Mechanism Of HNE-EGFR-FOXM1 Signaling Pathway Effect MUC5AC Expression In Chronic Rhinitis- Sinusitis

BACKGROUNDChronic rhinitis- sinusitis(Chronic rhinosinusitis, CRS) is a mucus hypersecretion disorders with plurality of mucin secretion, particularly MUC5 AC, but the exact mechanism is unclear. Inflammatory stimuli human neutrophil elastase(Human neutrophilelastase, HNE) may lead to airway epithelial cell hyperplasia and metaplasia, in turn, causes increased secretions. Epidermal growth factor receptor(Epidermal growth factor receptor, EGFR) activating the stimulator under the action again after the adoption of the downstream transcription factors promote metaplasia and mucus cells increased. Transcription factor FOXM1(Forkhead box M1) are closely related to airway inflammatory diseases, can promote the secretion of goblet cell proliferation and expression of a large number of MUC5 AC. Whether HNE-EGFR-FOXM1 signaling pathway can promote expression of MUC5 AC still needs further study. ObjectiveComparing different expression level of EGFR, FOXM1 and MUC5 AC in normal mucosa and nasal mucosa CRS tissues; Further detecting m RNA and protein level of EGFR, FOXM1 and MUC5 AC in primary human nasal epithelial cells after HNE stimulation. Further investigating molecular mechanism of CRS increased mucus. MethodsImmunoblotting and immunohistochemical were used to detect expression level of EGFR, p EGFR and FOXM1 in tissue samples of three groups: 10 samples from normal control group, 30 samples from CRS patients with polyps and 30 samples from CRS without polyps. Primary human nasal epithelial cells were stimulated with different concentrations of HNE(0,10, 20, 50,100 n M), cell lysates were collected after 24 h, fluorescence quantitative PCR experiments were used to detect MUC5 AC m RNA expression to get the optimal concentration of HNE concentration. The cell lysates were collected at different time points(0 h,8h,12 h, 24 h,48h) with optimal concentration irritation of HNE.The fluorescence quantitative PCR was used to detect MUC5 AC m RNA expression and get the best stimulation time. EGFR inhibitor AG1478 and FOXM1 inhibitor thiostrepton were used to suppress aim protein expression and finally detect m RNA level of EGFR,FOXM1 and MUC5 AC and Protein level of p EGFR, EGFR and FOXM1. Results(1)Immunohistochemistry results show that EGFR, FOXM1 and MUC5 AC expression level was not significant differenct between tissues with or without polyps CRS sinus mucosa,, but were significant higher than the normal control group.(2) Immunoblotting results show that expression level of EGFR, p EGFR and FOXM1 in tissues of with or without polyps CRS were significant higher than normal control group.(3) The m RNA level of EGFR, FOXM1 and MUC5 AC were significant higher than control group after stimulation of HNE primary human nasal mucosa cells, same tendency to protein expression level of p EGFR,EGFR and FOXM1. After thiostrepton inhibition, the m RNA level of EGFR and FOXM1 show no different to control groups contrast to significant lower expression of MUC5 AC. Protein expression level of p EGFR, EGFR and FOXM1 show no significantly distinguished either. After S + HNE stimulation show significantly increased m RNA of EGFR and FOXM1 compared to DMSO group contrast to decreased of MUC5 AC expression. But higher protein expression of p EGFR, EGFR and FOXM1 compared to control groups.(4) The m RNA level of EGFR, FOXM1 and MUC5 AC show significantly reduced after AG1478 stimulation,this is consistent with p EGFR, EGFR and FOXM1 protein expression. But after A + HNE stimulation, m RNA expression level EGFR and FOXM1 were significantly reduced compared to control group, consistent with significantly decreased protein expression of p EGFR, EGFR and FOXM1.Different stimulation factors can cause different expression level of p EGFR, EGFR and FOXM1. Histological results show that the expression level of EGFR and FOXM1 in CRS with or without sinus polyp mucosa were significantly higher than normal control group. These results suggest that the expression level of EGFR and FOXM1 is closely related to the development of sinusitis.