目的:探讨银杏内酯B(Ginkgolide B,GKB)对高糖低氧所致人视网膜内皮细胞(human retinal endothelial cel ls,HREC)损伤的保护作用.方法:建立高糖低氧诱导HREC损伤模型,然后经CCK-8法筛选GKB的最佳作用时间与浓度.ELISA检测TNF-α,ICAM-1,IL-6的水平来评估炎症反应;流式细胞术检测细胞的凋亡情况;Western印迹检测PI3K/AKT/mTOR信号通路的活性.结果:与正常对照组相比,高糖低氧模型组细胞活力显著降低,GKB处理可逆转高糖低氧所致的内皮细胞活力降低.高糖低氧模型组细胞的凋亡率显著增加,且细胞内炎症因子TNF-α,ICAM-1,IL-6的表达明显升高;而给予GKB处理后,细胞的凋亡率降低,TNF-α,ICAM-1,IL-6的水平部分回落.此外,高糖低氧模型组细胞中p-AKT,p-PI3K和p-mTOR表达降低;经GKB处理后细胞中p-PI3K,p-AKT和p-mTOR表达升高与蛋白的表达部分下降;给予AKT信号通路激动剂胰岛素样生长因子(insulin-like growth factor-1,IGF-1)可促进GKB的保护作用,并增加细胞活力.结论:GKB可显著对抗高糖低氧所致的视网膜内皮细胞凋亡损伤与炎症反应,其损伤保护作用可能与活化PI3K/AKT/mTOR信号通路有关.%Objective: To investigate the effect of Ginkgolide B (GKB) on human retinal endothelial cell (HREC) injury induced by high concentration of glucose and hypoxia. Methods: The high glucose and hypoxia-induced HREC injury cell model were established, then the optimal GKB concentration and time point were selected by CCK-8 assay. The inflammatory reaction was evaluated by the levels of TNF-α, ICAM-1 and IL-6 which were detected with ELISA. Cell apoptosis was determined by flow cytometry. Finally, activation of PI3K/AKT/mTOR pathway was detected with Western blot. Results: Compared with the normal control group, cells in high glucose and hypoxia model group have a lower cell viability, and GKB could reverse the effect of high glucose and hypoxia. High glucose and hypoxia led to a rise of early cell apoptotic ratio and higher expression of TNF-α, ICAM-1 and IL-6; however, after treatment with GKB, cell apoptotic ratio and the expression level of TNF-α, ICAM-1 and IL-6 decreased compared with high glucose and hypoxia model group. Furthermore, high glucose and hypoxia downregulated the protein expression of p-AKT, p-PI3K and p-mTOR; while compared with high glucose and hypoxia model group, the protein expression of p-AKT, p-PI3K and p-mTOR were partly increased, after treatment with GKB. Incubation with IGF-1 (AKT pathway agonist) could promote the activation effect of GKB on PI3K/AKT/mTOR pathway and up-regulate cell viability. Conclusion: Ginkgolide B could remarkably alleviate high glucose and hypoxia-induced HREC apoptotic injury and inflammatory reaction, and its protective effect may be related to the activation of PI3K/AKT/mTOR pathway.
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