内质网应激结合线粒体在高血糖加重脑缺血再灌注损伤中的作用

Unification of endoplasmic reticulum and mitochondrium in cerebral ischemia-reperfusion injury increased by hyperglycemia

目的探讨内质网和线粒体在高血糖加重局灶性脑缺血再灌注损伤中的联合作用。方法制备高血糖状态下局灶性脑缺血再灌注损伤模型。随机分为3组:高血糖组、正常血糖组及假手术组。采用Zea-Longa标准进行神经功能评分、原位缺口末端标记法(TUNEL法)检测细胞凋亡数、Western blot技术检测葡萄糖调节蛋白78(Glucose regulated protein 78,GRP78)及细胞色素C(Cytochrome C,CytC)蛋白表达水平,免疫组化法检测半胱氨酰天冬氨酸特异性蛋白酶(caspase)-9、caspase-12的表达。结果 (1)高血糖组的神经功能评分在各个时点均显著高于正常血糖组。(2)高血糖组的凋亡细胞数在各个时点均显著多于正常血糖组。(3)高血糖组的GRP78蛋白表达水平在各个时点均显著高于正常血糖组及假手术组,于再灌注3h时达高峰,随再灌注时间延长,蛋白表达逐渐减低。(4)高血糖组各个时点的CytC蛋白表达水平均显著高于正常血糖组及假手术组,并于再灌注12h时达峰值。(5)高血糖组各个时点的caspase-9、caspase-12表达均显著高于正常血糖组及假手术组,并于再灌注12h时达高峰。结论内质网和线粒体在高血糖加重脑缺血再灌注损伤过程中具有协同作用。

Objective To evaluate the unification of endoplasmic reticulum and mitochondrium in rats with focal cerebral ischemia-reperfusion injury under hyperglycemia. Methods The focal cerebral ischemia-reperfusion injury model under hyperglycemia was established. The samples were randomly divided into 3 groups ： hyperglycemia group, normalglyce- mia group and sham operation group. And then the scores of neurological deficit and the number of apoptotic neurons were estimated. Expression of GRP78 and Cytochrome C protein were detected by western blot technique （WB）. Expression of caspase-9 and caspase-12 were detected by the method of immunohistochemistry（IHC）. Results The.. scores of neurologi- cal deficit and the number of apoptotic neurons at corresponding time in hyperglycemia group was significantly higher than those in normalglycemia group. The expression of GRP78 at corresponding time in hyperglycemia group was significantly higher than that in normalglycemia group and sham operation group, and it reached the maximum at 3h of reperfusion. The expression of Cytochrome C in cytoplasm at corresponding time in hyperglycemia group was significantly higher than that in normalglycemia group and sham operation group, and reached the maximum at 12h of reperfusion. The expression of caspase-9 and caspase-12 at corresponding time in hyperglycemia group was significantly higher than those in normalglyce- mia group and sham operation group, their expression increased rapidly and reached the maximum at 12h of reperfusion. Conclusion The synergistic effect of endoplasmic reticulum and mitochondrium plays an important role in cerebral ische- mia-reperfusion injury aggravated by hyperglycemia.