高糖刺激下PKC/NADPH氧化应激途径对内皮细胞活性氧生成的影响及其机制探讨

Effects of PKC/NADPH Oxidase on ROS in HUVECs Cultured with High Glucose

目的:探讨高糖刺激下PKC/NADPH氧化应激途径对内皮细胞活性氧生成的影响。方法:实验分为正常对照组、NaOH对照组、DMSO对照组、20 mM葡萄糖处理4小时组(高糖组)、1 mol佛波酯预处理0.5小时再加入20 m M葡萄糖处理组(佛波酯组)和4 mol金丝桃素预处理0.5小时再加入20 mM葡萄糖处理组(金丝桃素组);Ⅱ型胶原酶消化法分离人脐静脉内皮细胞;流式细胞术检测细胞内活性氧;免疫荧光检测内皮细胞Ⅷ因子和NADPH氧化酶亚基p47phox定位。结果:1与正常对照组相比,高糖组细胞内活性氧增高(P<0.05,n=3),与正常对照组和高糖组相比,佛波酯组HUVECs内活性氧的产生显著增加(P<0.05,n=3),与正常对照组和高糖组相比,金丝桃素组HUVECs内活性氧的产生明显减少(P<0.05,n=3);2正常对照组和金丝桃素组中细胞NADPH氧化酶亚基p47phox主要位于胞浆,而佛波酯组和高糖组的NADPH氧化酶亚基p47phox位于胞膜。结论:高糖通过PKC信号通路调节内皮细胞NADPH氧化酶亚基p47phox的移位从而增加细胞内活性氧的生成。

Objective： To investigate effects of PKC/NADPH oxidase on reactive oxygen species（ROS） in Human umbilical vein endothelial cells（HUVECs） cultured with high glucose. Methods： The experiments were divided into six groups, including control group,Na OH control group, DMSO control group, group treated with glucose（20 m M） for 4 hours（high-glucose group）, group which was pretreated phorbol ester（1 mol） for half an hour then was stimulated by glucose（20 m M） for 4 hours（phorbol ester group） and group which was pretreated hypericin（4 mol） for half an hour then was stimulated by glucose（20 m M） for 4 hours（hypericin group）; Human umbilical vein endothelial cells was isolated by type Ⅱ collegease; flow cytometry was used to detect intracellular ROS production;immunofluorescence was used to investigate factor Ⅷ and p47 phox location in HUVECs. Results： 1 Compared with control group,intracellular ROS of high-glucose group were significantly increased（P〈0.05, n=3）, Compared with control group and, high-glucose group, intracellular ROS of phorbol ester group were significantly increased（P〈0.05, n=3）, Compared with control group and,high-glucose group, intracellular ROS of hypericin group were significantly decreased（P〈0.05, n=3）; 2 p47 phox mostly located cytoplasm for control group and hypericin group, p47 phox mostly located cell membrane for high-glucose group and phorbol ester group.Conclusion： PKC/NADPH oxidase can regulate ROS generation in HUVECs induced with high glucose by translocation of p47 phox.