内源性NO介导的Stargazin亚硝基化修饰在脑缺血再灌注后突触可塑性中的作用及机制

Endogenous NO Mediated Stargazin Nitroso Modification in Synaptic Plasticity after Cerebral Ischemia Reperfusion

目的研究Stargazin-亚硝基化修饰在脑缺血再灌注后突触可塑性中的作用,并探讨NO调控AMPAR"Trafficking"的分子机理。方法采用线栓法阻塞大脑中动脉复制局灶性脑缺血再灌注(MCAO/R)损伤大鼠作为模型组,分别在每只大鼠MCAO/R模型内给予NMDAR抑制剂MK801、氧化还原剂DTT干预作为实验组。采用mNSS评分标准检测大鼠神经功能,TTC染色检测脑部缺血损伤情况,TUNEL染色检测以及WB检测缺血侧海马神经元凋亡情况,Griess法检测缺血侧海马组织中NO的含量,此外Western Blot检测海马组织中Stargazin-亚硝基化修饰水平以及AMPAR蛋白的表达和活化情况。结果给予MK801和DTT处理后MCAO/R模型中Stargazin-亚硝基化修饰水平(P<0.01)以及NO含量(P<0.01)下降;AMPAR亚基GluR2磷酸化水平降低(P<0.0001);抑制Stargazin亚硝基化修饰能够改善MCAO/R引起的神经损伤与凋亡(P<0.001)。结论在MCAO/R模型中,抑制内源性NO和Stargazin亚硝基化水平可促进神经突触重塑,其机制可能是干预了Stargazin辅助蛋白与AMPAR亚基GluR2亲和力有关。

Objective To investigate the role of Stargazin-nitroso modification in synaptic plasticity after cerebral ischemia and reperfusion,and to investigate the molecular mechanism of NO regulating AMPAR"trafficking".Methods The rat model of focal cerebral ischemia—reperfusion injury was replicated by the middle cerebral artery occlusion(MCAO)method,and the animal model was interfered with NMDAR inhibitor MK801 and oxidative reductant DTT,respectively.mNSS score was used to detect the neurological function of rats.TTC staining and Western Blot were used to detect the ischemic injury of the brain;TUNEL staining was used to detect the apoptosis of hippocampal neurons in the ischemic side;Griess staining was used to detect the content of NO in the ischemic side of hippocampal tissue,and Western Blot was used to detect the stargazin-nitrogenization modification level and AMPAR protein expression and activation in the hippocampal tissue.Results After treatment with MK801 and DTT,the Stargazin-nitroso modification level(P<0.01)and NO content(P<0.01)in MCAO/R model were decreased.Phosphorylation of GluR2 in AMP AR subunit was decreased(P<0.0001);Inhibition of Stargazin,s nitrosylation modification improved MCAO/R-induced nerve damage and apoptosis(P<0.001).Conclusion Inhibition of endogenous NO and Stargazin nitrosylation levels promotes synaptic remodeling in the MCAO/R model,possibly by interfering with the GluR2 affinity between Stargazin helper proteins and AMP AR subunit.