高糖对人脐静脉内皮细胞NADPH氧化酶表达和活性氧生成的影响

摘要

Objective: To explore the effect of high glucose on NADPH oxidase (NOX) expression and intracellular reactive oxygen species (ROS) generation in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were divided into a control group, a mannite group, a glucose group and aglucose plus diphenylene iodonium (DPI) group. Intracellular ROS was detected by lfow cytometry. RNA and protein expression of NOX in HUVECs was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot, respectively. Results: 1) Compared with the control group, the intracellular ROS were signiifcantly increased in the glucose group (P<0.05,n=3), but there were no signiifcant change in the glucose plus DPI group (P>0.05,n=3); 2) Compared with the control group, the mRNA and protein expression of NOX4 in the glucose group were signiifcantly increased (P<0.05), but there were no signiifcant change in the mRNA and protein expression of NOX2, p22phox, p67phox and rac (allP>0.05); 3) there were no signiifcant difference in the mRNA and protein expression of NOX2, NOX4, p22phox, p67phox and rac between the glucose plus DPI group and the control group (allP>0.05). Conclusion: High glucose may increases intracellular ROS generation by increasing the expression of NOX4 in HUVECs, which might mediate the oxidative stress.%目的：探讨高糖对内皮细胞活性氧生成和NADPH氧化酶(nicotinamide adenine dinucleotide phosphate oxidase， NOX)表达的影响。方法：本实验以人脐静脉内皮细胞为研究对象，分为正常对照组、20 mmol/L甘露醇处理4 h高渗对照组、20 mmol/L葡萄糖处理2 h组、20 mmol/L葡萄糖处理4 h组、20 mmol/L葡萄糖处理6 h组和NOX特异性抑制剂二苯基碘(diphenylene iodonium，DPI)5μmol/L预处理0.5 h再加入20 mmol/L葡萄糖处理组(DPI预处理组)。用流式细胞仪检测各组细胞内活性氧的浓度；反转录聚合酶链反应及Western印迹法分别检测各组细胞内NOX各亚基mRNA和蛋白的表达变化。结果：1)与正常对照组相比，20 mmol/L葡萄糖处理2，4和6 h组活性氧增高(均P<0.05)，而DPI组活性氧生成与正常对照组比较差异无统计学意义(P>0.05)；2)与正常对照组相比，20 mmol/L葡萄糖处理4 h组NOX4 mRNA和蛋白的表达明显增加(均P<0.05)，NADPH氧化酶亚基NOX2，P67phox，P22phox和Rac表达差异均无统计学意义(均P>0.05)；3)与正常对照组相比，DPI预处理组NOX4，NOX2，P67phox，P22phox和Rac表达差异均无统计学意义(均P>0.05)。结论：高糖可能通过上调NOX4表达以增加活性氧生成而发挥氧化应激作用。