亚低温对局灶性脑缺血再灌注大鼠皮层内质网IRE1-XBP1信号通路的影响

Effect of mild hypothermia on IRE1-XBP1 signaling pathway in endoplasmic reticulum in cortex in a rat model of focal cerebral ischemia-reperfusion

目的评价亚低温对局灶性脑缺血再灌注大鼠皮层内质网肌醇酶1-X盒连接蛋白1(IRE1-XBP1)信号通路的影响。方法清洁级健康雄性SD大鼠54只,8~10周龄,体重200~230 g,采用随机数字表法分成3组(n=18):假手术组(S组)、脑缺血再灌注组(I组)和亚低温组(T组)。采用线栓阻断大脑中动脉2 h后恢复灌注的方法制备大鼠局灶性脑缺血再灌注损伤模型。T组于再灌注即刻行全身体表喷洒酒精降温,维持直肠温度32~34℃3 h。S组仅暴露血管,不阻断。于再灌注24 h时行神经功能缺陷评分,随后处死大鼠取脑组织皮层缺血区,透射电镜下观察细胞超微结构,TUNEL染色法确定神经细胞凋亡率,采用Western blot法检测IRE1和XBP1的表达,qRT-PCR法测定IRE1和XBP1 mRNA的表达。结果与S组比较,I组和T组神经功能缺陷评分升高,皮层缺血区神经细胞凋亡率、IRE1和XBP1及其mRNA表达水平升高(P<0.05),神经细胞胞核变性肿胀,核膜破碎缺损,染色质固缩边集,内质网扩张成囊池状;与I组比较,T组神经功能缺陷评分降低,皮层缺血区神经细胞凋亡率降低,IRE1和XBP1及其mRNA表达水平升高(P<0.05),神经细胞超微结构损伤减轻。结论亚低温减轻大鼠局灶性脑缺血再灌注损伤的机制与进一步激活神经细胞IRE1-XBP1信号通路,缓解内质网应激反应有关。

Objective To evaluate the effect of mild hypothermia on inositol requiring enzyme 1-X-box binding protein 1(IRE1-XBP1)signaling pathway in endoplasmic reticulum in cortex in a rat model of focal cerebral ischemia-reperfusion(I/R).Methods Fifty-four clean-grade healthy male Sprague-Dawley rats,aged 8-10 weeks,weighing 200-230 g,were divided into 3 groups(n=18 each)using a random number table method:sham operation group(group S),cerebral I/R group(group I)and mild hypothermia group(group T).Cerebral I/R was induced by inserting a nylon thread with rounded tip into the internal carotid artery which was occluded for 2 h and then released for reperfusion.The surface cooling was started immediately after reperfusion,and the rectal temperature was maintained at 32-34℃for 3 h in group T.Blood vessels were only exposed,without occlusion in group S.The neurologic deficit was assessed and scored at 24 h of reperfusion.The animals were then sacrificed and the ischemic area of the cerebral cortex was removed for examination of the ultrastructure of the cells(with a transmission electron microscope),for determination of nerve cell apoptosis(using TUNEL),for detection of the expression of IRE1 and XBP1(by Western blot)and for determination of the expression of IRE1 and XBP1 protein mRNA(using quantitative real-time polymerase chain reaction).Results Compared with group S,the neurologic deficit scores were significantly increased,nerve cell apoptosis in the ischemic area of the cerebral cortex was increased,the expression of IRE1,XBP1 protein and mRNA was up-regulated(P<0.05),the neuronal nuclei was degenerated and swollen,the nuclear membrane was fragmented and defective,the chromatin was pyknotic and marginalized,and the endoplasmic reticulum was dilated and cisternal in group I and group T.Compared with group I,the neurologic deficit scores were significantly decreased,nerve cell apoptosis in the ischemic area of the cerebral cortex was decreased,the expression of IRE1,XBP1 protein and mRNA was up-regulated(P<0.05),and the damage to the ultrastructure of nerve cells was reduced in group T.Conclusion The mechanism by which mild hypothermia alleviates focal cerebral I/R injury is associated with further activation of neuronal IRE1-XBP1 signaling pathway and alleviation of endoplasmic reticulum stress response in rats.