SIRT6调节NOX2/ROS/NF–kB信号通路对脂多糖诱导的肺上皮细胞氧化应激和炎症改善的作用机制

黄秋杰; 谢辉; 冯俊; 叶邦旺; 范华贵; 杨健

SIRT6调节NOX2/ROS/NF–kB信号通路对脂多糖诱导的肺上皮细胞氧化应激和炎症改善的作用机制

The action mechanism of SIRT6 regulating the NOX2/ROS/NF kB signaling pathway on lipopolysaccharide induced oxidative stress and inflammation improvement in pulmonary epithelial cells

摘要

摘要:
目的： 探究SIRT6调节NOX2/ROS/NF–κB信号通路对脂多糖诱导的肺上皮细胞氧化应激和炎症改善的作用机制。
方法： 将A549细胞随机分为Col组（未转染任何质粒）、LPS组（未转染任何质粒）、Ad–NC组（转染阴性对照质粒）和Ad–SIRT6组（SIRT6过表达腺病毒），除Col组外，其余各组A549细胞均给予10μg/mL的LPS刺激24h。CCK–8检测细胞增殖能力；流式细胞仪检测细胞凋亡率；ELISA检测细胞中炎性因子白介素–1β（IL）–1β、IL–6、肿瘤坏死因子α（TNF–α）水平和氧化应激指标超氧化物歧化酶（SOD）、丙二醛（MDA）水平；DCFH–DA探针检测活性氧（ROS）水平；蛋白质印迹法检测细胞中还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶2（NOX2）、核因子κB（NF–κB） p65、p–NF–κB p65、SIRT6蛋白表达。
结果： 与Col组相比，LPS组和Ad–NC组细胞存活率、细胞中SOD活性均降低（P ＜ 0.05），细胞凋亡率、细胞中TNF–α、IL–6、IL–1β含量、MDA含量、ROS相对荧光强度、细胞中NOX2、SIRT6蛋白及p–NF–κB p65/NF–κB p65比值均增加（P ＜ 0.05）；和LPS组相比，Ad–SIRT6组细胞存活率、细胞中SOD活性均增加（P ＜ 0.05），细胞凋亡率、细胞中TNF–α、IL–6、IL–1β含量、MDA含量、ROS相对荧光强度、细胞中NOX2、SIRT6蛋白及p–NF–κB p65/NF–κB p65比值均降低（P ＜ 0.05）。
结论： SIRT6过表达可减轻LPS诱导肺上皮细胞氧化应激和炎症反应，其作用机制可能和抑制NOX2/ROS/NF–κB途径有关。

Abstract:
Objective To explore the action mechanism of SIRT6 regulating the NOX2/ROS/NF-κB signaling pathway on lipopolysaccharide induced oxidative stress and inflammation improvement in pulmonary epithelial cells.
Methods A549 cells were randomly divided into the Col group (without transfection of any plasmid), LPS group (without transfection of any plasmid), Ad-NC group (transfected with negative control plasmid) and Ad-SIRT6 group (SIRT6 overexpressing adenovirus). Except for the Col group, A549 cells in the other groups were stimulated with 10μg/mL LPS for 24 hours. Cell proliferation ability was detected by CCK-8; The apoptosis rate of cells was detected by flow cytometry. ELISA was used to detect the levels of inflammatory factors interleukin-1 β (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) in cells, as well as the levels of oxidative stress indicatorssuperoxide dismutase (SOD) and malondialdehyde (MDA). The level of reactive oxygen species (ROS) was detected by DCFH-DA probe; Western blotting was used to detect the protein expressions of reduced nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), nuclear factor κB (NF-κB) p65, p-NF-κB p65 and SIRT6 in cells.
Results Compared with the Col group, the cell survival rate and SOD activity in the LPS group and Ad-NC group both decreased (P ＜ 0.05), and the apoptosis rate of cells, contents of TNF-α, IL-6, IL-1β in cells, content of MDA, relative fluorescence intensity of ROS, proteins of NOX2 and SIRT6 in cells andratio of p-NF-κB p65/NF-κB p65 all increased (P ＜ 0.05). Compared with the LPS group, the cell survival rate and SOD activity in the Ad-SIRT6 group both increased (P ＜ 0.05), and the apoptosis rate, contents of TNF-α, IL-6, IL-1β in cells, content of MDA, relative fluorescence intensity of ROS, proteins of NOX2 and SIRT6 and ratio of p-NF-κB p65/NF-κB p65 all decreased in cells (P ＜ 0.05).
Conclusions Overexpression of SIRT6 can alleviate LPS-induced oxidative stress and inflammatory responses in lung epithelial cells, and its mechanism of action may be related to the inhibition of the NOX2/ROS/NF-κB pathway

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