Expression And Activation Of IL-6/STAT3 Signaling Pathway In Airway: To Explore Its Role In A Mouse Model Of Allergic Asthma

Bronchial asthma(asthma in brief) was the most common disorder caused by chronic inflammation in the airways in children,Asthma has become much more common in recent decades,and it was a common problem with a major economic and health burden around the world. Asthma was the leading chronic disease among children in most industrialized countries.The past two decades have witnessed a dramatic increase in the prevalence of asthma worldwide, especially in children.The increasing rate of asthma in children in urban were also revealed by a research around the city in china.The pathogenesis of asthma were very complex and there were still many unanswered questions,in most cases the inappropriate immune response were the basic mechanism of asthma.Asthma was generally believed to be associated with chronic abnormalities in T-cell function,the two major of CD4+Th cells,termed Thl and Th2.These Th2 cells and their signature cytokines were thought to play a key pathogenic role in asthma.The therapeutic approach to the balance of Thl/Th2 in asthma attracted a lot of attentions.Cytokines play a pivotal role in the development of CD4+Th cells, and the current studies have implied that many signaling pathways including JAK/STAT,MAPK,Rho/Rock,PI3K play a important role in the pathogenesis of asthma via mediating those cytokines actions.Rencently studies have revealed that cytokine-mediated signals were primarily transduced by the JAK/STAT signaling cascade,the JAK/STAT signaling played a key role in asthma.A deep research in the signaling pathways may be helpful to the understanding of the mechanisms of asthma and novel treatment approach.IL-6 was a pleiotropic and proinflammatory cytokine that played a important biological role in inflammation,immunity,proliferation and differentiation of cells.IL-6 had a close link to the inflammatory diseases such as asthma,and IL-6 also contributed to the repair and fibrosis of tissues.IL-6 elicits cellular actions by binding to the membrane-bound IL-6 receptor(mIL-6R) or soluble IL-6 receptor,then recruiting 2 membrane-spanning glycoprotein130 (gp130) molecules into tetra-or hexametric structures, thereby forming the active IL-6R complex, Homodimerization of the 2 gp130 molecules leads to intracellular activation of the nonreceptor protein tyrosine kinases Src and JAK as well as members of the STAT family of transcription factors,all of IL-6 signal through signal transducers and activators of transcription 3(STAT3). STAT3 was another cytoplasmic peptide belonging to the STAT family,with diverse roles in biological processes including cell proliferation,survival,apoptosis and inflammation.It was activated by a large number of extracelluar stimuli including IL-6,various other cytokines, Granulocyte clony-stimulating factor,epidermal growth factor and IL-10. STAT3 was identified initially as an acute phase response gene in the liver and had a pivotal role in directing inflammatory responses by inducing the gene expression of cytokines, chemokines, and adhesion molecules. Determining the role of STAT3 in mediating inflammatory responses had been hindered by the fact that knocking out STAT3 results in embryonic lethality, STAT3 also plays a role in dendritic cell function and T cell survival,thus those studies implied its role in governing inflammation and immune responses. Compared with wild-type mice, a significant decrease in HDM-induced airway inflammation and AHR was seen in a STAT3 conditional knockout (KO) mice in the airway epithelium, the study demonstrated that airway epithelial STAT3 was essential for the development of chronic asthma. Ammit and colleagues had investigated that IL-6 increased eotaxin and VEGF release to signal through STAT3 via IL-6 trans-signaling in ASM,and has relevance to the development of airway remodeling. Finntto suggest that IL-6/STAT3 signaling pathway in the lung controled the critical balance between effector and regulatory T cell function,thus the inhibition of the IL-6 receptor signal may be a novel therapeutical target.Although the current studies had highlighted the pivotal role of IL-6/STAT3 signaling pathway in asthma,but much less is known about the expression and activation of IL-6/STAT3 in airway,and the role of IL-6/STAT3 signaling pathway in the pathophysiology of allergic asthma still remains unknown.Therefore a further research are reguired,the present study aim to explore the role of IL-6/STAT3 signaling pathway in asthma via a mouse model of allergic asthma,we analyzed the association between IL-6,STAT3 and related index,and investigated the effects of AG490 and BUD intervening on the airway inflammation and remodeling.Part 1:Expression and Activation of IL-6/STAT3 in Airway of Asthmatic Mouse Model and its Relationship with Airway InflammationObjective:To study the expression and activation of IL-6/STAT3 in airway of asthmatic mouse model and its association with airway inflammation, To explore the role of IL-6/STAT3 signaling pathway in allergic asthma.Methods:30 Balb/c mice were randomly divided into control group (n=10) and asthma group(n=10) and AG490 group(n=10). The mice were sensitized with ovalbumin to establish the asthmatic model. The histological changes were evaluated by HE staining, Bronchoalveolar lavage fluid (BALF) were collected,the total cell and the cell differentials were counted,the level of IL-4,IL-5and IL-6 in BALF were measured by ELISA. The expression of STAT3 and TARC in airway were detected by immunohistochemistry technique;lung tissue extracts were analyzed for total STAT3 and p-STAT3 by Western blot.SPSS13.0 software was used to analyze the data.Results:1 The histological changes by HE staining show that typical inflammatory changes in asthma group compared with AG490 group, The total cells,EOS amounts and levels of cytokine in BALF of asthma groups were significantly higher than in AG490 group (P< 0.05).2 The expression of STAT3,TARC and p-STAT3 in asthma group were significantly higher than in controll group(P< 0.05), The level of TARC and p-STAT3 in AG490 group were significantly lower than in asthma group (P< 0.05),3 The level of STAT3 in airway and level of p-STAT3 in lung tissue had positive correlations with IL-4,IL-5 and IL-6 in BALF(P< 0.05).4 There were close correlation between the expression of TARC and STAT3 in the airway epithelium via nonparametric rank correlating analysis.Conclusions:The expression and activation of STAT3 in airway were upregulated in OVA-induced experimental asthma model, its level were associated with Th2-biased airway Inflammation and increament of IL-6 in airway, inhibiting signal pathway of airway STAT3 can ameliorate airway allergic inflammation, IL-6/STAT3 signaling pathway of airway may paly an important role in the chemotaxis and recruitment of Th2 type Cells by up-regulation of airway epithelial TARC expression.Part 2:The Role of Airway IL-6/STAT3 Signaling Pathway in Airway Remodeling in a Mouse Model of Allergic AsthmaObjective:To study the expression and activation of IL-6/STAT3 in airway of asthmatic mouse model and its association with airway remodeling,the changes after inhibition the activation of STAT3, In order to explore the role of IL-6/STAT3 Signaling pathway in airway remodeling.Methods:30 Balb/c mice were randomly divided into control groups (n=10) and a sthma groups(n=10) and AG490 groups(n=10). The mice were sensitized with ovalbumin to establish the asthmatic model.The histological changes were evaluated by HE staining and masson's trichrome staining,Standard total brochial wall thickness(Wat/Pi)and Standard smooth muscle thickness(Wam/Pi),Standard smoth nucleus counts(N/Pi),the percentages of collagen deposition(Fib%) were measured by image analysis system. The level of IL-4,IL-5 and IL-6 in BALF were measured by ELISA.The expression of STAT3 in airway were detected by immunohistochemistry technique;lung tissue extracts were analyzed for total STAT3 and p-STAT3 by Western blot.SPSS13.0 software was used to analyze the data.Results:1 The histological changes of airway remodeling were significantly different between the three groups; 2The level of Wat/Pi,Wam/Pi,N/Pi,Fib% were significantly higher in asthma groups than in control groups, While the level of Wat/Pi,Wam/Pi were significantly lower in AG490 groups compared with asthma groups (P<0.05),3 The level of total cells,EOS,IL-4,IL-5 in the BALF of asthmatic mice had positive correlations with Wat/Pi,Wam/Pi,N/Pi (P<0.05), and The level of IL-6 and p-STAT3 in lung tissue had positive correlations with Wat/Pi,Wam/Pi,N/Pi (P< 0.05) respectively.Conclusions:The expression of IL-6 and STAT3 were up-regulated and constitutive activated,which were significantly correlated with airway remodeling,Inhibition the activation of STAT3 can ameliorate airway remodeling. IL-6/STAT3 signaling pathway may paly an important role in airway remodeling in asthma.Part3:Effects of Inhale Budesonide in Early Phase on the Airway Inflammation,Airway Remodeling and IL-6/STAT3 Signaling Pathway in AsthmaObjective:To investigate the effects of inhale budesonide (BUD) in early phase on the airway inflammation and remodeling in asthmatic mouse,and its effects on the IL-6/STAT3 signaling pathway in airway,in order to explore the therapeutic target of budesonide.Methods:30 Balb/c mice were randomly divided into control groups (n=10), asthma groups(n=10), budesonide groups(n=10) and AG490 groups(n=10). The mice were sensitized with ovalbumin to establish the asthmatic model. The histological changes were evaluated by HE staining and masson's trichrome staining.The level of IL-4,IL-5 and IL-6 in BALF were measured by ELISA. The expression of STAT3 in airway were detected by immunohistochemistry technique;lung tissue extracts were analyzed for total STAT3 and p-STAT3 by Western blot.SPSS13.0 software was used to analyze the data.Results:1 The level of inflammatory cells and Th2 cytokines in the BALF of BUD groups were significantly lower than asthma groups (P< 0.05),there were no significant difference compared with AG490 groups.2 The parameters of airway remodeling in BUD groups were significantly different from those in asthma group(P<0.05).3 The level of IL-6 and STAT3 in airway in BUD groups were significantly lower than asthma groups (P<0.05) 4 The level of total STAT3 and p-STAT3 in the lung of BUD groups were significantly lower than asthma groups (P<0.05).Conclusions:Inhaled corticosteroid (ICS) in early phase could downregulating the expression of IL-6 and STAT3 in airway,inhibiting the activation of STAT3 in lung via asthmatic mouse model. The IL-6/STAT3 signaling pathway of airway may be one of the potential therapeutical targets of ICS.