Effect Of Lyn On Early Stage Of Airway Remodeling In Bronchial Asthma And Its Mechanism

Objective:Bronchial asthma is one of the most common respiratory diseases in children.With the change of the global climate environment,the incidence rate of asthma has increased year by year,which has become a major problem seriously endangering social public health.Asthma is induced by various environmental factors and allergens,and gradually forms chronic airway inflammation,which is accompanied by mucus hypersecretion,varying degrees of airflow obstruction,bronchial hyperresponsiveness and airway remodeling.The study of airway remodeling in asthma is one of the hotspots of asthma research.It can appear at the early stage of asthma disease formation[1].Asthma generally starts from the abnormality of antigen presenting cells and bronchial epithelial cells.Pathological changes such as abnormal damage and repair of epithelial cells,epithelial mesenchymal transformation,abnormal migration and anti-apoptosis,subepithelial fibrosis and proliferation of airway smooth muscle cells gradually lead to the occurrence of airway remodeling.These changes interact with each other and cause the occurrence and development of asthma.With the gradual aggravation of pathological changes,children with asthma have progressive decline in lung function and maintain a continuous airway hyperresponsiveness state,leading to severe asthma and refractory asthma.Studies have shown that bronchial epithelial injury is an important component and initial link of the pathogenesis of asthma,and is closely related to airway remodeling and the severity of disease.And airway remodeling cannot be restored once it is formed,so it is particularly important to prevent the occurrence of early airway remodeling.In recent years,with the gradual deepening of the research on asthma,the analysis and research on the initiating events of the pathogenesis of asthma has also been increasingly valued.Extensive research and analysis have been carried out on the genes,transcription,proteins,secretome and other aspects related to asthma.These studies have made certain progress,but have not yet reached the expected results.Further research is needed to treat as soon as possible.The new tyrosine protein kinase Lyn is a member of the non-receptor tyrosine kinase family.It participates in the regulation of cell events and plays an important role in the growth,differentiation,survival,adhesion and migration of normal cells.When over-activated,it can lead to the occurrence and development of pathological processes.Lyn kinase can strongly affect the signal pathway of immune cells,so it can be an important sensor for autoimmune diseases and play an important role in tumors,asthma,psoriasis and other diseases.Lyn kinase can play a positive or negative role in the same or different cells.In the process of bronchial asthma,Lyn kinase regulates the disease progression of asthma.It regulates the chronic inflammation,airway hyperresponsiveness and airway remodeling of asthma through a variety of different signal pathways,through negative or positive activation of cell signals,thereby affecting the severity and duration of asthma attacks.Lyn kinase plays an important role in the occurrence and development of airway remodeling in bronchial asthma,but whether it is positive or negative regulation in the process of early airway remodeling and its specific regulatory mechanism need further experimental research and proof.PI3K/Akt signal pathway(phosphatidylinositol 3 kinase/protein kinase B signal pathway)widely exists in all kinds of cells.It is an important signal transduction pathway involved in cell growth,proliferation,apoptosis,differentiation regulation and glucose transport.Its abnormal activation can activate a variety of downstream substrates,leading to the transformation of normal cells to abnormal cells,enhancing the ability of cell proliferation,metastasis and invasion,and inhibiting cell apoptosis.In response to the above problems,this project aims to establish a mouse model of early airway remodeling in asthma to determine the cellular location and expression level of Lyn kinase during early airway remodeling.Then,in vitro experiments were conducted to further explore whether Lyn kinase has an effect on the process of mesenchymal transformation of airway epithelial cells and related cell biological behaviors.Finally,the potential mechanism of Lyn kinase affecting the biological function of airway epithelial cells was preliminarily discussed.Methods:1.To establish a model of early airway remodeling in mice with bronchial asthma,and detect the expression of Lyn and various functional proteins.