慢性机械压力诱导气道上皮黏蛋白5AC的表达

Chronic mechanical stress induces mucin 5AC protein expression in rat airway epithelial cells in vitro

目的探讨慢性机械压力能否诱导气道上皮黏蛋白(mucin,MUC)5AC表达及通过的相关信号通路。方法培养大鼠气道上皮细胞建立气液相界面(air liquid interface,ALI),通过对Transwell培养板上的气道上皮细胞每天施加10、20、30 cmH2O水平的气体压力1 h并持续7 d来模拟慢性机械压力作用,并以表皮生长因子受体(epidermal growthfactor receptor,EGFR)特异性抑制剂AG1478、细胞外信号调节激酶(extracellular signal regulated kinase,ERK)激酶抑制剂PD98059分别作为干预因素处理细胞。将细胞分为空白对照组、单纯压力组、AG1478+30 cmH2O压力组、PD98059+30 cmH2O压力组,单纯压力组分为10、20、30 cmH2O3个压力水平。RT-PCR检测MUC5AC mRNA表达水平,ELISA法检测MUC5AC蛋白含量,Western blot检测磷酸化ERK(p-ERK)、磷酸化c-Jun氨基末端激酶(phosphorylated c-Jun N-terminalkinase,p-JNK)和磷酸化P38(p-P38)水平。结果与空白对照组比较,单纯压力组MUC5AC蛋白及mRNA表达水平随压力水平的升高而升高(P<0.05),呈压力依赖性,同时伴随p-ERK蛋白含量增加(P<0.05),但p-JNK和p-P38含量没有明显变化(P>0.05)。30 cmH2O压力水平MUC5AC蛋白及mRNA表达水平[(0.65±0.09)、(0.62±0.04)]明显高于空白对照组[(0.28±0.04)、(0.20±0.05),P<0.01],p-ERK蛋白含量(0.68±0.05)较空白对照组(0.27±0.07)显著增加(P<0.01)。应用AG1478和PD98059分别预处理后,MUC5AC蛋白、mRNA表达水平及p-ERK蛋白水平在AG1478+30 cmH2O压力组分别为(0.39±0.08)、(0.40±0.06)、(0.25±0.04),PD98059+30 cmH2O压力组分别为(0.36±0.09)、(0.38±0.13)、(0.26±0.11),与单纯压力组30 cmH2O水平比较均显著降低(P<0.05)。结论慢性机械压力能诱导气道上皮MUC5AC表达增加,这一作用可能通过机械压力压缩侧面胞间隙来实现,而EGFR及ERK信号通路参与其中。

Objective To study the effect of chronic mechanical stress on mucin （MUC）5AC expression in airway epithelial cells of rats and its possible signaling pathway. Methods Airway epithelial cells from rats were cultured on transwell membrane at an air-liquid interface, stimulated by chronic mechanical stress from air pressure and pre-treated with EGFR kinase inhibitor AG1478 and specific inhibitor of the ERK pathway PD-98059, respectively. The cells were divided into negative control group, stress treatment group, AG1478 pre-treatment group, and PD98059 pre-treatment group. Chronic mechanical stress was induced by 10, 20 and 30 cmH2O, respectively in stress treatment group. MUC5AC protein was detected by enzyme-linked immunosorbent assay. MUC5AC mRNA level was analyzed by RT-PCR. Protein levels of p-JNK, p-ERK and p-P38 were measured by Western blotting. Results The protein level of MUC5AC, mRNA and p-ERK was significantly higher in stress group than in control group （P 〈 0.05 ）. However, no significant difference was found in protein level of p-JNK and p-P38 between stress and control groups （ P 〉 0.05 ）. The expression level of MUC5AC protein and mRNA was significantly higher in 30 cmH2O stress group than in control group （0.65 ± 0.09 and 0.62 ±0.07 vs 0. 28 ±0.04 and 0.20 ±0.05, P 〈0.01 ）. The expression level of p-ERK was also significantly higher in 30 cmH20 stress group （0.68 ± 0.05） than in control group （0.27 _＋ 0.07, P 〈0. 01 ）. After pre-treatment with AG1478 and PD98059, the protein and mRNA expression levels of MUC5AC, and protein expression level of p-ERK was 0.39 ±0.08, 0.40 ±0.06, 0.25 ± 0.04 in AG1478 pre-treatment ＋ 30 cmH2O group, and 0.36 ±0.09,0.38±0.13,0.26 ±0.11 in PD98059 pre-treatment ＋30 cmH2O group, which was significantly lower than in 30 cmH20 stress group （P 〈 0.05）. Conclusion Chronic mechanical stress can significantly up-regulate the expression of MUC5AC protein and mRNA by compressing the lateral intercellular space. EGFR and ERK signal pathways play a key role in the process.