【摘要】 目的 探究NaNO₂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NaNO₂染毒处理，CCK-8检测细胞存活率，确定NaNO₂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）、NaNO₂组（HDM预处理后进行NaNO₂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NaNO₂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，NaNO₂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 NaNO₂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。更多还原

【Abstract】 Objective To investigate the effects and underlying mechanisms of NaNO₂ 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 NaNO₂. Cell viability was assessed using CCK-8 assay to determine the optimal concentration and treatment duration of NaNO₂. Then 16HBE cells were randomly divided into normal control group, HDM group（pretreated with HDM）, and NaNO₂ group（pretreated with HDM followed by NaNO₂ 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-α were 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 NaNO₂ was 500 μmol/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 NaNO₂ group exhibited a significant reduction in KLF2 protein and mRNA levels, increases in HIF-1α protein 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 NaNO₂ may exacerbate HDM-induced oxidative stress injury in human bronchial epithelial cells by regulating the expression of KLF2.更多还原