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

Objective: The 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. Methods: 16HBE 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. Results: The 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% and 50% (P<0.01) . No significant change was observed in p-JNK protein expression between the chloroacetic acid-treated and control groups. In comparison with the vehicle control and the exposed group, p-p38, p-ERK1/2, p-JNK protein expressions significantly declined in the inhibitor controls and inhibitor groups. Compared with the controls, the cell survival rates had significant reductions of 28%, 18%, 36% and 26% respectively in chloroacetic acid treated group, SB203580 group, U0126 group and SP600125 group, and the apoptosis rates in the abovementioned groups were 7, 4, 8 and 7 times. Compared with chloroacetic acid-treated group, the cell viability increased by 14% in SB203580 group and decreased by 11% in U0126 group, and the cell apoptosis rates decreased by 36% in SB203580 group and increased by 18% in U0126 group (P<0.05) . But no significant changes were observed in cell viability and apoptosis between SP600125 and chloroacetic acid-treated group. Conclusion: Chloroacetic acid might activate p38 MAPK signaling pathway and inhibit ERK1/2 MAPK signaling pathway. The signaling pathways of p38 and ERK1/2 MAPK are involved in 16HBE cell apoptosis induced by chloroacetic acid, but JNK is not involved in chloroacetic acid-induced 16HBE cell apoptosis.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，差异有统计学意义（P<0.01）；与氯乙酸染毒组比较，SB203580组细胞活力增加了14%，U0126组降低了11%，差异有统计学意义（P<0.05），SB203580组细胞凋亡率下降了36%，U0126组上升了18%，差异有统计学意义（P<0.05），SP600125组未见明显变化。 结论： 氯乙酸能激活p38 MAPK信号通路而抑制ERK1/2信号通路，且p38通路的激活和ERK1/2通路的失活参与氯乙酸诱导的16HBE细胞凋亡过程，而JNK信号通路未参与氯乙酸所致16HBE细胞凋亡。.