NADPH氧化酶在人真皮成纤维细胞氧化应激损伤中的作用

摘要

目的：探讨NADPH氧化酶在人真皮成纤维细胞氧化应激损伤中的作用。方法 H2O2构建氧化应激模型，将实验分为正常组、氧化损伤组和NADPH氧化酶抑制剂（DPI）组，MTT检测细胞活力，DCFH-DA荧光探针检测细胞内活性氧（ROS）改变， Western blot分析NADPH氧化酶胞膜亚基gp91phox表达变化。结果 H2O2对成纤维细胞的氧化损伤呈时间和浓度依赖，H2O2700μmol/L处理24 h后细胞活力下降约40%（P<0.05），ROS升高2倍（P<0.05）。而抑制剂组细胞活力较氧化损伤组增加20%（P<0.05），ROS下降至正常水平（P<0.05）。Western blotting结果显示氧化损伤组gp91phox表达随H2O2浓度逐渐升高，而抑制剂组表达接近正常水平。结论 H2O2可通过影响NADPH氧化酶特别是胞膜亚基gp91phox引发成纤维细胞氧化损伤。%Objective To investigate the role of NADPH oxidase (Nox) in the oxidative stress injury of human dermal fibroblasts (HFbs). Methods An oxidative stress injury model was established in HFbs by exposure to H2O2. Normal HFbs and HFbs exposed to H2O2 with and without pretreatment with NADPH oxidase inhibitor were tested for cell viability using MTT assay, and the intracellular reactive oxygen species (ROS) were determined with a DCFH-DA fluorescent probe. Western blotting was used to measure the protein expressions of membrane-bound subunit gp91phox of NADPH oxidase in the cells. Result H2O2 time-and concentration-dependently induced oxidative stress injury in the fibroblasts, causing a reduction of the cell viability to 40%after a 24-h exposure at 700μmol/L (P<0.05) and an increase of ROS by 2 folds after a 2-h exposure at 700μmol/L (P<0.05). Compared with the cells with oxidative stress injury, the cells with NADPH oxidase inhibitor pretreatment showed a 20%higher cell viability (P<0.05) and normal ROS level (P<0.05) following H2O2 exposure. Western blotting demonstrated increased expression of gp91phox in the cells exposed to increasing H2O2 concentrations, but gp91phox expression remained normal in cells pretreated with NADPH oxidase inhibitor. Conclusion H2O2 can induce oxidative stress injury in the fibroblasts by affecting NADPH oxidase, especially its membrane-bound subunit gp91phox.