NOX1在TNF-α诱导的肺泡上皮细胞氧化损伤和炎症中的作用研究

目的:探索NADPH氧化酶1(NOX1)在肿瘤坏死因子α(TNF-α)诱导的肺泡上皮细胞A549氧化损伤和炎症中的作用。方法:以real-time PCR和Western blot法测定TNF-α处理后肺泡上皮细胞中NOX1的m RNA和蛋白表达水平。在肺泡上皮细胞中转染NOX1 si RNA及其阴性对照,以TNF-α诱导以后,用real-time PCR和Western blot测定细胞中NOX1水平。硫代巴比妥酸法检测细胞中丙二醛(MDA)含量,黄嘌呤氧化法检测细胞中超氧化物歧化酶(SOD)活性,ELISA法检测细胞培养液中的白细胞介素4(IL-4)、白细胞介素6(IL-6)和白细胞介素1β(IL-1β)含量,流式细胞术检测细胞凋亡率,Western blot检测凋亡蛋白cleaved caspase-3的水平。结果:TNF-α诱导处理后A549细胞中NOX1 m RNA和蛋白水平均升高(P 0. 05),NOX1 si RNA转染可以下调细胞中NOX1的m RNA和蛋白表达(P 0. 05),转染si RNA阴性对照对细胞中NOX1的m RNA和蛋白表达没有影响。TNF-α处理后细胞中MDA含量升高,SOD活性降低,细胞分泌的IL-4、IL-6和IL-1β增多,细胞凋亡率和凋亡蛋白cleaved caspase-3水平均升高,与没有经TNF-α处理的细胞比较,差异有统计学意义(P 0. 05)。下调NOX1的细胞经TNF-α诱导后,细胞中MDA含量降低,SOD活性升高,细胞分泌的IL-4、IL-6和IL-1β减少,凋亡率及凋亡蛋白cleaved caspase-3水平降低,与只经过TNF-α诱导处理的细胞比较,差异有统计学意义(P 0. 05)。结论:TNF-α诱导肺泡上皮细胞表达NOX1。下调NOX1表达可减少细胞氧化损伤和炎症因子分泌,减少细胞凋亡。

Role of NOX1 in TNF-α-induced oxidative damage and inflammation in alveolar epithelial cells

AIM: To explore the role of NADPH oxidase 1 (NOX1) in tumor necrosis factor-α (TNF-α)-induced oxidative damage and inflammation in alveolar epithelial cells.METHODS: The mRNA and protein expression levels of NOX1 in alveolar epithelial cells after TNF-α treatment were determined by real-time PCR and Western blot. NOX1 siRNA and its negative control were transfected into the alveolar epithelial cells. After the induction of TNF-α, NOX1 levels in the cells were measured by real-time PCR and Western blot, and the content of malondialdehyde (MDA) in the cells was detected by thiobarbituric acid method. Xanthine oxidation assay was used to detect the activity of superoxide dismutase (SOD) in the cells. The contents of interleukin-4 (IL-4), IL-6 and IL-1β in cell culture medium were examined by ELISA. The rate of apoptosis was analyzed by flow cytometry. Western blot was used to detect the level of apoptotic protein cleaved caspase-3.RESULTS: The expression of NOX1 at mRNA and protein levels in TNF-α-induced cells was increased after induction (P<0.05). After transfection of NOX1 siRNA, the expression of NOX1 at mRNA and protein levels in the cell was downregulated (P<0.05). Transfection of siRNA negative control had no effect on the expression level of NOX1 in the cells. The content of MDA in the cells after TNF-α treatment was increased, the activity of SOD was reduced, the releases of IL-4, IL-6 and IL-1β by the cells were increased, and the apoptotic rate and the level of apoptotic protein cleaved caspase-3 were increased as compared with the cells that were not treated with TNF-α (P<0.05). The content of MDA in the cells with NOX1 knockdown induced by TNF-α was reduced, the activity of SOD elevated, and the releases IL-4, IL-6 and IL-1β, the apoptotic rate and the level of apoptotic protein cleaved caspase-3 decreased, as compared with the cells only treated with TNF-α induction (P<0.05).CONCLUSION: TNF-α induces the expression of NOX1 in the alveolar epithelial cells. Knockdown of NOX1 expression reduces cellular oxidative damage, releases of inflammatory factors, and cell apoptosis.