NaNO_(2)对HDM预处理的气道上皮细胞氧化损伤的影响及其机制

Effect of NaNO_(2) on oxidative damage in house dust mite pretreated airway epithelial cells and its underlying mechanism

目的探究NaNO_(2)对经屋尘螨(house dust mite,HDM)预处理的人支气管上皮细胞氧化损伤的影响及其机制。方法体外培养人支气管上皮细胞16HBE,经HDM预处理后用不同浓度NaNO_(2)染毒处理,CCK-8检测细胞存活率,确定NaNO_(2)最适浓度及处理时间;将细胞接种于6孔板中随机分为3组(n=3):正常对照组、HDM组(HDM预处理)、NaNO_(2)组(HDM预处理后进行NaNO_(2)染毒处理)。Western blot和RT-PCR检测各组细胞Krüppel样锌指转录因子2(Krüppel-like transcription factors 2,KLF2)、低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)和Notch1的表达水平;ELISA法检测细胞炎症因子白介素-6(interleukin-6,IL-6)和肿瘤坏死因子-α(tumor necrosis factor,TNF-α)的表达水平;荧光探针法检测细胞活性氧(reactive oxygen species,ROS)水平;微量法测定丙二醛(malondialdehyde,MDA)与谷胱甘肽(glutathione,GSH)含量。结果CCK-8实验确定NaNO_(2)最适干预方式为500μmol/L处理24 h。与正常对照组比较,HDM组KLF2蛋白和mRNA水平减少,HIF-1α蛋白和mRNA水平(P<0.01)、Notch1的蛋白(P<0.05)、mRNA水平和ROS含量升高(P<0.01),MDA、TNF-α和IL-6含量均增高,GSH含量降低(P<0.05);与HDM组比较,NaNO_(2)组KLF2蛋白和mRNA水平明显减少,HIF-1α蛋白和mRNA水平(P<0.01)、Notch1的蛋白(P<0.05)和mRNA水平、ROS含量显著升高(P<0.01),MDA(P<0.05)、TNF-α(P<0.001)和IL-6含量显著增高,GSH含量明显减少(P<0.05)。结论NaNO_(2)可能通过调控KLF2的表达加重HDM诱导的人支气管上皮细胞氧化应激损伤。

Objective To investigate the effects and underlying mechanisms of NaNO_(2)on oxidative damage in human bronchial epithelial cells pretreated with house dust mite(HDM).Methods Human bronchial epithelial cell line 16HBE were pretreated with HDM and then exposed to varying concentrations of NaNO_(2).Cell viability was assessed using CCK-8 assay to determine the optimal concentration and treatment duration of NaNO_(2).Then 16HBE cells were randomly divided into normal control group,HDM group(pretreated with HDM),and NaNO_(2)group(pretreated with HDM followed by NaNO_(2)exposure).The expression of Krüppel-like transcription factors 2(KLF2),hypoxia-inducible factor-1α(HIF-1α),and Notch1 at protein and mRNA levels in each group were measured by Western blotting and RT-PCR,respectively.The contents of the inflammatory cytokines IL-6 and TNF-αwere assessed using ELISA.The production of reactive oxygen species(ROS)were measured using a fluorescence probe detection.The contents of malondialdehyde(MDA)and glutathione(GSH)were quantified by micro-methods.Results CCK-8 assay determined that the optimal intervention of NaNO_(2)was 500μmol/L for 24 h.Compared with the normal control group,the HDM group showed reduced protein and mRNA expression of KLF2,but increased protein and mRNA(P<0.01)expression of HIF-1α,and Notch1 at protein(P<0.05)and mRNA levels,elevated production of ROS(P<0.01),and increased contents of MDA,TNF-α,and IL-6,with a decrease in GSH content(P<0.05).Compared with the HDM group,the NaNO_(2)group exhibited a significant reduction in KLF2 protein and mRNA levels,increases in HIF-1αprotein and mRNA levels,Notch1 protein(P<0.05)and mRNA levels,elevated ROS content(P<0.01),and significantly increased contents of MDA,TNF-α(P<0.001),and IL-6,with a notable decrease in GSH content(P<0.05).Conclusion NaNO_(2)may exacerbate HDM-induced oxidative stress injury in human bronchial epithelial cells by regulating the expression of KLF2.