PKCγ对异氟烷预处理抗大鼠脑缺血再灌注损伤的影响

The Changes of PKCγ in the Protective Effect of Isoflurane Preconditioning Against Cerebral Ischemic Reperfusion Injury in Rats

目的探讨异氟烷预处理抗脑缺血再灌注损伤过程中蛋白激酶γ(gamma of protein kinase C,PKCγ)在细胞膜和细胞浆内转位中的动态变化规律。方法采用大脑中动脉线拴方法制备大鼠局灶性脑缺血再灌注模型。72只SD大鼠随机分为3组:假手术组24只,根据再灌注后各时间点不同又分为再灌注后4、24、72 h组,每组8只。缺血再灌注组、异氟烷预处理组,每组24只,根据缺血再灌注后时间点不同又分为缺血再灌注后4、24、72 h组,每组8只。假手术组分别在再灌注后4、24、72 h处死大鼠,取大鼠额叶皮质;缺血再灌注组大鼠前脑缺血2 h后,分别在再灌注后4、24、72 h处死大鼠,取大脑额叶皮质;异氟烷预处理组于缺血前1.5 h经半密闭的吸入箱吸入1.5%异氟烷,持续1、0.5 h后制备脑缺血再灌注模型,其余同缺血再灌注组。各亚组大鼠大脑额叶皮质细胞浆和细胞膜上的PKCγ含量用免疫印迹(Western blot)法测定。结果与假手术组的再灌注后4、24、72 h组比较,缺血再灌注组和异氟烷预处理组的同时间点组细胞膜上PKCγ水平均有显著升高(P均<0.05)、在细胞浆中的PKCγ水平均有显著减少(P均<0.05)。异氟烷预处理组的缺血再灌注后4、247、2 h组PKCγ水平均较缺血再灌注组的同时间点组在细胞膜上显著升高(P均<0.05)、在细胞浆中的PKCγ水平均有显著减少(P均<0.05),且异氟烷预处理组的缺血再灌注后24 h组PKCγ水平为最高。结论异氟烷预处理可增加大鼠大脑额叶皮质PKCγ向细胞膜转位;PKCγ膜转位可能有利于大鼠脑的缺血再灌注损伤。

Objective To explore the changes with time in the distribution and levels of protein kinase Cγ （PKCγ） in cytosolic and cell membrane fractions in the course of isoflurane preconditioning＇s neuroprotection. Methods Seventy-two rats were randomly allocated to three experiment groups, sham-operated, ischemia-reperfusion and isoflurane-preconditioning （n = 24 for each）. The rats in sham-operated were divided into three different sub-groups： reperfusion at 4, 24,72 h （n=8 for each）. The rats in ischemia-reperfusion and isoflurane-preconditioning groups were divided respectively into three different sub-groups ： ischemia-reperfusion at 4,24,72 h （n= 8 for each）. The focal cerebral ischemic-reperfusion rat model was established with thread ligation method. The rats in sham-operated group were killed at 4,24 and 72 hour after reperfusion respectively,and samples of frontal cortex were taken. After 2 hours of ischemia, the rats in ischemic-reperfusion group were killed at 4, 24 and 72 hour after reperfusion respectively,and samples of frontal cortex were taken. The concentration of PKCγ in cytosolic and cell membrane fractions in the frontal cortex of the brain was examined by western blot analysis. In the isoflurane-preconditioning group, 1.5% isoflurane was administered at 90 min before the onset of ischemia and last for 60 rain. The rats＇ vessels were dissected but the suture was not introduced into the lumen of the internal carotid artery in sham-operated group. Results The levels of PKCγ in isoflurane-preconditioning and ischemia-reperfusion groups increased in membrane but decreased in cytosolic fractions after 4, 24, 72 hour reperfusion compared with that in sham-operated group（all P〈 0.05）. The levels of PKCγ in isoflurane-preconditioning group were higher in membrane and lower in cytosolic fractions than those in ischemia-reperfusion group at 4, 24 and 72 hour of reperfusion respectively（all P〈0.05）. And the amount of translocated PKCγ reached the peak at 24 hour after reperfusion in isoflurane-preconditioning group. Conelusion Isoflurane-preconditioning enhances the translocation of PKCγ from the cytosol to the membrane fraction, thereby preventing beneficial cell signaling possibly during reperfusion injury.