丝裂原活化蛋白激酶信号通路在氯乙酸诱导人支气管上皮细胞凋亡中的作用  

Effect of mitogen-activated protein kinase signaling pathway on apoptosis induced by chloroacetic acid in human normal bronchial epithelial 16HBE cells

作　　者：孟涛[1] 杨墨[3] 李云霞[1] 贾强[2] 于功昌[2] 戴宇飞[1] Meng Tao Yang Mo Li Yuxia Jia Qiang Yu Gongchang Dai Yufei(Key Laboratory of Chemical Safety and Health, National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing 100050, China)

机构地区：[1]中国疾病预防控制中心职业卫生与中毒控制所化学污染与健康安全重点实验室,北京100050 [2]山东省医学科学院职业卫生与职业病防治研究院 [3]山西医科大学公共卫生学院

出　　处：《中华劳动卫生职业病杂志》2017年第5期321-327,共7页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：公益性行业科研专项（201402021）;十二五国家科技支撑计划资助项目（2014BA112B02）

摘　　要：目的探讨丝裂原活化蛋白激酶（MAPK）信号通路在氯乙酸诱导的人正常支气管上皮细胞16HBE凋亡过程中的作用。方法用0.5、1.0、1.5、2.0、2.5、3.0、3.5 mmol/L氯乙酸处理16HBE细胞24 h后，应用CCK-8和LDH法检测细胞存活情况，采用Annexin V-FITC/PI双染法检测细胞凋亡情况，并用免疫印迹法检测MAPK信号通路蛋白p38、ERK1/2、JNK的磷酸化。用MAPK通路特异性抑制剂（SB203580、U0126、SP600125）预处理16HBE细胞1 h，再用2.5 mmol/L氯乙酸染毒24 h后，检测加入抑制剂后p-p38、p-ERK1/2、p-JNK的表达水平及细胞活力和凋亡的变化。结果随着氯乙酸染毒浓度的增加，用CCK-8法和LDH法检测的细胞存活率逐渐降低，相关系数分别为-0.902和-0.825（P〈0.05），均具有较好的剂量效应关系，且CCK-8法检测效能明显优于LDH法，差异有统计学意义（P〈0.05）。氯乙酸可诱导16HBE细胞凋亡，1.5、2.0、2.5 mmol/L氯乙酸所致细胞凋亡率分别为17.2%±4.0%、24.6%±4.2%和39.3%±5.7%，均明显高于对照组（5.6% ± 3.0%），差异有统计学意义（P〈0.05）。氯乙酸染毒组p38和ERK1/2蛋白磷酸化水平存在时间、剂量依赖性关系；与对照组比较，16、24 h组p-p38蛋白水平分别是对照的2.1和2.6倍，差异有统计学意义（P〈0.01），p-ERK1/2蛋白水平分别比对照降低了37%和52%，差异有统计学意义（P〈0.05），而p-JNK蛋白各时间点均无明显变化；染毒24 h后，2.0、2.5 mmol/L组p-p38蛋白水平分别是对照组的1.9和2.6倍，差异有统计学意义（P〈0.01），p-ERK1/2蛋白水平分别比对照组降低了40%和50%，而JNK蛋白各组未见明显磷酸化。与对照组和氯乙酸染毒组比较，抑制剂对照组和抑制剂组p-p38、p-ERK1/2及p-JNK蛋白均明显减少；与对照组比较，氯乙酸染毒组和p-38、ERK及JNK通路抑制剂组细胞活力分别下降了28%、18%、36%和26%，凋亡率分别为对照组的7、4、8和7倍，ObjectiveThe aim of this study was to investigate the effect of mitogen-activated protein kinase （MAPK） signaling pathway on apoptosis induced by chloroacetic acid in human normal bronchial epithelial 16HBE cells.Methods16HBE cells were exposed to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mmol/L chloroacetic acid for 24 h in vitro. The cytotoxicity induced by chloroacetic acid was assessed by CCK-8 and LDH assays. Cell apoptosis was detected by Annexin V-FITC and PI staining. The protein expression levels of phosphorylation of p38, ERK1/2 and JNK were determined by western blotting. 16HBE cells were pretreated with MAPK signaling pathway specific inhibitors including SB203580, U0126 and SP600125 for 1 h, and these cells were subsequently treated with 2.5 mmol/L chloroacetic acid for 24 h. The expressions of p-p38, p-ERK1/2 and p-JNK as well as the changes of cell viability and apoptosis were measured after pretreated with inhibitors for 1 h.ResultsThe cell viability by CCK-8 and LDH methods gradually reduced in a dose-dependent manner when chloroacetic acid concentrations elevated （P〈0.05） , and their correlation coefficients were -0.902 and -0.825, respectively. The detection efficiency of CCK-8 assay significantly increased compared with LDH assay （P〈0.05） . The cell apoptosis rates, which were （17.2±4.0） %, （24.6± 4.2） %, （39.3 ± 5.7） % in 1.5, 2.0, 2.5 mmol/L chloroacetic acid-treated groups, were higher than that of the control group[ （5.6 ± 3.0） %] （P〈0.05） . There was a time-or dose-dependent change in the protein expressions of p-p38, p-ERK1/2 and p-JNK. Compared with the control, the levels of p-p38 had 2.1 and 2.6-fold increases in 16 and 24 h treated groups （P〈0.01） , while the levels of p-ERK1/2 distinctly decreased by 37% and 52% （P〈0.01） . In comparison with the control group, the expressions of p-p38 had 1.9 and 2.6-fold increases in 1.5 and 2.5 mmol/L treatment groups （P〈0.01） , whereas the expressions of p-ERK1/2 significantly decreased by 40

关 键 词：氯乙酸 丝裂原活化蛋白激酶 上皮细胞 

分 类 号：R135[医药卫生—劳动卫生]