GSTM5参与的氧化／抗氧化失衡对支气管上皮细胞炎症的调节作用  

Role of oxidative/antioxidant ambalance induced by inhibition of GSTM5 in the regulation of inflammation in bronchial epithelial cell

作　　者：张佳坷 李理[2] 袁伟锋[2] 郭振辉[3] 黄文杰[2] Zhang Jiake;Li Li;Yuan Weifeng;Guo Zhenhui;Huang Wenjie(Department of Respiratory Medicine,the First Affiliated Hospital of Guangdong Pharmaceutical University,Guangzhou 510080,China)

机构地区：[1]广东药科大学附属第一医院呼吸内科,广州510080 [2]广州军区广州总医院呼吸内科,510010 [3]广州军区广州总医院MICU科,510010

出　　处：《国际呼吸杂志》2018年第13期961-967,共7页International Journal of Respiration

基　　金：国家自然科学基金面上项目（81370173）;广州市科技计划项目（201701010020）;广东省自然科学基金（2014A030313597）;广东省广州市科研条件建设项目[穗科信字（2012）224-5号]

摘　　要：目的沉默人支气管上皮细胞谷胱甘肽硫转移酶mu5（GSTM5）的表达，探讨GSTM5参与的氧化／抗氧化失衡对支气管上皮细胞炎症的调节作用。方法肿瘤坏死因子α（TNF-α）（10μg／L，24h）刺激建立人支气管上皮细胞炎症损伤模型，转染小干扰RNA（siRNA）沉默人支气管上皮细胞GSTM5的表达，反转录聚合酶链反应（RT-PCR）法检测烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶（NOX）家族NOXl、NOX2、NOX3、NOX4、NOX5、双重氧化酶1（DUOXl）、DUOX2相关基因转录水平；蛋白质印迹法检测NOX1和NOX2蛋白表达水平；比色法检测细胞内丙二醛（MDA）及总抗氧化能力（T-AOC）；酶联免疫吸附测定（ELISA）法检测细胞培养上清液中自介素1β（IL-1β）、IL-6、IL-10、γ干扰素（IFN-γ）浓度；噻唑兰（MTT）法检测细胞存活率。结果合成的siRNA～GSTM5序列2组可明显下调人支气管上皮细胞GSTM5mRNA的表达（P〈0．05）；沉默GSTM5组NOXl、NOX2基因转录强度与蛋白表达水平较TNF-α组及阴性对照组相比均增高（P〈0．05），而NOX3、NOX4、NOX5、DUOXl、DUOX2基因表达强度差异无统计学意义（P均〉0．05）；沉默GSTM5组较TNF-α组细胞内MDA的增高（P〈O．05），T-AOC能力减弱（P〈0．05），上清液中IL-18、IL-6、IL-10、IFN-γ浓度均较TNF-α组升高（P均〈0．05），细胞存活率降低（P〈O．05）。结论GSTM5对炎症诱导的支气管上皮细胞炎症损伤有保护作用，下调GSTM5加剧支气管上皮细胞氧化／抗氧化失衡，上调细胞炎症水平，加重细胞损伤。Objective Synthesis small interfering RNA （siRNA） to inhibit the expression of glutathione S-transferase mu5 （GSTM5） in human bronchial epithelial cells （16HBE） and investigate the effects of inhibition of glutathione S-transferasemu 5 （GSTMS） on the inflammation in acute lung injury （ALI）.Methods 16HBE cells were treated with tumor necrosis faetor-α （TNF-α）（10 μg/L,24 h） in the absence or presence of the siRNA. The transcription level of nicotinamide-adenine dinucleotide phosphate oxidase-1 （NOX1）, NOX2, NOX3, NOX4, NOX5, dual oxidase-1 （DUOX1） and DUOX2 were evaluated by reverse transcription polymerase chain reaction （RT-PCR）. Western blot was performed to investigate the protein levels of NOX1 and NOX2. The concentration of malondialdehyde （MDA） and total antioxidationcapacity （T-AOC） were detected by eolorimetric method. The concentration of IL-1β, γ-IFN, IL-6 and IL-10 in the culture supernatant were measured by enzyme linked immunosorbent assay （ELISA）. The survival rate of cells was assessed by the methyl thiazolyl tetrazolium （MTT） assay. Results The siRNA significantly inhibited the expression of GSTM5 （ P〈0.05）. When transfected with the siRNA,The 16HBE cells had a higher transcription and protein levels of NOX1 and NOX2 （all P〈 0.05）. Consequently, the concentration of MDA significantly increased （ P 〈0.05）, and the activation of T-AOC decreased remarkably （ P〈0.05）. The concentration of INF-γ, IL-1β, IL-6 and IL10 in the siGSTM5 group were higher than those in the TNF-a group and control[ group （P〈 0.05 ）. Consequently, the survival rate of 16HBE in the siGSTM5 group decreased dramatically （P〈0.05）. Conclusions Inhibition of GSTM5 may aggravate the oxidative/antioxidant imbalance, up-regulates the inflammation and aggravates cell injury in bronchial epithelial cells.

关 键 词：谷胱甘肽硫转移酶mu 5 RNA干扰 氧化/抗氧化失衡 炎症 

分 类 号：R563[医药卫生—呼吸系统]