肺炎支原体对支气管上皮细胞黏蛋白分泌的影响

Effect of Mycoplasma pneumoniae on the mucin secretion in bronchial epithelial cells

目的探讨肺炎支原体对支气管上皮细胞黏蛋白分泌的影响及机制。方法将人支气管上皮细胞(HBEC)分为接种肺炎支原体的感染组和未接种肺炎支原体的对照组。采用免疫荧光标记法观察2组细胞中黏蛋白5AC(MUC5AC)和黏蛋白5B(MUC5B)的表达情况;荧光定量聚合酶链反应检测2组细胞中MUC5B、MUC5AC、叉头框蛋白A2(FOXA2)、信号传导与转录激活因子3(STAT3)、信号传导与转录激活因子6(STAT6)、表皮生长因子受体(EGFR) mRNA表达水平; Western blot法检测感染组和对照组细胞中MUC5B、MUC5AC、FOXA2、磷酸化STAT3(pSTAT3)、磷酸化STAT6(p-STAT6)、磷酸化EGFR(p-EGFR)蛋白表达; Western blot法检测分别加入STAT3、STAT6、EGFR的抑制剂IX Cpd188、山柰酚、AG1478预处理以后各组细胞中MUC5B、MUC5AC、FOXA2蛋白表达。结果感染组细胞中MUC5B和MUC5AC荧光信号较对照组明显增强。感染组细胞中MUC5B、MUC5AC mRNA和蛋白相对表达量显著高于对照组(P <0. 05),而FOXA2 mRNA和蛋白相对表达量显著低于对照组(P <0. 05)。感染组细胞中STAT3、STAT6、EGFR mRNA和磷酸化蛋白p-STAT3、p-STAT6、p-EGFR相对表达量均显著高于对照组(P <0. 05)。感染组及IX Cpd188、山柰酚、AG1478预处理组细胞中FOXA2蛋白相对表达量均显著低于对照组(P <0. 05),而IX Cpd188、山柰酚、AG1478预处理组细胞中FOXA2蛋白相对表达量均显著高于感染组(P <0. 05)。感染组及IX Cpd188、山柰酚、AG1478预处理组细胞中MUC5B蛋白相对表达量高于对照组(P <0. 05);与感染组比较,IX Cpd188、山柰酚、AG1478预处理细胞中MUC5B、MUC5AC蛋白相对表达量均显著降低(P <0. 05)。结论肺炎支原体感染可能通过STAT/EGFR-FOXA2信号通路促进支气管上皮细胞黏蛋白分泌,从而引发呼吸道相关疾病。

Objective To investigate the effect and mechanism of Mycoplasma pneumoniae on the mucin secretion in bronchial epithelial cells.Methods The human bronchial epithelial cells （HBEC） were divided into infection group and control group.The HBECs in the infection group were inoculated with Mycoplasma pneumoniae ,while the HBECs in the control group were not inoculated with Mycoplasma pneumoniae .The expression of mucin 5AC （MUC5AC） and mucin 5B （MUC5B） in the HBECs was observed by immunofluorescence staining.The expression of MUC5B,MUC5AC,forkhead box protein A2 （FOXA2）,signal transducers and activators of transcription-3（STAT3）,signal transducers and activators of transcription-6 （STAT6） and epidermal growth factor receptor （EGFR） mRNA were detected by fluorescence quantitative polymerase chain reaction.The expression of MUC5B,MUC5AC,FOXA2,phosphorylated STAT3 （p-STAT3）,phosphorylated STAT6 （p-STAT6） and phosphorylated EGFR （p-EGFR） were detected by western blot.The expression of MUC5B,MUC5AC and FOXA2 protein after the pretreatment of STAT3,STAT6,EGFR inhibitor IX Cpd188,kaempferol and AG1478 were detected by western blotting.Results The fluorescence signals of MUC5B and MUC5AC in HBECs of the infection group were significantly higher than those in the control group.Compared with the control group,the relative expression of MUC5B,MUC5AC mRNA and protein in the HBECs increased significantly （ P 〈0.05）,while the relative expression of FOXA2 mRNA and protein in the HBECs decreased significantly in the infection group （ P 〈0.05）.The relative expression of STAT3,STAT6,EGFR mRNA and p-STAT3,p-STAT6,p-EGFR protein in HBECs of the infection groups were significantly higher than those in the control group （ P 〈0.05）.The relative expression of FOXA2 protein in HBECs of the infection group and IX Cpd188,kaempferol and AG1478 pretreatment group was significantly lower than that in the control group （ P 〈0.05）;while the relative expression of FOXA2 protein in the HBECs of the IX pd188,kaempferol and AG1478 pretreatment groups was significantly higher than that in infection group （ P 〈0.05）.The relative expression of MUC5B protein in the HBECs of the infection group and the IX Cpd188,kaempferol and AG1478 pretreatment groups was higher than that in the control group （ P 〈0.05）;the relative expression of MUC5B and MUC5AC protein in the HBECs of the IX Cpd188,kaempferol and AG1478 pretreatment groups was significantly lower than that in the infection group （ P 〈0.05）.Conclusion Mycoplasma pneumoniae infection may promote the mucin secretion in bronchial epithelial cells through STAT/EGFR-FOXA2 signaling pathway,which may lead to respiratory related diseases.