银杏内酯诱导的缺血耐受及相关机制探讨

目的 研究银杏内酯（Gin）预处理诱导PC12细胞对缺血的耐受及相关机制。方法将大鼠肾上腺嗜铬细胞瘤PC12株细胞随机分成对照组、缺血9h组、缺血预处理（IP）1.5h＋缺血9h组和银杏内酯预处理＋缺血9h组,用噻唑蓝比色分析、细胞形态学观察、Western印迹法以及凝胶电泳迁移实验等方法比较银杏内酯预处理对PC12细胞缺血模型的影响。结果在PC12细胞缺血模型中,9h缺血可明显降低PC12细胞活力,细胞存活率为（49．3±2．8）％。而银杏内酯24h预处理能显著提高9h缺血细胞的存活率（65．9±2．8）％（P〈0．01）;细胞形态学显示银杏内酯预处理后再缺血处理9h,可明显减轻缺血9h所致的细胞损害,较多细胞保留突起,突起网络依然存在,胞体完好。与对照组比,银杏内酯预处理能明显增加缺氧诱导因子-1d（HIF-1d）蛋白表达（P〈0．01）;银杏内酯预处理诱导表达的HIF-1具有生物学活性,它能与DNA结合,并激活下游基因促红细胞生成素（EPO）蛋白表达（P〈0．01）。结论银杏内酯预处理可以诱导PC12细胞对缺血的耐受,其诱导耐受的作用可能与银杏内酯预处理诱导PC12细胞HIF-1α稳定表达、提高HIF-1与DNA结合活性以及增加其下游基因EPO蛋白表达有关。

Ginkgolides induced ischemic tolerance and its possible molecular mechanism: experiment with rat pheochromocytoma cell line PC12

OBJECTIVE: To explore the ischemic tolerance induced by Ginkgolides in PC12 cells and its possible molecular mechanism. METHODS: An ischemic model was developed in PC12 cell line with deprivation of oxygen-glucose (OGD). PC12 cells was randomly divided into four groups: 9 hours ischemia group, 1.5 hours ischemic preconditioning + 9 hours ischemia group, Ginkgolides preconditioning + 9 hours ischemia group and control group. Cells viability was examined by MTT assay and cellular morphology was analyzed under the phase-contrast microscope. The molecular mechanism of Ginkgolides induced ischemic tolerance was pinpointedby analyzing the expression of hypoxia-inducible factor-1 alpha (HIF-1alpha) and erythropoietin (EPO). The DNA binding activities of HIF-1 in PC12 cells were examined by electrophoretic mobility shift assay. RESULTS: In ischemic model, the viability of PC12 cells was decreased (49.3 +/- 2.8)% after OGD for 9 hours. However, Ginkgolides pretreatment could remarkably increase the viability of PC12 cells (65.9 +/- 2.8)% (P < 0.01). Pretreatment of Ginkgolides for 24 hours could largely rescue the morphology of PC12 cells to the damage of subsequent exposure to 9 hours ischemia insult, many cellular bodies were intact and many neurites and network of PC12 cells were still exist. At molecular level, the expression of HIF-1alpha was greatly induced by Ginkgolides treatment after compared with the control group (P < 0.01). The DNA binding activities of HIF-1 in PC12 cells pretreated with Ginkgolides was also increased. And it activates its downstream target EPO, the protein expression (P < 0.01). CONCLUSION: The pretreatment of Ginkgolides could induce tolerance against ischemia in PC12 cells. The molecular mechanism of this process may involve in the activation of HIF-1alpha and the DNA binding activity of HIF-1 and its downstream target EPO.