Inflammatory Factors Regulates The O-Glycosylation Of Airway Mucous Through Cosmc

Backgroud:The human respiratory system is in close contact with the external environment,and is vulnerable to the invasion of harmful inhalation,bacteria and viruses in the air,which causes various acute and chronic airway inflammation diseases.But long term chronic inflammation of respiratory tract can cause a series of pathological changes in the lung,such as goblet cell metaplasia,airway mucus hypersecretion,bronchial tortuosity obstruction,respiratory function damage,etc.the airway mucus hypersecretion is of great significance to the development and return of chronic respiratory diseases.Airway mucus is a kind of mucin composed of mixed mucin such as water,ion and plasma protein exudate.It can protect and lubricate the respiratory tract by sticking to the particles and microorganisms inhaled in the airway.However,excessive secretion of airway mucus can damage the function of mucociliary clearance and local defense,which leads to the difficulty of bacterial colonization,inflammation infection control and gas exchange dysfunction.Many airway inflammatory diseases such as chronic bronchitis,pulmonary cystic fibrosis,asthma,bronchiectasis and so on all have airway mucus hypersecretion.Mucin,which is composed of mucin,is mucin 5AC(MUC5AC)secreted by goblet epithelial cells in airway.Repeated airway inflammation can often cause goblet cells to metabolize and secrete a large amount of MUC5 AC.MUC5AC is a highly O-glycosylated macromolecular glycoprotein,which is composed of the main chain of serine,threonine and proline rich protein and the side chain of oligosaccharide branch.As the product of MUC5 AC gene,MUC5 AC gene has complex molecular structure.O-glycosylation is the main post translation modification method of MUC5 AC,which makes it form complex branching network molecular structure,and gives MUC5 AC unique structural and rheological characteristics.Because MUC5 AC is of great significance in the pathogenesis of airway inflammation,many researches are devoted to improving the airway mucus hypersecretion.It is hoped that airway obstruction and bacterial colonization can be improved by reducing the mucin produced and secreted by airway epithelial cells during inflammation,which is conducive to the clinical treatment of sputum discharge,anti infection and respiratory function.But in the actual clinical work,we found that patients with respiratory inflammatory diseases often have cough phlegm difficulties.This phenomenon is not only related to respiratory muscle fatigue,inflammation secretion located in the end of the respiratory tract,damage of the cilia clearance function of the bronchi epithelium,loss of airway water,and other factors,but also related to the excessive viscosity of the airway endocrine mucus texture,which makes it difficult to remove the mucus.The increase of the viscosity of respiratory secretions can aggravate airway obstruction and bacterial colonization.The mucin glycosylation modification affects the texture of mucus,especially the abnormal glycosylation of MUC5 AC can cause the viscosity of mucus texture to be difficult to be removed.Meanwhile,the secretion potential of airway mucus is highly related to the mucin glycosylation state.Therefore,it is of great significance to improve the mucus texture by interfering with glycosylation modification of mucin,so that it can be easily discharged by the airway cilia clearance system,as well as inhibiting the high secretion of mucus.The mobility of mucin O-glycan through the matrix pore is determined by the length and charge density of mucin O-glycan.Cosmc is the companion molecule of t-synthase,an important glycosyltransferase in the process of glycosylation modification of MUC5 AC.It helps t-synthase oligopeptides form dimers through disulfide bonds,and helps the molecular structure fold to form an active glycosyltransferase,which promotes the formation of glycosylated product T antigen and the extension of side branch sugar chain,which is of great significance for the smooth progress of glycosylation modification.It was found that activation of signaling pathway molecules,such as akt/erk,is closely related to the expression of cosmc.These signaling pathway molecules are often activated under the stimulation of airway inflammatory factors,which promotes the increase of MUC5 AC synthesis in airway epithelial cells.Therefore,we hope to explore the inflammatory factor stimulated and