Xiong Tao, Chen Hong-Ju, Qu Yi, Luo Bi-Ru, Mu De-Zhi
Key Laboratory of Obstetic & Gynecologic and Pediatric Diseases and Birth Defects of Ministy of Education, Department of Pediatrics, West China Second Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2013 Mar;44(2):274-9.
To investigate the activity of protein kinase B (Akt) and its downstream protein, glycogen synthase kinase-3beta (GSK-3beta) under hypoxia-ischemia (HI), and the possible regulation for axonal density.
Postnatal day 10 SD rats were suffered the right common carotid artery ligation and 8% mixture of oxygen and nitrogen hypoxia 2.5 h to produce HI model. The expression of total and phosphorylated Akt and GSK-3beta was detected by western blot after HI. After pretreatment of Akt inhibitor, wortmannin or LY294002, Western blot detect the expression of total and phosphorylated of Akt, GSK-3beta at 4 h and 24 h after HI. After pretreatment of wortmannin, axonal density was determined by Bielschowsky silver impregnation, and histological injury was evaluated by hematoxylin and eosin (HE) staining.
The expression of total Akt and GSK-3beta remained unchanged after HI. p-Akt protein significantly decreased at 0.5 h, increased at 2 h and reached the highest at 4 h, returned to baseline at 8 h, declined at 24 and 48 h after HI, and finally returned to baseline again at 72 h compared with that of sham controls, p-GSK-3beta protein decreased at 0. 5 h, increased at 2 h, reached the highest at 4 h, returned to baseline at 8 and decreased at 24 h, reached the lowest at 48 h, and returned to baseline at 72 h. Wortmannin or LY294002 intervention didn't change the expression of total Akt and GSK-3beta, while decrease the p-Akt and p-GSK-3beta expression. HI cause decreased axonal density, and the histological injury of brain. Wortmannin pretreatment could aggravate the histological injury and decrease axonal density after HI.
The Akt pathway is involved in axonal density and histological brain injury after HI in neonatal rat.
探讨缺氧缺血（HI）状态下蛋白激酶B（Akt）及其下游蛋白糖原合酶激酶-3β（GSK-3β）的活性，以及对轴突密度的可能调控作用。
出生后10天的SD大鼠行右侧颈总动脉结扎并置于8%氧氮混合气体中缺氧2.5小时以建立HI模型。HI后通过蛋白质免疫印迹法检测总Akt和磷酸化Akt以及GSK-3β的表达。用Akt抑制剂渥曼青霉素或LY294002预处理后，通过蛋白质免疫印迹法检测HI后4小时和24小时总Akt、磷酸化Akt以及总GSK-3β、磷酸化GSK-3β的表达。用渥曼青霉素预处理后，通过 Bielschowsky 银染法测定轴突密度，并用苏木精-伊红（HE）染色评估组织学损伤。
HI后总Akt和GSK-3β的表达保持不变。与假手术对照组相比，磷酸化Akt蛋白在HI后0.5小时显著降低，2小时升高，4小时达到最高，8小时恢复至基线水平，24小时和48小时下降，最终在72小时再次恢复至基线水平；磷酸化GSK-3β蛋白在0.5小时降低，2小时升高，4小时达到最高，8小时恢复至基线水平，24小时下降，48小时达到最低，72小时恢复至基线水平。渥曼青霉素或LY294002干预未改变总Akt和GSK-3β的表达，但降低了磷酸化Akt和磷酸化GSK-3β的表达。HI导致轴突密度降低以及脑的组织学损伤。渥曼青霉素预处理可加重HI后的组织学损伤并降低轴突密度。
Akt信号通路参与新生大鼠HI后的轴突密度和脑组织学损伤。
