The Regulation Mechanism Of IL-4 Inducing 16HBE Mucin MUC1 Secretion

Chronic bronchitis,asthma,chronic obstructive pulmonary disease(COPD),and cystic fibrosis(CF)are all airway inflammatory diseases with mucus hypersecretion as the main pathological feature.These diseases are characterized by inflammatory cytokine-mediated inflammatory responses.A large number of inflammatory stimulating factors can stimulate the airway to secrete a large amount of mucus,and the excessive mucus cannot be discharged normally,which causes airway obstruction.Some pathogenic microorganisms cannot be discharged and cause repeated infection of the airway,which seriously affects the development of the disease course.These are the main causes of higher morbidity and mortality of respiratory diseases.The main component of mucus is mucin,and the production of excessive mucus is accompanied by the production of increased mucin.The regulation mechanism of inflammatory stimulating factors on mucin is complicated.At present,there are relatively few related studies and most of these studies are about the regulation mechanism of airway secretory mucin MUC5 AC affected by Th2 type inflammatory factor(IL-13).Inflammatory factor IL-4 and other mucins,such as membrane-bound mucin MUC1,are less studied.Membrane-bound mucin MUC1 can help cilia to swing,which is essential for mucus cilia clearance.Therefore,related research on its secretion mechanism will provide a new theoretical reference for the treatment of airway inflammatory diseases.In this experiment,the human bronchial epithelial cell line 16 HBE was induced by Th2 type inflammatory stimulating factor IL-4 to develop an inflammatory response,and then high-throughput transcriptome sequencing was used to screen out the differential genes involved in the inflammatory response and the signaling pathways where the differential genes are located.Then real-time fluorescence quantitative PCR and Western Blot experiments were performed to study the regulatory mechanism of mucin MUC1 secretion in human bronchial epithelial cell line 16 HBE induced by IL-4.The experimental results are as follows:1.Using qRT-PCR method to analyze the expression of TMEM16 A in human bronchial epithelial cells induced by different concentrations IL-4.The expression of TMEM16 A increased with the increase of IL-4 concentration,and the effect was the strongest when stimulated by IL-4 at 20ng/m L.2.Through high-throughput transcriptome sequencing technology,we find that 6965 differential genes were generated when 20ng/m L IL-4 induced inflammatory response,of which 4264 differential genes were up-regulated and 2701 genes down-regulated.3.Analysis find that most of the differential genes are involved in the signal transduction mechanism,including 305 signal pathways,among which the signals related to immune and inflammatory response are Jak/Stat Signaling(Janus kinase signal transduction transcriptional activator signal pathway);NF-?B Signaling(Nuclear factor-?B signaling pathway);MAPK signaling pathway(mitogen-activated protein kinase signaling pathway);Toll-like Receptors(TLRs)Pathway(Toll-like receptor signaling pathway);B Cell Receptor Signaling(B cell antigen receptor signaling pathway);T Cell Receptor Signaling(T cell antigen receptor signaling pathway).The first four are the most important signaling pathways of the inflammatory response.Channel proteins in these channels may be involved in the regulation of airway mucin MUC1.4.Through qRT-PCR experimental results,we can conclude that the changed mucins are MUC1,MUC2,MUC3,MUC15,MUC16,MUC20 from human bronchial epithelial cells 16 HBE in inflammatory response induced by 20ng/m L IL-4.Among them,MUC1,MUC2 expression was up-regulated,but MUC3,MUC15,MUC16 and MUC20 expression were down-regulated.5.Using qRT-PCR and Western Blot methods to detect the expression of TMEM16 A and MUC1 in human bronchial epithelial cells 16 HBE induced by 20 ng / m L IL-4,we find that TMEM16 A and MUC1 expressions were up-regulated.TMEM16 A may be involved in the expression of Mucin MUCI in human bronchial epithelial cells 16 HBE induced by IL-4.After knocking out TMEM16 A by si RNA,IL-4 induced a decrease in MUC1 secretion in 16 HBE cells.This shows that TMEM16 A plays an important role in regulating the secretion of mucin MUC1 induced by IL-4.6.Using qRT-PCR and Western Blot methods to detect the expressions of STAT6 and p-STAT6 in human bronchial epithelial cells 16 HBE induced by 20ng/m L IL-4,we find that the expression of p-STAT6 protein was up-regulated under the effect of IL-4.This indicates that the phosphorylation of STAT6 plays a role in the response and STAT6 may be involved in the expression of TMEM16 A and MUCI protein in human bronchial epithelial cells 16 HBE induced by IL-4.The ASAL1517499,STAT6 specific inhibitor,significantly reduced the expression of TMEM16 and MUC1 proteins in human bronchial epithelial cells 16 HBE induced by IL-4,which further confirmed that STAT6 participates in the expression of TMEM16 A and MUC1 in human bronchial epithelial cells 16 HBE induced by IL-4.7.Using Western Blot to detect the expression of ERK1/2 and p-ERK1/2 in human bronchial epithelial cells 16 HBE induced by 20ng/m L IL-4,it showed that the expression of p-ERK1/2 protein was up-regulated under the effect of IL-4,indicating that the phosphorylation of STAT6 is involved in the response and ERK1/2 is involved in the secretion of mucin MUC1 induced by IL-4.8.The PD98059,ERK1/2 specific inhibitor,does not change the expression of TMEM16 A,but it reduces the increased expression of MUC1 protein in human bronchial epithelial cells 16 HBE induced by IL-4.It indicates that ERK1/2,as a downstream signal of TMEM16 A,is involved in the expression of mucin MUC1 in human bronchial epithelial cells 16 HBE induced by IL-4.Conclusion: This experiment found that IL-4 can increase the expression of TMEM16 A and MUC1 in human bronchial epithelial cell line 16 HBE,and the expression of airway mucin MUC1 affected by IL-4 is through the signal pathways where the STAT6 and ERK1/2 signaling proteins are located.However,TMEM16 A may participate in the regulation process as an intermediate signal molecule.The study of these signal molecules can provide new strategies and therapeutic targets for airway inflammatory mucus hypersecretion diseases.