| BackgroundBronchial asthma (asthma) is one of common and frequently-occurring diseases in the respiratory system, and it’s a great threat to human health. According to the statistical results of the world health organization, there are about300million asthma patients all over the world. About fifteen million people lose labor ability because of this disease every year. According to incomplete statistics, there are at least15million to20million asthma patients in our country.The morbidity and mortality of bronchial asthma all over the world are increasing year by year, this kind of situation causes enormous economic losses every year, asthma has become a serious public health problem. Asthma episodes frequently and the cause is complex. Under the interaction of host susceptibility and environmental factors,bronchial asthma is a syndrome which basic pathophysiological change is chronic airway inflammation and airway remodeling.It’s a kind of disease that can be controlled but difficult to cure. Now, more and more researchers study about asthma’s pathogenesis,do their best to prevent and control the allergic inflammation and the development about asthma. In the development of airway inflammation in asthma, the naive T cells in CD4+T cells become ThO cells by the allergen-induced activation. ThO cells continue to differentiate in the direction of the Th2cells and generate a lot of Th2cells. Th2cells in the dominant status, they can induce Th2inflammation on a large scale.,the inflammation mediate by the downstream of the IgE and/or not the IgE.In the onset process of asthma, the infiltration, aggregation, activation and release of inflammatory cells in airway tissue are the main features, cytokines play an important role in the process. The repeated stimulation of chronic inflammation and the destruction of the epithelial structure can cause asthma airway remodeling. At present, the airway inflammation and airway remodeling in asthma as two pathological features are gradually accepted by people. Various cytokines and inflammatory mediators can lead to the airway remodeling.but the mechanism is not fully clear.Interleukin21(IL-21) is a cytokine found in2000, belongs to a type I cytokine family, mainly secreted and synthesed by CD4+Th2cell, and have homology with IL-2, IL-4and IL-15. Since IL-21is found, about its biological functions has become a research hot spot. The effect of IL-21is various, mainly due to IL-21receptors are widely distributed. IL-21is considered to be secretion of cytokines activated by peripheral blood T cell, now be considered to be synthesized from a series of differentiated CD4+Th cells. Biological functions mainly to stimulate T cell proliferation, NK cell proliferation and differentiation and CD40specific response of B cell proliferation. IL-21receptors (IL-21R) express in lymphatic hematopoietic cells, fibroblasts, keratinocytes, intestinal epithelial cells. Experiments show that IL-21plays an important role in innate immunity, adaptive immunity and cellular immune response.IL-21is associated with a variety of autoimmune diseases and inflammatory diseases, such as rheumatoid disease, systemic lupus erythematosus, systemic sclerosis disease, inflammatory bowel disease, allergic disease, it plays an important role in immune regulationfor these diseases.IL-21plays a key role in Th cell differentiation, bronchial asthma is a kind of allergic disease caused by immune abnormalities, immune dysfunction plays an important role in asthma incidence.T cell differentiate in different direction to determine whether asthma disease occurs or not, Th1/Th2imbalance and imbalance of Th17/Treg theory have always been research hot spot. Th1/Th2cells are in a state of reciprocal inhibition. Under normal circumstances, Th1/Th2cells in a constant state, but in patients with asthma,the Th1cells function decline and the function of Th2cells increase abnormally. When the mumber of treg cells decline, the inhibition of inflammatory response function is reduced.The overexpression of Th17cells mediate airway inflammation. IL-21is a cytokine secreted by Th2CD4+cells, can promote the secretion of Th17cells, some people think that Th17cells are an important generation source of IL-21, thus infer that IL-21may involved in the pathogenesis of asthma disease. In2009, Rajshekhar Chatterjee for the first time reported IL-21gene polymorphism was significantly associated with asthma.The functions of IL-21depend on its receptor shared gamma