Akt∕GSK-3β信号通路在曲古霉素A减轻小鼠脑缺血再灌注损伤中的作用

Role of Akt/GSK-3β signaling pathway in trichostatin-A-induced reduction of cerebral ischemiareperfusion injury in mice

目的 评价蛋白激酶B∕糖原合成酶激酶-3β(Akt∕GSK-3β)信号通路在曲古霉素A(TSA)减轻小鼠脑缺血再灌注损伤中的作用.方法 清洁级健康成年雄性Balb∕c小鼠40只,体重18~22 g,采用随机数字表法分为4组(n=10):假手术组(S组)、缺血再灌注组(I∕R组)、TSA组和TSA+Akt抑制剂LY294002组(TL组).采用大脑中动脉闭塞法(缺血1 h再灌注24 h)建立小鼠脑缺血再灌注损伤模型.TSA组于模型建立前连续3 d腹腔注射TSA 5 mg∕kg,TL组于模型建立前连续3 d腹腔注射TSA 5 mg∕kg,并于模型建立前30 min尾静脉注射LY29400215 nmol∕kg.再灌注24 h时取脑组织,采用TTC法测定脑梗死体积,采用比色法测定SOD、ROS活性和MDA含量,采用TUNEL法确定细胞凋亡指数,采用Western blot法测定Akt、磷酸化Akt(p-Akt)、GSK-3β和磷酸化GSK-3β(p-GSK-3β)的表达,计算p-Akt∕Akt比值和p-GSK-3β∕GSK-3β比值.结果 与S组比较,I∕R组脑梗死体积增加,脑组织SOD活性降低,MDA含量、ROS活性和细胞凋亡指数升高,p-Akt∕Akt比值和p-GSK-3β∕GSK-3β比值降低(P<0.05);与I∕R组比较,TSA组脑梗死体积减小,脑组织SOD活性升高,MDA含量、ROS活性和细胞凋亡指数降低,p-Akt∕Akt比值和p-GSK-3β∕GSK-3β 比值升高(P<0.05);与TSA组比较,TL组脑梗死体积增加,脑组织SOD活性降低,MDA含量、ROS活性和细胞凋亡指数升高,p-Akt∕Akt和p-GSK-3β∕GSK-3β比值降低(P<0.05).结论 TSA减轻小鼠脑缺血再灌注损伤的机制与激活Akt∕GSK-3β信号通路有关.

Objective To evaluate the role of protein kinase B/glycogen synthase kinase-3 beta (Akt/GSK-3β)signaling pathway in trichostatin-A (TSA)-induced reduction of cerebral ischemia-reperfusion (I/R)injury in mice.Methods Forty pathogen-free healthy male Balb/c mice,weighing 18-22 g, were divided into 4 groups (n=10 each)using a random number table method:sham operation group (S group),I/R group,TSA group and TSA plus Akt inhibitor LY294002group (TL group).Cerebral I/R was induced by middle cerebral artery occlusion (1-h ischemia followed by 24-h reperfusion).TSA 5 mg/kg was intraperitoneally injected for 3consecutive days before establishing the model in TSA group.TSA 5 mg/kg was intraperitoneally injected for 3 consecutive days before establishing the model,and LY 29400215 nmol/kg was injected via the caudal vein at 30 min before establishing the model.Brain tissues were obtained at 24h of reperfusion for determination of cerebral infarct size (by TTC ),activities of superoxide dismutase (SOD)and reactive oxygen species (ROS)and malondialdehyde (MDA)content (by colorimetric assay),cell apoptosis (by TUNEL)and expression of Akt,phosphorylated Akt (p-Akt),GSK-313 and phosphorylated GSK-3β(p-GSK-3β).The apoptosis index and ratios of p-Akt/Akt and p-GSK-3β/ GSK-3β were calculated.Results Compared with S group,the cerebral infarct size was significantly increased,the activity of SOD in brain tissues was decreased,the MDA content and ROS activity in brain tissues and apoptosis index were increased,and the ratios of p-Akt/Akt and p-GSK-3β/GSK-3β were decreased in I/R group (P<0.05).Compared with I/R group,the cerebral infarct size was significantly decreased,the activity of SOD in brain tissues was increased,the MDA content and ROS activity in brain tissues and apoptosis index were decreased,and the ratios of p-Akt/Akt and p-GSK-3β/GSK-3β were decreased in TSA group (P<0.05).Compared with TSA group,the cerebral infarct size was significantly increased,the activity of SOD in brain tissues was decreased,the MDA content and ROS activity in brain tissues and apoptosis index were increased,and the ratios of p-Akt/Akt and p-GSK-3β/GSK-3β were decreased in TL group (P<0.05).Conclusion The mechanism by which TSA attenuates cerebral I/R injury is related to activating Akt/GSK-3β signaling pathway in mice.