银杏内酯B对缺血缺氧性脑损伤新生大鼠脑神经元凋亡及P-AKT（ser473）表达的影响  

Effects of ginkgobalide B on neurocyte apoptosis and the expression of protein kinase B after experimental hypoxic-ischemic brain injury

作　　者：王军[1] 杜江华[1] 程萍萍[1] 朱登纳[1] 张勇[1] 熊华春[1] 袁俊英[1] 张轶 王宝珍[1] Wang Jun Du Jianghua Cheng Pingping Zhu Dengna Zhang Yong Xiong Huachun Yuan Junying Zhang Yi Wang Baozhen(The Department of Rehabilitation for Children, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Chin)

机构地区：[1]河南郑州大学第三附属医院儿童康复科,郑州450052 [2]郑州大学第一附属医院

出　　处：《中华物理医学与康复杂志》2017年第9期646-650,共5页Chinese Journal of Physical Medicine and Rehabilitation

基　　金：河南省教育厅科学技术研究重点项目（14A320004）

摘　　要：目的 观察银杏内酯B（GB）对缺血缺氧性脑损伤（HIBD）新生大鼠脑皮质神经元凋亡及P-AKT（ser473）蛋白表达的影响。 方法 采用随机数字表法将90只SPF级新生7日龄SD大鼠分为假手术组、缺血缺氧（HI）组及GB组，每组30只。采用经典Rice法建立HIBD动物模型，其中GB组分别于术后0h、24h按每千克体重10mg经腹腔注射GB。各组均于术后不同时间点（包括术后6h、12h、24h及48h）随机处死6只大鼠，采用荧光定量PCR技术检测凋亡关键执行分子Caspase-3 mRNA表达水平，发现GB抑制凋亡最显著的时间点为缺血缺氧后24h；然后增加大鼠并纳入GB＋LY294002组，于制模后给予GB及PI3K-AKT通路抑制剂LY294002预干预，进一步探讨缺血缺氧后24h时PI3K-AKT通路在GB抗凋亡中的作用。采用HE染色法观察各组大鼠脑皮质结构变化，采用TUNEL法检测脑皮质神经元凋亡情况，采用免疫组化法检测P-AKT（ser473）蛋白表达情况。 结果 HI组和GB组Caspase-3 mRNA表达于缺血缺氧后6h开始增高，于缺血缺氧后12h进一步上升，于缺血缺氧后24h时达到峰值，随后开始下降；与假手术组比较，HI组和GB组Caspase-3 mRNA表达于缺血缺氧后6h、12h、24h、48h均明显增高，另外GB组Caspase-3 mRNA表达均明显低于HI组，组间差异具有统计学意义（P〈0.05）。与假手术组比较，缺血缺氧后24h时HI组、GB组及GB＋LY294002组脑皮质神经元Caspase-3表达增强、凋亡阳性细胞增多、P-AKT（ser473）表达减弱，其中HI组和GB＋LY294002组Caspase-3表达及凋亡细胞数量均明显超过GB组，P-AKT（ser473）蛋白表达则明显弱于GB组，组间差异均具有统计学意义（P〈0.01）。 结论 银杏内酯B具有明显抗神经元凋亡作用，且以缺血缺氧后24h时对神经细胞凋亡的抑制作用最强；此外PI3K-AKT信号通路在GB抗缺血缺氧诱导脑神经元凋亡中发挥重要作用。Objective To observe the effect of ginkgobalide B （GB） on neurocyte apoptosis and protein kinase B expression in neonatal rats after hypoxic-ischemic brain damage （HIBD）. Methods Ninety seven-day-old Sprague-Dawley rats were randomly divided into a sham group, an HIBD group and a GB group, each of 30. HIBD was induced in the HIBD and GB groups using the classical Rice method, while the sham group was given a sham operation. GB （10 mg/kg） was injected intraperitoneally to the rats in the GB group at 0 h and 24 h after the modeling. Then 6 rats were killed 6 h, 12 h, 24 h and 48 h after the modeling, and the expression of caspase-3 mRNA was detected using a real-time PCR to find the time point of maximum effectiveness. Then to further explore the role of the PI3K-AKT pathway in the anti-apoptosis effect of ginkgolide B, a a GB＋LY294002 group of 6 rats, which was injected with PI3K-AKT pathway inhibitor LY294002 （1.8 mg/kg） intraperitoneally at 30 min before the modeling and with GB（10 mg/kg） at 0 h and 24 h after the modeling, was added to the experiment. Hematoxylin-eosin staining, terminal-deoxynucleotidyl transferase-mediated nick end labeling and immunohistochemical staining were then used to observe any morphological changes in the cortex, to detect neuronal apoptosis and to quantify the expression of P-AKT protein. Results The expression of caspase-3 in the HI and GB groups began to increase 6 hours after the HIBD and reached a peak after 24 hours, followed by a gradual decline. The expression of caspase-3 in the GB group was significantly lower than in the HI group throughout, while that of both of those groups was significantly higher than in the sham group. Apoptosis-positive cells and the expression of caspase-3increased had significantly in the HI, GB and GB＋LY294002 groups 24 hours after the HIBD compared with the sham group, while the expression of P-AKT protein had decreased significantly. Moreover, the apoptosis-positive cells and the expression of caspase-3 of the HI and GB�

关 键 词：银杏内酯B 缺血缺氧性脑损伤 细胞凋亡 

分 类 号：R742[医药卫生—神经病学与精神病学]