Trx in Rat Hippocampal CA1 Regions

目的研究探讨全脑缺血再灌注诱导大鼠海马CA1区硫氧还蛋白(Trx)巯基去亚硝基化的机制。方法将SD大鼠随机分为假手术组(Sham组)、全脑缺血再灌注组(I/R组)、SNP组(腹腔注射)、GSNO组(侧脑室注射)及溶剂对照组(生理盐水组腹腔注射/侧脑室注射);采用四动脉结扎去结扎法构建大鼠全脑缺血再灌注模型,运用生物素转化法检测蛋白质的巯基亚硝基化,聚丙烯酰胺凝胶电泳、免疫印迹方法、焦油紫染色方法对Trx巯基去亚硝基化水平以及大鼠海马神经元的损伤进行分析研究。结果与Sham组和其他I/R组相比,I/R6h组Trx巯基亚硝基化水平明显降低(P<0.05);与I/R组相比,外源性NO供体GSNO与SNP组的Trx巯基亚硝基化水平显著增多(P<0.05),但生理盐水组差异无统计学意义。结论在全脑缺血再灌注作用时间为6h时,全脑缺血再灌注诱导大鼠海马CA1区Trx巯基去亚硝基化程度最强;外源性NO供体能够抑制Trx巯基去亚硝基化从而对神经元起保护作用。

Objective To investigate the denitrosylation of Trx induced by cerebral ischemia/reperfusion in the CA1 area of rat hippocampus.Methods The rats were randomly divided into 4 groups:sham group,ischemia/reperfusion group(I/R group),SNP(intraperitoneal injection),GSNO(unilateral intracerebroventricular injections)and solvent control group(intraperitoneal injection/lateral ventricle injection in saline group).The rats were intraperitoneally injected with SNP(5 mg/kg,dissolved in physiological saline)three times at an interval of 1.5 h,with the first SNP performed 30 min prior to cerebral ischemia(SNP group).We perform the study mainly by immunoblotting and Cresyl violet staining.S-nitrosylation is examined mainly by the biotin-switch assay.Results The denitrosylation of Trx is significantly noticeable in the I/R group than in the Sham group.The denitrosylation of Trx is lightly observed in the GSNO group and SNP group than in the I/R group,meanwhile the saline group is almost the same with I/R group.Cresyl violet staining shows the neuroprotective effect of inhibiting Trx denitrosy-lation against neuronal injury induced by cerebral ischemia-reperfusion in hippocampal CA1 pyramidal neurons.Conclusion The denitrosylation of Trx have an important role in the cell apoptosis induced by I/R in the rats hippocampal CA1 regions.