海人藻酸诱导大鼠海马CA1区Procaspase3巯基去亚硝基化机制的研究

The research of KA injection-induced denitrosylation of Procaspase3 in the rat hippocampal CA1 regions

目的:探讨海人藻酸(Kainic acid,K A)注射诱导大鼠海马CA1区半胱氨酸天冬氨酸蛋白酶3酶原(cysteine aspartate-specific proteasezymogen,Procaspase3)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)、KA注射组(KA组)、给药组(NS102组,DNCB组,GSNO组)及溶剂对照组(生理盐水组,DMSO组)。采用KA侧脑室注射构建大鼠癫痫模型。运用生物素转化法检测蛋白质巯基亚硝基化,聚丙烯酰胺凝胶电泳、免疫印迹方法对Procaspase3巯基去亚硝基化进行分析,焦油紫染色法观察大鼠成活海马神经元的数量。结果:与sham组相比,KA组Procaspase3巯基亚硝基化水平明显降低,与KA组相比,NS102组、DNCB组和GSNO组Procaspase3巯基亚硝基化水平显著上升,差异均有统计学意义(P<0.05),但DMSO组和生理盐水组差异无统计学意义。说明注射浓度为0.6μg/10μL的KA能够诱导大鼠海马CA1区Procaspase3巯基去亚硝基化;KA通过激活KA受体导致大鼠海马CA1区Procaspase3巯基去亚硝基化,KA受体抑制剂NS102能够抑制Procaspase3巯基去亚硝基化;TrxR参与调节KA诱导的大鼠海马CA1区Procaspase3巯基去亚硝基化;外源性NO供体能够抑制Procaspase3巯基去亚硝基化。焦油紫染色结果显示,与KA组相比,NS102组、DNCB组和GSNO组神经元损伤降低。结论:KA注射通过激活KA受体诱导大鼠海马CA1区Procaspase3巯基去亚硝基化,Procaspase3巯基去亚硝基化受TrxR和外源性NO调节,抑制Procaspase3巯基去亚硝基化对海马神经元具有保护作用。

Objective:To investigate the denitrosylation of Procaspase3 induced by KA injection on the apoptosis in the CA1 area of rat hippocampus.Methods:KA is injected at concentrations of 0.6μg/10μL in SD rats.The rats were decapitated after 6 hours.The rats were randomly divided into 7 groups:Sham group,KA injection group(KA group),the unilateral intracerebroventricular injections NS102,DNCB and GSNO before KA injection group(NS102 group,DNCB group,GSNO group),the unilateral intracerebroventricular injections 10%DMSO or saline group(DMSO group,saline group).The study was performed mainly by immunoblotting and Cresyl violet staining.S-nitrosylation is examined mainly by the biotin-switch assay.Results:The denitrosylation of Procaspase3 is significantly more noticeable in the KA group(0.6μg/10μL 6 h)than in the Sham group.The denitrosylation of Procaspase3 is less observed in the NS102 group than in the KA group,the same situation appears in the DNCB group and in the GSNO group,and meanwhile the DMSO group and the saline group are almost the same with the KA group.Cresyl violet staining shows the neuroprotective effect of inhibiting Procaspase3 denitrosylation against neuronal injury induced by KA injection in hippocampal CA1 pyramidal neurons.Conclusion:KA(0.6μg/10μL 6 h)injection induced the significantly noticeable of denitrosylation of the Procaspase3 in hippocampal CA1 region of rats,via the activation of KA recepeor.KA-induced Procaspase3 denitrosylation can be attenuated by exogenous NO and TrxR.NS102,DNCB and GSNO could provide neuroprotection of the pyramidal neurons of CA1 regions by attenuating Procaspase3 denitrosylation.