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目的探讨p38丝裂原激活蛋白激酶(P38)信号通路参与高渗应激诱导黏蛋白5AC(MUC5AC)高表达的作用机制。方法利用545、695、855、1055和1280mOsm/L高渗氯化钠溶液培养人气道上皮NCl-H292细胞。同时以1280m Osm/L高渗氯化钠为刺激因素,以P38特异性抑制剂SB203580、c-jun氨基末端激酶(JNK)特异性抑制剂SP600125及细胞外信号调节蛋白激酶(ERK)特异性抑制剂U0126为干预因素,采用RT-PCR技术及ELISA观察培养细胞MUC5AC转录水平和MUC5AC蛋白水平改变。采用Western blotting法检测1280mOsm/L高渗氯化钠培养上清液中细胞匀浆磷酸化P38(p-P38)、磷酸化JNK(p-JNK)及磷酸化ERK(p-ERK)蛋白水平。结果分别以545、695、855、1055和1280mOsm/L高渗氯化钠刺激后,培养细胞中MUC5AC mRNA及蛋白含量显著高于对照组(P<0.01),且呈现出与渗透浓度相关的趋势。SB203580显著下调1280mOsm/L高渗氯化钠所致的p-P38含量升高,同时显著下调MUC5AC mRNA及蛋白含量(P<0.05);SP600125则明显下调p-JNK的含量,并下调MUC5AC mRNA转录及蛋白含量(P<0.05),但作用弱于SB203580;1280m Osm/L高渗氯化钠对ERK的含量无影响(P>0.05),U0126对MUC5AC mRNA转录及蛋白含量也无明显影响(P>0.05)。结论高渗应激可呈浓度依赖性地从转录水平诱导气道上皮细胞呈现黏液高分泌状态,且P38信号通路起主要作用。
Objective To investigate the mechanism of P38 mitogen-activated protein kinase (P38) signaling pathway involved in the hyperosmotic stress-induced mucin 5AC (MUC5AC) overexpression. Methods Human epithelial NCl-H292 cells were cultured in 545, 695, 855, 1055 and 1280 mOsm / L hypertonic sodium chloride solution. At the same time, 1280 m Osm / L hypertonic sodium chloride was used as the stimulus to specifically inhibit P38-specific inhibitor SB203580 and JNK-specific inhibitor SP600125 and extracellular signal-regulated protein kinase (ERK) U0126 was used as an intervention factor. The transcription level of MUC5AC and the level of MUC5AC protein in cultured cells were detected by RT-PCR and ELISA. Western blotting was used to detect the protein levels of phosphorylated P38 (p-P38), phosphorylated JNK (p-JNK) and phosphorylated ERK (p-ERK) in 1280mOsm / L hypertonic sodium chloride culture supernatant. Results The mRNA and protein levels of MUC5AC in cultured cells were significantly higher than those in control group (P <0.01) after stimulation with 545, 695, 855, 1055 and 1280 mOsm / L hypertonic sodium chloride respectively and showed a trend related to osmotic concentration . SB203580 significantly downregulated the content of p-P38 induced by hypertonic sodium chloride at 1280 mOsm / L, and significantly decreased the mRNA and protein level of MUC5AC at the same time (P <0.05); SP600125 significantly decreased the content of p-JNK and down-regulated MUC5AC mRNA transcription (P <0.05), but the effect was weaker than that of SB203580. The hypertonic sodium chloride at 1280m Osm / L had no effect on the content of ERK (P> 0.05), but U0126 had no significant effect on the transcription and protein content of MUC5AC > 0.05). Conclusion Hypertonic stress can induce mucosal hypersecretion in airway epithelial cells in a concentration-dependent manner from transcriptional level, and P38 signaling pathway plays a major role.