(1)To establish a model of early airway remodeling in mice with bronchial asthma.Sixteen Balb/c mice were divided into two groups:normal control group and OVA asthma group.In the asthma group,mice were intraperitoneally injected 50 μ GOVA and 0.8mg aluminum hydroxide gel were combined to sensitize mice.On the 14th,17th,21st,24th,27th and 28th days,2%OVA was given to mice for 30 minutes every day.In the control group,mice were sensitized and stimulated with normal saline instead of OVA.(2)Collect the blood and bronchoalveolar lavage fluid of model mice,detect the level of serum ovalbumin-specific immunoglobulin E,count the total number of white blood cells and the classification of white blood cells in BALF,and determine the level of cytokines in BALF.(3)The lung tissue samples of model mice were collected for HE staining and Masson staining to observe the airway inflammation and collagen deposition;(4)Collect lung tissue samples of model mice for immunohistochemical staining to observe the expression of Lyn in airway epithelial cells,and use Western blot to detect the expression level of Lyn protein in lung tissue of model mice.(5)Lyn and its inhibitor Bafetinib interfere with the early airway remodeling model of asthma in mice.Forty Balb/c mice were divided into four groups:(1)normal saline control group(2)OVA asthma group(3)OVA+Lyn intervention asthma group(4)OVA+Bafetinib intervention asthma group.The sensitization and stimulation methods of each group are the same as before.In OVA+Lyn group and OVA+Bafetinib group,Lyn(OVA+Lyn group)and Bafetinib(OVA+Bafetinib)were given intranasal drip 2 hours before challenge,respectively.In N group and OVA group,normal saline was given intranasal drip.(6)Collect the blood and bronchoalveolar lavage fluid of mice in each group,detect the level of serum ovalbumin-specific immunoglobulin E,count the total number of white blood cells and the classification of white blood cells in BALF,and determine the level of cytokines in BALF.(7)The expression of Lyn,E-cadherin,N-cadherin and Vimentin in airway epithelial cells was observed by immunohistochemical staining of lung tissue samples of mice in each group.(8)The lung tissues of mice in each group were collected,and the protein expression levels of Lyn,E-cadherin,N-cadherin,Vimentin,Beclin-1,p62,bax,bcl-2,clearedcapase-3 and caspase-3 were detected by Western blot.2.To study the effect of Lyn on the function of bronchial epithelial cells.(1)Grouping and intervention:(1)normal saline control group(2)IL-5 group(3)IL5+Lyn group(4)IL-5+Bafetinib group.In each intervention group,human bronchial epithelial cell line Beas-2B was pretreated with IL-5,and Lyn recombinant protein or its inhibitor Bafetinib was added for intervention.In the control group,normal saline was used instead of pretreatment and intervention.The migration,proliferation and apoptosis of cells were detected by CCK8,Transwell and Annexin V+PI methods,and the mitochondrial autophagy was observed by transmission electron microscopy to determine whether Lyn can affect the proliferation,migration,percentage of apoptosis and autophagy of cells.(2)Western blot method was used to detect the expression level of Lyn,E-cadherin,N-cadherin,Vimentin,Beclin-1,p62,bax,bcl-2,cleaved-capase-3,caspase-3 in cells,to determine whether Lyn can affect the changes of EMT,autophagy and apoptosis indicators in cells.3.To explore the mechanism of Lyn reducing early airway remodeling by affecting the proliferation,migration,apoptosis of bronchial epithelial cells,autophagy and EMT process.(1)The changes of PI3K,Akt and pAkt protein expression in lung tissue of asthmatic mice and human bronchial epithelial cell line Beas-2B pretreated with IL-5 were detected.(2)The human bronchial epithelial cell line Beas-2B was pretreated with IL-5,and Lyn recombinant protein or its inhibitor Bafetinib was added.The protein expression levels of PI3K,Akt and pAkt were detected by Western blot to determine whether Lyn affected the activation of PI3K/Akt signal pathway.(3)In the pretreatment of human bronchial epithelial cell line Beas-2B with IL-5,the inhibitor of Lyn Bafetinib and PI3K inhibitor LY294002 were added at the same time.The migration,proliferation and apoptosis of the cells were detected by CCK8,Transwell and Annexin V+PI methods.The changes of mitochondrial autophagy were observed by transmission electron microscopy.Lyn,E-cadherin,N-cadherin,Vimentin,Beclin-1,p62,bax,bcl-2 were detected by Western Blot method The protein expression level of cleaved caspase-3 and caspase-3,to determine whether Lyn’s influence on EMT,proliferation,migration,apoptosis and autophagy is partially dependent on PI3K/Akt signaling pathway.Results:1.The expression of Lyn in bronchial epithelial cells and lung tissue of asthmatic mice decreased,and Lyn can reduce the early airway remodeling in asthmatic mice.