activated intracellular signaling pathway factors,especially neutrophil elastase through this study,NE)and its downstream signal factors,and the relationship between the modification of mucopalbumin,are helpful to further improve the consistency of airway secretion and promote the treatment of airway mucus removal.This study is carried out in the following three parts.Part 1 Inflammation effect the expression of Cosmc and T antigenChapter 1 Expression of Cosmc and T antigen in airway epithelium of rat model with acute airway inflammation and human beenObjective: We hired immunohistochemistry to detect the expression of cosmc and T antigen in airway epithelium of rats with airway inflammation and patients with pulmonary inflammation,to verify whether inflammatory factors can cause the expression of cosmc and T antigen in airway epithelial cells in vitro,and the relationship between airway inflammation and mucous O-glycosylation.Methods: 1.The rat airway inflammation model was established by LPS,the paraffin seal of rat lung tissue was separated,and the expression of Cosmc and T antigen in paraffin section of rat lung tissue was detected by immunohistochemistry.2.To collect the paracancerous lung tissue of lung cancer patients with pulmonary inflammatory disease and detect the expression of cosmoc and T antigen in the inflammatory lung tissue by immunohistochemistry.Results: The expression of cosmoc and T antigen in airway epithelium of rats with airway inflammation and human inflammatory lung tissue was higher than that of rats in control group and patients without pulmonary inflammatory disease.Conclusion: Inflammation can affect the expression of Cosmc and T antigen in rat and human airway epithelium.Inflammatory factors may be related to airway mucous glycosylation.Chapter 2 Expression of Cosmc in BEAS-2B cells after NE stimulationObjective: To investigate the relationship between inflammatory factors and airway mucous glycosylation,we detected the expression of Cosmc and T antigen in BEAS-2B cells after NE interfering.Methods: BEAS-2B cells were cultured in vitro and stimulated by inflammatory factor NE.Western blot,PCR and Laser Confocal were used to detect the expression level of Cosmc,lectin-related electrophoresis and fluorescence immunoassay were hired to detected the expression of T antigen.Results: Compared with the control group,the expression of Cosmc and T antigen in BEAS-2B cells increased after NE stimulation.Conclusion: NE affected the expression of Cosmc and T antigen in airway epithelial cells,further confirming the relationship between inflammatory factors and airway mucous glycosylation.Part 2 NE regulates the expression of T antigen through effect the expression of Cosmc and T-synthase activityObjective: We inhibited the expression of Cosmc to explore the important role of Cosmc in NE induced increase of T antigen expression and the mechanism of NE affecting T antigen expression through Cosmc.Methods: We transfected BEAS-2B cells with sh RNA to inhibit the expression of Cosmc.Western blot,PCR and fluorescence analysis were used to detect the expression of T-synthase protein,gene transcription and activity in the transfected cells.The expression of T antigen was detected by lectin related electrophoresis and fluorescence immunoassay.Results: In transfected BEAS-2B cells,the activity of T-synthase decreased and the expression of T antigen also decreased after the stimulation of NE.Conclusion: NE regulates T-synthase activity and subsequent synthesis of T antigen by influencing the expression of Cosmc.Part 3 NE regulate the expression of Cosmc and T antigen through PI3 K signaling pathwayObjective: Eexploring the important role of PI3 K in the expression of Cosmc induced by NE stimulation,in order to understand the regulation of signal pathway on the expression of Cosmc and the effect on T-synthase activity and T antigen production.Methods: We hired the agonists and inhibitors of EGFR/Ras/PI3 K respectively as interfering factors to culture BEAS-2B cells.Western blot was used to detect the expression of p-EGFR,ras,P85? and Cosmc in BEAS-2B cells.Results: Fluorescence detection,lectin related electrophoresis and fluorescence immunoassay were used to detect the level of T-synthase activity and T antigen expression in the cells after the interfering the cells with NE and PI3 K inhibitor.Conclusion: NE affect the expression of Cosmc by regulating the signal pathway molecule PI3 K,thus interfering with mucous glycosylation.