NF-κB/NOX1信号通路在TNF-α诱导A549细胞凋亡中的作用

Effects of NF-κB/NOX1 signaling pathway in TNF-α-induced apoptosis of A549 cells

目的探讨肿瘤坏死因子-α(TNF-α)诱导的A549细胞凋亡中核转录因子-κB(NF-κB)/烟酰胺腺嘌呤二核苷酸磷酸氧化酶1(NOX1)信号通路的作用。方法 (1)以浓度为0.00、0.25、0.50和1.00 nmol/L的TNF-α刺激A549细胞,采用CCK-8实验检测细胞存活率,筛选TNF-α的最优刺激剂量。(2)取对数生长期A549细胞随机分为4组,对照组、TNF-α组、BAY11-7082(NF-κB抑制剂)组和TNF-α+BAY11-7082组。对照组细胞不予任何处理;TNF-α组和BAY11-7082组分别予浓度为0.50 nmol/L的TNF-α和浓度为5μmol/L的BAY11-7082刺激;TNF-α+BAY11-7082组细胞先后加入上述浓度的TNF-α和BAY11-7082刺激。培养24 h后,采用CCK-8实验检测细胞存活率,流式细胞术检测细胞凋亡率,蛋白印迹法检测NF-κB(p65)和NOX1蛋白的相对表达水平。结果 (1)以浓度为0.50 nmol/L的TNF-α刺激A549细胞时,可降低其增殖活性并促进细胞的凋亡,选择该浓度作为后续实验的TNF-α刺激剂量。(2)与对照组比较,TNF-α组A549细胞存活率下降(P<0.05),细胞凋亡率和NF-κB(p65)、NOX1蛋白的表达均升高(P值均<0.05)。与对照组比较,BAY11-7082组A549细胞存活率和NF-κB(p65)、NOX1相对表达水平均下降(P值均<0.05),细胞凋亡率均升高(P<0.05)。与TNF-α组比较,TNF-α+BAY11-7082组A549细胞由长梭形变为不规则形,细胞存活率升高(P<0.05),细胞凋亡率和NF-κB(p65)、NOX1相对表达水平均下降(P值均<0.05)。结论 NF-κB/NOX1信号通路参与了TNF-α诱导的A549细胞凋亡。

Objective To investigate the effects of nuclear transcription factor-κB(NF-κB)/amide adenine dinucleotide phosphate oxidase 1(NOX1) signaling pathway in tumor necrosis factor-α(TNF-α) induced apoptosis of A549 cells. Methods i) A549 cells were stimulated with TNF-α at the concentrations of 0.00, 0.25, 0.50, and 1.00 nmol/L. CCK-8 assay was used to detect the cell viability to screen the optimal stimulating concentration of TNF-α. ii) A549 cells at logarithmic growth stage were randomly divided into four groups, the control group, the TNF-α group, the BAY11-7082(NF-κB inhibitor) group and the TNF-α+BAY11-7082 group. The cells in the control group were not treated. The TNF-α and BAY11-7082 groups were stimulated with 0.50 nmol/L TNF-α and 5 μmol/L BAY11-7082, respectively. The TNF-α+BAY11-7082 group was stimulated by both TNF-α and BAY11-7082. After 24 hours of culture, the cell survival rate was detected by CCK-8 assay. Flow cytometry was used to detect cell apoptotic rate, and Western blotting was used to detect the relative expression of NF-κB(p65) and NOX1 proteins. Results i) When A549 cells were stimulated with TNF-α at the concentration of 0.50 nmol/L, the cell proliferative activity was reduced and the cell apoptosis was promoted. This concentration was selected as the stimulation dose of TNF-α in subsequent experiments. ii) The survival rate of A549 cells in the TNF-α group decreased(P<0.05), the apoptotic rate and the protein expressions of NF-κB(p65) and NOX1 increased in TNF-α group(all P<0.05) compared with the control group. In BAY11-7082 group, the survival rate and the relative expression of NF-κB(p65) and NOX1 of A549 cells were decreased(all P<0.05), and the apoptotic rate of A549 cells was increased(P<0.05) compared with the control group. A549 cells in TNF-α+BAY11-7082 group changed from a long spindle shape to an irregular one. The cell survival rate increased(P<0.05), the apoptotic rate and the relative expression of NF-κB(p65) and NOX1 decreased(all P<0.05) compared with the TNF-α group. Conclusion NF-κB/NOX1 signaling pathway is involved in A549 cells apoptosis induced by TNF-α.