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目的:研究柚皮苷(naringin,Nar)对高糖(high glucose,HG)诱导人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)损伤的作用及对PI3K/AKT/e NOS信号通路的影响。方法:利用HG(33 mmol/L glucose)培养基孵育HUVECs不同时间(6、12、24、48、72 h)后,用胰岛素(5 m IU/L)刺激细胞15 min,建立内皮细胞损伤模型,分别测定各组上清液中NO水平、细胞中e NOS和磷酸化e NOS(p-e NOS)的水平。对损伤的HUVECs用不同浓度的Nar(5、10、25、50、100 mg/L)孵育不同时间(6、12、24、48和72 h),用胰岛素(5 m IU/L)刺激细胞15min,检测细胞上清中的NO水平,观察Nar对HG诱导HUVECs损伤的作用。对损伤细胞先分别用PI3K抑制剂LY294002(10μmol/L)和AKT抑制剂AKT inhibitorⅣ(0.5μmol/L)预处理24 h,再用50 mg/L的Nar处理24 h,用胰岛素(5 m IU/L)刺激细胞15 min,检测细胞上清中NO的水平,以及e NOS、p-e NOS、PI3K、AKT及p-AKT的蛋白水平,探讨Nar减轻HG对HUVECs损伤作用的机制。结果:Nar的量效和时效结果显示,50 mg/L Nar预处理HUVECs 24 h后,其减轻HG对HUVECs损伤的作用最为显著,表现为NO和p-e NOS的水平升高(P<0.05)。加入PI3K和AKT抑制剂后,Nar减轻HG致内皮细胞的损伤及上调p-e NOS和NO水平的作用完全取消(P<0.05)。结论:Nar能减轻HG诱导的血管内皮细胞损伤,其作用机制可能是通过PI3K/AKT/e NOS信号通路介导的。
AIM: To investigate the effect of naringin (Nar) on the injury of human umbilical vein endothelial cells (HUVECs) induced by high glucose (HG) and the effect on the PI3K / AKT / e NOS signal pathway . Methods: HUVECs were incubated with HG (33 mmol / L glucose) for different times (6, 12, 24, 48, 72 h) and then stimulated with insulin (5 m IU / L) for 15 min to establish endothelial cell injury model The levels of NO, eNOS and pe NOS in the supernatants were determined. Injured HUVECs were incubated with different concentrations of Nar (5, 10, 25, 50 and 100 mg / L) for different times (6, 12, 24, 48 and 72 h) and cells stimulated with insulin (5 m IU / L) 15min, detect the level of NO in the cell supernatant to observe the effect of Nar on HG-induced HUVECs injury. The injured cells were pre-treated with PI3K inhibitor LY294002 (10 μmol / L) and AKT inhibitor Ⅳ (0.5 μmol / L) for 24 h, then treated with 50 mg / L Nar for 24 h, / L) for 15 min. The levels of NO, eNOS, peNOS, PI3K, AKT and p-AKT in the supernatant of the cells were measured to explore the mechanism by which Nar reduced HG injury to HUVECs. Results: The dose-effect and time-effect results of Nar showed that HUVECs were significantly attenuated by 50 mg / L Nar pretreated with HUVECs for 24 h, indicating the increased levels of NO and p-e NOS (P <0.05). After addition of PI3K and AKT inhibitors, the effect of Nar on HG-induced endothelial cell injury and up-regulation of p-e NOS and NO levels was abolished (P <0.05). Conclusion: Nar can reduce HG-induced vascular endothelial cell injury, and its mechanism may be mediated through the PI3K / AKT / e NOS signaling pathway.