谷胱甘肽硫转移酶M5通过p38MAPK/NF-κB途径下调肿瘤坏死因子α介导的人支气管上皮细胞炎症水平  

GSTM5 Down-regulates TNF-α-Mediated Inflammation Levels in Human Bronchial Epithelial Cells by p38MAPK / NF-κB Pathway

作　　者：黄云[1] 袁伟锋[2] 李理[2] 黄文杰[2] 

机构地区：[1]广州医科大学研究生学院,广东广州510182 [2]广州军区广州总医院呼吸内科,广东广州510010

出　　处：《中国呼吸与危重监护杂志》2016年第2期171-175,共5页Chinese Journal of Respiratory and Critical Care Medicine

基　　金：国家自然科学基金面上项目(编号:81370173;81070003);国家自然科学基金青年科学基金(编号:81200002)

摘　　要：目的探讨急性肺损伤炎症状态下谷胱甘肽硫转移酶M5(GSTM5)对人支气管上皮细胞16HBE炎症水平的影响及其可能的分子机制。方法肿瘤坏死因子α(TNF-α;10 ng/m L)诱导16HBE,建立急性肺损伤细胞炎症模型,分组处理如下:空白对照组、TNF-α组(TNF-α)、GSTM5组(GSTM5+TNF-α)、阴性对照组(阴性对照质粒+TNF-α)。GSTM5组及阴性对照组分别采用脂质体Lipofectamine2000转染导入GSTM5-GFP质粒及阴性对照质粒;酶联免疫吸附试验检测各组细胞上清液中IL-6、IL-8、IL-10的含量,反转录聚合酶链反应检测NF-κB mRNA表达水平,蛋白质印迹法检测NF-κB、P-NF-κB、p38、P-p38蛋白表达水平。结果荧光显微镜检查证实成功构建GSTM5-GFP真核表达质粒并转染成功。TNF-α诱导后,细胞培养上清液中IL-6、IL-8、IL-10浓度较空白对照组升高(P<0.05),GSTM5组细胞上清液中IL-6、IL-8、IL-10均较TNF-α组降低(P<0.05),差异有统计学意义;同时,TNF-α刺激后各组细胞总NF-κB mRNA转录水平、NF-κB、p38磷酸化水平均较空白对照组增高(P<0.05),而GSTM5组较TNF-α组与阴性对照组降低(P<0.05)。结论过表达GSTM5可下调TNF-α诱导的人支气管上皮细胞16HBE炎症水平,其机制与GSTM5抑制p38MAPK、NF-κB磷酸化密切相关。Objective To investigate the effects of glutathione S-transferase M5（ GSTM5） on the inflammation in human bronchial epithelial 16 HBE cells and its possible molecular mechanisms. Methods Acute lung injury cell model was constructed with 16 HBE cells induced by tumour necrosis factorα（ TNF-α,10 ng / m L）. The cells were devided into a control group,a TNF-α group（ TNF-α）,a GSTM5 group（ GSTM5 ＋ TNF-α）,a negative control group（ negative control plasmid ＋ TNF-α）. GSTM5-GFP plasmid and negative control plasmid were respectively transfected to the cells of the GSTM5 group and the negative control group using Lipofectamine2000. The contents of interleukin-6（ IL-6）,IL-8,IL-10 in the cell supernatant were measured by ELISA. The expression of nuclear factor-κB（ NF-κB） mRNA was detected by RT-PCR,and the expression of NF-κB,phospho-NF-κB,p38,phospho-p38 protein were detected by Western blot. Results The GSTM5-GFP eukaryotic expression vector was successfully constructed and transfected successfully confirmed by fluorescence microscope. The contents of IL-6,IL-8,IL-10 in the TNF-α-induced cell supernatant were significantly higher than those in the control group（ P〈0. 05）,and the contents of IL-6,IL-8,IL-10 in the GSTM5 group were lower than those in the TNF-α group（ P〈0. 05） with statistically significant difference. At the same time,the total NF-κB mRNA,phospho-NF-κB and phospho-p38 protein were increased in TNF-α stimulated cells compared with the control group（ P〈0. 05）,while the GSTM5 group was lower than that in the TNF-α group and the negative control group（ P〈0. 05）. Conclusion Overexpression of GSTM5 inhibits the phosphorylation of p38 MAPK and NF-κB and down-regulates the inflammation of TNF-α-induced human bronchial epithelial 16 HBE cells.

关 键 词：谷胱甘肽硫转移酶M5 急性肺损伤 炎症因子 核因子-ΚB P38丝裂原活化蛋白激酶 

分 类 号：R563[医药卫生—呼吸系统]