脂多糖通过Nrf2信号通路诱导人气道上皮细胞MUC5AC表达

Lipopolysaccharide induces expression of MUC5AC in human airway epithelial cells via Nrf2 signaling pathway

目的:探讨核因子E2相关因子2(Nrf2)信号通路在脂多糖(LPS)上调气道黏蛋白5AC(MUC5AC)表达中的作用。方法:LPS干预体外培养的人气道上皮细胞系A549细胞,以诱导MUC5AC表达。分别转染Nrf2小干扰RNA(siRNA)和Nrf2过表达质粒(pcDNA3-Myc3-Nrf2),敲减或过表达A549细胞中的Nrf2,再加入10 mg/L LPS干预24 h,RT-qPCR和Western blot法分别检测细胞MUC5AC、Kelch样环氧氯丙烷相关蛋白1(Keap1)、Nrf2及其下游抗氧化因子[NAD(P)H:醌氧化还原酶1(NQO1)、谷氨酸-半胱氨酸连接酶催化亚基(GCLC)、谷胱甘肽S-转移酶pi(GST-pi)和血红素加氧酶1(HO-1)]的mRNA及蛋白表达情况。结果:(1)LPS上调A549细胞黏蛋白MUC5AC表达(P<0.01),上调Nrf2及其下游抗氧化因子NQO1、GCLC及GST-pi表达,下调HO-1表达(P<0.05)。(2)与阴性对照组相比,转染Nrf2 siRNA能有效抑制A549细胞Nrf2的表达;敲减Nrf2的A549细胞经LPS干预后,MUC5AC的表达显著增加(P<0.05)。(3)与对照组相比,转染过表达质粒pcDNA3-Myc3-Nrf2明显增加A549细胞Nrf2的表达;增强Nrf2表达后,LPS诱导的MUC5AC表达被抑制(P<0.05)。结论:Nrf2信号通路参与调控LPS诱导的气道上皮细胞MUC5AC表达。

AIM:To investigate the role of nuclear factor E2-related factor 2(Nrf2)signaling pathway in lipolysaccharide(LPS)-induced mucin 5AC(MUC5AC)expression in human airway epithelial cells(A549 cells).METHODS:The A549 cells were stimulated with LPS to induce MUC5AC expression.Furthermore,the A549 cells were transfected with Nrf2 siRNA,pcDNA3-Myc3-Nrf2 or negative control for 24 h,and then were harvested for Nrf2 mRNA and protein assay to estimate the knockdown and overexpression effects.After transfection,the cells were stimulated with LPS,and the mRNA and protein expression levels of MUC5AC,Kelch-like ECH-associated protein 1(Keap1),Nrf2 and downstream antioxidant factors,such as NAD(P)H:quinone oxidoreductase 1(NQO1),glutamate-cysteine ligase catalytic subunit(GCLC),glutathione S-transferase-pi(GST-pi)and heme oxygenase-1(HO-1),were detected by RT-qPCR and Western blot.RESULTS:(1)After stimulation with LPS for 24 h,the mRNA and protein expression levels of MUC5AC,Nrf2 and downstream antioxidant factors,such as NQO1,GCLC and GST-pi,in A549 cells were all increased significantly(P<0.01).However,the mRNA and protein expression of HO-1 was down-regulated(P<0.05).(2)After transfection with Nrf2 siRNA,the Nrf2 expression in A549 cells was knocked down,and the MUC5AC expression induced by LPS was significantly augmented(P<0.05).(3)On the other hand,after transfection with pcDNA3-Myc3-Nrf2,the Nrf2 expression of A549 cells were up-regulated,and the MUC5AC expression induced by LPS was suppressed(P<0.05).CONCLUSION:This study suggested that Nrf2 signaling pathway is involved in the regulation of LPS-induced MUC5AC expression in human airway epithelial cells.