脑缺血/再灌注通过神经性一氧化氮合酶诱导KA受体亚基GluR6巯基亚硝基化的研究

Cerebral ischemia-reperfusion induce GluR6 S-nitrosylation through neuronal NO synthase

目的探讨脑缺血/再灌注是否通过神经性一氧化氮合酶(n NOS)诱导KA受体亚基Glu R6巯基亚硝基化。方法将28只SD大鼠随机分成4组,每组7只:假手术组、脑缺血/再灌注6 h组、脑缺血/再灌注6 h合并给药组、脑缺血/再灌注6 h溶剂对照组。采用四动脉结扎法构建大鼠全脑缺血模型,给予SD大鼠腹腔注射n NOS的抑制剂7-硝基吲唑。主要应用"生物素转化法"、SDS-PAGE、免疫印迹等方法对Glu R6的蛋白表达及其巯基亚硝基化水平进行研究。结果脑缺血/再灌注组与假手术组相比Glu R6的巯基亚硝基化水平显著升高,差异有统计学意义(P<0.05);脑缺血/再灌注联合n NOS的抑制剂7-NI组与脑缺血/再灌注组相比,Glu R6的巯基亚硝基化水平显著降低,差异有统计学意义(P<0.05);各组Glu R6的蛋白表达水平比较,差异无统计学意义(P>0.05)。结论在脑缺血/再灌注早期n NOS诱导Glu R6的巯基亚硝基化,为缺血性脑损伤疾病的治疗提供了新的理论依据。

Objective To discuss whether neuronal nitric oxide synthase induce KA receptor subunit Glu R6 S-nitrosylation during ischemia-reperfusion.Methods 28 SD rats were randomly divided into 4 groups,each group had 7 rats：sham operation group,cerebral ischemia reperfusion 6 h combination with neuronal nitric oxide synthase inhibitor 7-NI group,cerebral ischemia reperfusion group,cerebral ischemia/reperfusion 6 h solvent control group.Transient cerebral ischemia was induced by a four-vessel occluded（4-VO） method.SD rats were treated intraperitoneally with 7-Nitroindazole, an inhibitor of n NOS（7-NI,25 mg/kg）.Biotin switch assay,SDS-PAGE and western blot was performed to study Snitrosylation and expression levels of Glu R6.Results Glu R6 S-nitrosylation levels in cerebral ischemia-reperfusion group increased significantly versus than that of sham group（P〈0.05）.Glu R6 S-nitrosylation levels in cerebral ischemiareperfusion combination with neuronal nitric oxide synthase inhibitor 7-NI group descended significantly versus cerebral ischemia-reperfusion group,it was significant difference（P〈0.05）;however,Glu R6 protein expression level was no significant difference among groups.Conclusion n NOS induce Glu R6 S-nitrosylating during the early stages of ischemia-reperfusion,which can be a new theory basis for stroke therapy.