Lyn Kinase Regulate STAT6 Pathways Involved In The Mechanism Of High Mucus Secretion In Asthma

Objective:Recent years,many studies have shown that Lyn plays an important role in the hypersecretionof Mucus in asthma,but the specific mechanism is unclear.In this research,we mainly studiedthe effect of Lyn on the expression of MUC5 AC in bronchial epithelial cells and investigate the related mechanisms.Methods:1.Cell experiment:(1)The structure of pLV.Ex3d.P/puro-EF1A-hLYN-eGFP virus:this part completed by Cyagen Biotechnology Company.(2)Human bronchial epithelial cells(16HBE)were cultured in six 35mm culture dishes.Three culture dishes were infected by pLV.Ex3d.P/puro-EF1A-hLYN-eGFP virus and the surplus dishes infected by pLV.Ex3d.P/puro-EF1A-eGFP virus were as control.Then cells were stimulated respectively by IL-4 and IL-13 at the concentration of lng/ml for 24h.Cells without stimulation were used as control.After 24h,protein was extracted from cells by cell lysis buffer and used to test the expression of Stat6,pSTAT6,Lyn in each group by western-blot.(3)Human bronchial epithelial cells(16HBE)were cultured in six confocal dishes.Three culture dishes were infected by pLV.Ex3d.P/puro-EF1A-hLYN-eGFP virus and the surplus dishes infected by pLV.Ex3d.P/puro-EF1A-eGFP virus were ascontrol.Then cells were stimulated respectively by IL-4 and IL-13 at the concentration of 1ng/ml for 24h.Cells without stimulation were used as control.After 24h,expression of MUC5AC in cells was detected by immunofluorescence and observed under confocal microscope.2.Animal experiment:16 female wide type mice(WT)(6-8w)were randomly divided into asthma group and control group,with 8rats in each group.16 female Lyn overexpression mice(Lyn+)(6-8w)were randomly divided into asthma group and control group,with 8rats in each group.Asthma group were sensitized by i.p.injection with ova-Aluminium hydroxide and challenged with intranasal instillation of 1%ova,control group received the equal dose of normal saline.24 hours afterlast challenge each group were bronchoalveolar lavage fluid(BALF)and countcell,lung tissue was given HE staining,Masson's staining and PAS staining,immunofluo-rescence was used to measure the expression level of MUC5AC,western blotting detection of STAT6,pSTAT6,Lyn,ELISA was used to detectcon-centrations IL-4,IL-13 and IL-17 in mice lung tissue.Results:1 cell experiments:(1)compared with the sequence of the human pLV.Ex3d.P/puro-EF1A-hLYN-eGFP virus Lyn sequences,that showed the construction sequence and the expected base sequence were exactly matched.(2)Western-blot showed the expressions of STAT6 and pSTAT6 were reduced when cells were infected by virus with high expression of Lyn.(3)Immunefluorescent showed that the expression of MUC5AC was reduced when cells were infected by virus with high expression of Lyn.2.animal experiment:(1)Cytological changes in BALF:Compared with control group,white blood cell,percentage of eosinophils(EOS)and neutrophils in the asthma group were significantly higher thanit(P<0.05),while lyn+ asthma group was lower than wt asthmagroup(P<0.05).(2)Pathological changes in lung tissue of mice:HE staining showed that WT asthma group has a large number of inflammatory cells infiltration around bronchial,blood vessels and the surrounding lung tissue,part of epithelial cell necrosis,goblet cells proliferation,while Lyn+ asthma group significantly reduced and there was few inflammation in control group.PAS staining reveals that there was a large number of glycoproteins expressed in bronchial of WT asthma group,while Lyn+ asthma group has a small amount and control group has few expression.Masson's staining showed that compared with WT asthma group,there was lower smooth muscle cell proliferation in Lyn+asthma group.Immunofluorescence showed that the fluorescence intensity of MUC5 AC in WT asthma group was higher than Lyn+asthma group,and the lowest was control group.(3)Westem-blot:Compared with control group,the expression level of STAT6,PSTAT6 in asthma group were significantly higher than it,while the expression level in lyn+asthma group was lower than wt asthma group.(4)ELISA for the detection of lung tissue levels of IL-4,IL-13,IL-17:compared with control group,asthma group was significantly higher,while lyn+asthma group was lower than wt asthma group,the difference was statistically significant(P<0.05).Conclusions:1.cell experiments:Lyn kinase can reduce IL-4,IL-13 induced MUC5 AC production in human bronchial epithelial cells,its mechanism of action may be to regulate the expression of MUC5AC through reducing STAT6.2.animal experiments:Lyn kinase can relieve asthma airway inflammation in mice,reducing bronchial mucus secretion,and the role of the mechanism may be reduced by the action of STAT6 signaling pathway to relieve airway inflammation and bronchial mucus secretion in mice.