(1)In the asthma mouse model,the number of inflammatory cells in bronchoalveolar lavage fluid increased,mainly eosinophilic airway inflammation,and the inflammatory mediators increased.Pathological smears showed a large number of inflammatory cells infiltrating around bronchus and blood vessels,slight thickening of airway wall,slight thickening of smooth muscle layer around bronchus,increased deposition of collagen around bronchus,and early airway remodeling.(2)The expression of Lyn in bronchial epithelial cells was decreased by immunohistochemistry in bronchial and lung tissues of asthmatic mice.The expression level of Lyn protein in lung bronchus and tissues was lower than that in control group.(3)The expression of airway inflammation and airway remodeling in asthmatic mice treated with Lyn recombinant protein was reduced,while the expression of airway inflammation and airway remodeling in asthmatic mice was further aggravated after the intervention of Lyn inhibitor Bafetinib.Western blot results also showed that Lyn recombinant protein could increase the expression of E-cadherin,Beclin-1 and bcl-2 protein in the bronchial lung tissue of asthmatic mice,and decrease the expression of N-cadherin,Vimentin,PI3K,Akt,pAkt,p62,bax,cleared-capase-3 and caspase-3 protein.Bafetinib can reduce the expression of E-cadherin,Beclin-1,bcl-2 protein and increase the expression of N-cadherin,Vimentin,PI3K,Akt,pAkt,p62,bax,cleaved caspase-3 and caspase-3 protein in the bronchial lung tissue of asthmatic mice.2.In bronchial epithelial cells,Lyn can reduce EMT changes,promote cell proliferation and autophagy,and reduce cell migration,invasion and apoptosis.(1)The expression of Lyn protein in Beas-2B cells pretreated with IL-5 decreased,which proved that IL-5 has biological activity.Lyn recombinant protein can reduce EMT changes,increase proliferation and autophagy,and reduce invasion and apoptosis of Beas-2B cells pretreated with IL-5.Bafetinib can aggravate the EMT changes,reduce the proliferation and autophagy,and increase the invasion and apoptosis of Beas-2B cells pretreated with IL-5.(2)Western blot also showed that Lyn recombinant protein could increase the expression of E-cadherin,Beclin-1 and bcl-2 in Beas-2B cells pretreated with IL-5,and decrease the expression of N-cadherin,Vimentin,p62,bax,cleaved-capase-3 and caspase-3.However,Bafetinib can reduce the protein indexes of E-cadherin,Beclin-1 and bcl-2 in Beas-2B cells pretreated with IL-5,and increase the expression of Ncadherin,Vimentin,p62,bax,cleaved caspase-3 and caspase-3.3.Lyn alleviates the EMT changes,invasion and apoptosis induced by IL-5 and promotes cell proliferation and autophagy by regulating PI3K/Akt signaling pathway.(1)In IL-5-induced Beas-2B cells,Western blot showed that Lyn recombinant protein could reduce the expression of PI3K,Akt and pAkt;Bafetinib can increase the expression of PI3K,Akt and pAkt.(2)In IL-5-induced Beas-2B cells,Western blot results showed that PI3K inhibitor LY294002 could partially reduce the increased expression of PI3K,Akt and pAkt caused by Lyn inhibitor Bafetinib,and the increased EMT changes and enhanced invasion and apoptosis functions partially reduced and decreased,and the weakened proliferation and autophagy functions were partially restored.Western blot results showed that the expression of E-cadherin,Beclin-1 and bcl-2 in the group with PI3K inhibitor LY294002 increased,and the expression of N-cadherin,Vimentin,p62,bax,cleaved-capase-3 and caspase-3 decreased,compared with the group with Bafetinib alone.The degree of increase or decrease of each index was lower than that of the group with LY294002 alone.Conclusion:1.Lyn is expressed in bronchial epithelial cells of asthmatic mice.Lyn can reduce the early airway remodeling in asthmatic mice.2.In Beas-2B cells,Lyn can inhibit cell migration,apoptosis and EMT process,promote cell proliferation and autophagy process;Lyn inhibitor Bafetinib can promote migration,apoptosis and EMT process,inhibit cell proliferation and autophagy process.3.Lyn can inhibit cell migration,apoptosis and EMT process by inhibiting PI3K/Akt signal pathway,and promote cell proliferation and autophagy process.PI3K inhibitor can partially restore and block the cell migration,apoptosis and EMT process caused by Lyn,and partially restore and block the cell proliferation and autophagy process improved by Lyn.