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

作　　者：李熙炫 彭东红[1,2] LI Xixuan;PENG Donghong(Department of Respiratory Diseases,National Clinical Research Center for Child Health and Disorders,Key Laboratory of Child Development and Disorders of Ministry of Education,China International Science and Technology Cooperation Base for Child Development and Critical Disorders,Chongqing Key Laboratory of Pediatrics,Children’s Hospital of Chongqing Medical University,Chongqing,400014;Jiangxi Children’s Medical Center,Jiangxi Hospital Affiliated to Children’s Hospital of Chongqing Medical University,Nanchang,Jiangxi Province,330108,China)

机构地区：[1]重庆医科大学附属儿童医院呼吸内科,国家儿童健康与疾病临床医学研究中心,儿童发育疾病研究教育部重点实验室,儿童发育重大疾病国家国际科技合作基地,儿童代谢与炎症性疾病重庆市重点实验室,重庆400014 [2]重庆医科大学附属儿童医院江西医院,南昌330108

出　　处：《陆军军医大学学报》2024年第12期1395-1402,共8页Journal of Army Medical University

摘　　要：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.

关 键 词：氧化应激 KLF2 气道上皮细胞 哮喘 氮氧化物 

分 类 号：R363.21[医药卫生—病理学] R562.25[医药卫生—基础医学] X511[环境科学与工程—环境工程]