NOX1与NF-κB对TNF-α诱导A549细胞氧化损伤的影响

The effects of NOX1 and NF-κB on TNF-α induced oxidative damage in A549 cells

目的探讨酰胺腺嘌呤二核苷酸磷酸氧化酶1(NOX1)和核转录因子-κB(NF-κB)在肿瘤坏死因子-α(TNF-α)诱导A549细胞发生氧化损伤中的关系。方法①以质量浓度为0.0、10.0、25.0和50.0μg/L的TNF-α刺激A549细胞,采用CCK-8实验检测各组细胞抑制率,选择合适的TNF-α刺激浓度。②取对数生长期A549细胞分为空白对照组、溶剂对照组、TNF-α组、二亚苯基碘(DPI)组和TNF-α+DPI组进行NOX1抑制实验。取对数生长期A549细胞分为空白对照组、TNF-α组、BAY11-7082组和TNF-α+BAY11-7082组进行NF-κB抑制实验。以蛋白印迹法检测各组细胞NOX1和p65相对表达水平,以流式细胞术检测细胞内活性氧(ROS)相对表达水平。结果①随着TNF-α剂量的增加,A549细胞的抑制率增加(P<0.05),选择25.0μg/L作为后续实验TNF-α刺激剂量。②TNF-α组细胞NOX1、p65蛋白及ROS相对表达水平分别高于空白对照组、溶剂对照组和DPI组(P<0.05);TNF-α+DPI组细胞上述指标均低于TNF-α组(P<0.05),但高于DPI组(P<0.05)。TNF-α组细胞NOX1、p65蛋白及ROS相对表达水平分别高于空白对照组和BAY11-7082组(P<0.05),TNF-α+BAY11-7082组细胞上述指标均低于TNF-α组(P<0.05)。结论抑制NOX1或NF-κB均可减轻TNF-α诱导的A549细胞氧化损伤。

Objective To investigate the effects of nicotinamide adenine dinucleoside phosphate oxidases 1(NOX1)and nuclear factor-kappa B(NF-κB)in tumor necrosis factor-α(TNF-α)-induced oxidative damage in A549 cells.Methods i)TNF-αwas used to stimulated A549 cells at the concentrations of 0.0,10.0,25.0 and 50.0μg/L.Cell inhibition rate in each group was tested by CCK-8 assay to select the appropriate concentration.ii)A549 cells in logarithmic growth phase were divided into blank control group,solvent control group,TNF-αgroup,diphenylene iodine(DPI)group and TNF-α+DPI group for NOX1 inhibitor experiment.Logarithmic growth phase A549 cells were divided into blank control group,TNF-αgroup,BAY11-7082 group and TNF-α+BAY11-7082 group for NF-κB inhibitor experiment.The relative expression of NOX1 and p65 protein in each group was detected by Western blot method.The relative expression of intracellular reactive oxygen species(ROS)were detected by flow cytometry.Results i)The inhibition rate of A549 cells increased with the increase of TNF-αdose(P<0.05),and 25.0μg/L was selected as the stimulation dose of TNF-αin subsequent experiments.ii)The relative expression of NOX1,p65 protein and ROS in the TNF-αgroup was higher than that in the blank control group,solvent control group and DPI group,respectively(P<0.05).The above indexes in TNF-α+DPI group were lower than that in TNF-αgroup(P<0.05),but higher than that in DPI group(P<0.05).The relative expression of NOX1,p65 protein and ROS in the TNF-αgroup were higher than that in the blank control group and the BAY11-7082 group(P<0.05),while the above indicators in the TNF-α+BAY11-7082 group were lower than that in the TNF-αgroup(P<0.05).Conclusion Inhibition of NOX1 or NF-κB can alleviate the oxidative damage induce by TNF-αin A549 cells.