一氧化氮清除剂Carboxy-PTIO对短暂性局灶性脑缺血大鼠PERK/p-eIF2α通路的调节作用

Regulatory effect of nitric oxide scavenger Carboxy-PTIO on PERK/p-eIF2α pathway in rats with transient focal cerebral ischemia

目的观察一氧化氮(nitric oxide,NO)清除剂Carboxy-PTIO(C-PTIO)对大脑中动脉梗死(middle cerebral artery occlusion,MCAO)模型大鼠再灌注后24 h脑组织中PERK/p-eIF2α通路介导的细胞死亡的影响,探究NO的产生对大鼠脑内内质网应激(endoplasmic reticulum stress,ERS)介导的神经元凋亡的影响。方法将18只健康雄性SD大鼠依据数字表法随机分为假手术(Sham)组、MCAO组和MCAO+C-PTIO组,每组6只。大鼠大脑中动脉缺血再灌注模型制作采用改良线栓法,并于缺血90 min后拔出线栓进行再灌注。术中监测大鼠肛温,使其维持在正常范围。于再灌注后24 h处死大鼠,迅速取脑,用免疫荧光染色检测大鼠脑组织冰冻切片缺血半暗带区p-eIF2α和C/EBP homologous protein(CHOP)的表达水平,用免疫荧光双标法将NO介导的蛋白质损伤标志物3-硝基酪氨酸(3-nitrotyrosine,3-NT)分别与p-eIF2α和CHOP进行定位。结果1)Sham组大鼠没有p-eIF2α和3-NT染色阳性细胞,偶见CHOP染色阳性细胞,MCAO组大鼠脑组织缺血半暗带区3-NT、p-eIF2α、CHOP均大量表达(P<0.05)。2)与MCAO组相比,经腹腔注射给予C-PTIO后,缺血再灌注大鼠脑组织半暗带区3-NT、p-eIF2α、CHOP表达均明显减少(P<0.05)。3)缺血再灌注大鼠脑组织半暗带区,3-NT分别与p-eIF2α和CHOP免疫荧光染色共定位。结论在脑缺血再灌注过程中,NO介导的氧化应激损伤引起ERS,并激活PERK/eIF2α通路引起细胞凋亡;抑制NO的产生,可以减弱这一过程,起到神经保护作用。

Objective To observe the effect of nitric oxide(NO)remover Carboxy-PTIO(C-PTIO)on cell death mediated by PERK/p-eIF2αpathway in the brain tissues of middle cerebral artery occlusion(MCAO)model rats after 24 hours reperfusion,and to explore the effect of NO production on endoplasmic reticulum stress(ERS)-mediated neuronal apoptosis.Methods Eighteen healthy male Sprague-Dawley rats were divided into sham operation group,MCAO group and MCAO+C-PTIO group(n=6)according to random number table.Rat model of middle cerebral artery ischemia-reperfusion was made by modified suture method,the rats were underwent 90 min of right MCAO,and then reperfusion by withdrawing the filament.The anus temperature of the rats was monitored during the operation.The rats were sacrificed 24 h after reperfusion,and the brain was collected quickly.The expression of p-eIF2αand CHOP in the ischemic penumbra of frozen sections of the brain tissues were detected by immunofluorescence staining.The co-localization of NO-mediated protein damage marker 3-nitrotyrosine(3-NT)with p-eIF2αand CHOP,respectively,was observed with immunofluorescence double labeling.Results 1)No p-eIF2αand 3-NT positive cells were observed in the sham group,while CHOP staining positive cells were occasionally observed.In the MCAO group,the expression of 3-NT,p-eIF2αand CHOP in the ischemic penumbra of the brain tissue increased significantly(P<0.05).2)Compared with the MCAO group,the expressions of 3-NT,p-eIF2αand CHOP in the penumbra of the ischemia-reperfusion rats were significantly decreased after intraperitoneal injection of C-PTIO(P<0.05).3)In the penumbra of brain tissue of ischemia-reperfusion rats,3-NT was co-localized with p-eIF2αand CHOP as observed with immunofluorescence staining.Conclusion NO-mediated oxidative stress damage induces ERS and activates PERK/eIF2αpathway to induce apoptosis during cerebral ischemia-reperfusion.Inhibition of NO production attenuates this process and plays a neuroprotective role.