银杏内酯B对损伤神经干细胞内HIF-1α及PI3K/Akt信号通路的影响

摘要

目的：探讨银杏内酯B（GKB）对谷氨酸损伤神经干细胞（NSCs）内的磷脂酰肌醇-3激酶（PI3K）、磷酸化丝氨酸-苏氨酸蛋白激酶（p-Akt）、低氧诱导因子-1α（HIF-1α）活性的影响，以进一步明确银杏内酯B神经保护作用的分子机制。方法体外培养新生大鼠海马神经干细胞，并建立体外谷氨酸神经干细胞损伤模型。将其分为：（1）对照组；（2）GKB组（终浓度40 mg/L）；（3） wortmannin＋GKB组；（4）wortmannin组（终浓度10 nmol/L）。Western blot 印迹法检测各组NSCs中 PI3K、p-Akt、HIF-1α蛋白水平的表达。结果从新生大鼠海马区分离培养出具有自我更新、增殖的神经球，并在细胞球中可以检测到NSCs表面标记物巢蛋白（nestin）及5-溴-2-脱氧尿嘧啶（BrdU）的表达。100～400μmol/L谷氨酸作用NSCs 30 min，培养基中乳酸脱氢酶含量随着谷氨酸浓度的提高而明显增高[分别为（26.94±4.75）U/L，100μmol/L GLU；（38.22±5.86）U/L，200μmol/L GLU；（45.64±5.35）U/L，400μmol/L GLU]。经wortmannin处理后PI3K、p-Akt、HIF-1α蛋白活性较对照组明显下降，差异有统计学意义（P＜0.05）。wortmannin＋GKB组和wortmannin组比较，PI3K、p-Akt、HIF-1α表达明显上调。施加GKB后，PI3K、p-Akt、HIF-1α蛋白表达均较对照组增加，差异有统计学意义（P＜0.05）。结论银杏内酯B处理诱导损伤神经干细胞HIF-1α蛋白的表达可能与PI3K/Akt信号通路的激活有关。%Objective To explore the effects of ginkgolide B (GKB) on the activity of the PI3K, phosphorylated Akt (p-Akt), hypoxia-inducible factor-1α (HIF-1α) in the cultured neuronal stem cells induced by glutamate, in order to provide the further evidence for the molecular mechanism of neuroprotection of ginkgolide B. Methods The neural stem cells from neonatal rat hippocampus were cultured and the model of injured NSCs by glutamate acid was built up. The cells were divided randomly into:(1) the control group;(2) GKB group (final concentration of 40 mg/L);(3) wortmannin+GKB group;(4) wortmannin group (final concentration of 10 nmol/L). Western blot was used to detect the protein levels of PI3K, p-Akt, HIF-1α in the 4 groups. Results The cells cultured from the hippocampus of neonatal rats grew with the self-renewal and proliferation of neural ball and the expression of nestin which was the surface marker of NSCs and the BrdU (5-bromo-2'-deoxyuridine) could be detected by immunocytochemical fluorescent staining. After the 100-400μmol/L range of glutamate dosage were used to intervene for 30 minutes, the lactate dehydrogenase in the neural stem cell damage model obviously improved with increased of the glutamate dosage, (26.94±4.75)U/L, 100μmol/L GLU;(38.22±5.86)U/L, 200 μmol/L GLU; (45.64±5.35)U/L, 400 μmol/L GLU. The expression of PI3K, p-Akt and HIF-1α was significantly decreased after treated by wortmannin, the difference was statistically significant (P<0.05). Wortmannin+GKB group compared with the control group, the expression of PI3K, p-Akt and HIF-1αhad no significant difference. The protein expression of PI3K, p-Akt and HIF-1αincreased compared with the control group after applying to GKB, the difference was statistically significant (P<0.05). Conclusion The expression of HIF-1αprotein in the cultured neural stem cells injured by glutamate induced by ginkgolide B treatment may be relate with the activation of P13K/Akt signal pathway.