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

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。

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

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+LY294002 groups were significantly higher than those of the GB group, while their expression of P-AKT protein was significantly lower after 24 hours. Conclusion Ginkgobalide B can decrease neurocyte apoptosis caused by hypoxic-ischemic brain damage, especially at 24 h after the damage. The PI3K-AKT signaling pathway plays an important role in this effect.