chain, IL-21mainly transmit signal through JAK-STAT signal pathway. IL-21R protein is composed of538amino acids, and have homology with IL-2R beta chain, IL-4R alpha chain and IL-9R, N side has four highly conservative Cys residue, transmembrane region has "WSXWS sequence, cytoplasm area exists classic signaling subunits boxl and box2motif, show that the receptor has signaling function.IL-21combines with IL-21R,mainly through coupling with gamma c, after that,they activate the JAK1and JAK3(especially JAK3), activation of the JAK tyrosine kinase phosphorylate STAT1and STAT3, phosphorylation STAT transfers to the nucleus, have the effect of activating T cell nucleus factor (NFAT) and induce expression of IL-21gene. JAK-STAT signal participates in transduction pathways of many cytokines, influences Th cell differentiation and Th cell immune deviation,it’s an important signaling pathways in asthma. But the expression of IL-21and IL-21R in lung tissue of asthmatic mice is not reported. We set up animal model of chronic asthma mice and observe the expression of IL-21,IL-21R, STAT3, p-STAT3in lung tissue of asthmatic mice, intervene the asthmatic mice by using dexamethasone and tyrosine kinases (JAK, janus Kinnase) inhibitors AG490.We study the influence of IL-21on asthmatic airway inflammation and airway remodeling, discusse the mechanism of IL-21/STAT3signal pathway in the pathogenesis of asthma.Objectives1To observe the expression of IL-21and IL-21R in lung tissue of mice and asthmatic mice with different experimental methods, observe the relationship between IL-21, its receptor and airway inflammation, airway remodeling.2To observe the expression of STAT3and activation of STAT3(p-STAT3) in asthmatic mice,observe the relationship between these index and airway inflammation, airway remodeling.3After intraperitoneal injection of JAK pathway inhibitor AG490,To observe the changes in asthma airway inflammation and airway remodeling index, and observe the expression changes of total STAT3and p-STAT3in lung tissue.4To observe the changes of airway inflammation in asthma mice and the influence on IL-21/STAT3signal pathways by using of dexamethasone to intervent the asthmatic mice.Methods1The establishment and group of animal models44SPF BALB/C female mice were randomly divided into4groups, control group, asthma group, AG490group and dexamethasone group,11mice each group. In asthma group, mice were sensitized by means of intraperitoneal injection of OVA (10μg) precipitated with aluminum hydroxide (100μg) on days1and14,from21st day,25g/L of OVA solution were atomized inhalation for30min, three times a week for8weeks. The control group with normal saline instead of OVA. In dexamethasone group,0.5h before aerosolized OVA, each mouse was given10mg/kg dexamethasone by means of intraperitoneal injection. In AG490group,each mouse was injected AG490, per mouse450ug/each time, three times a week, every5mg AG490with0.5mL dimethyl phosphite maple (0.05%),9.5mL dual steaming water dissolves.2The expression of IL-21and IL-21R in lung tissue of chronic asthma miceTweenty-four hours after the last atomization inhalation, the left lungs were lavaged, the bronchoalveolar lavage fluid(BALF) were collectted. The mice were sacrificed,right lung tissue were putted in liquid nitrogen tank that can be used to extract total RNA and protein. Left lung tissue paraffin embedding sectioning lines for HE staining.The expression of IL-21and IL-21R in the lung tissue were analyzed by immunohistochemical method. The mRNA expression of IL-21and IL-21R were detected by fluorescent quantitative PCR method.Western blotting method were used to detect the protein expression of IL-21and IL-21R.3The relationship between IL-21,IL-21R and airway inflammation,airway remodeling.After centrifugation of alveolar lavage fluid, collected sediment to classifyt and count the cells by blood cells count plate.Observed mainly the total amount of cells and the count of eosinophils and neutrophils. Pathology change of airway and lung tissue were observed by HE staining. After HE staining,we used IPP image analysis software6.0to analyse unit length basement membrane airway wall area (airway wall thickness, total bronchial wall area/basement membrane perimeter, WAt/Pbm), area per unit length basement membrane of airway smooth muscle (airway smooth muscle thickness,the area of the wall occupied by smooth muscle/basement membrane perimeter, WAm/Pbm). The correlation between the index of IL-21,IL-21R and WAt/Pbm,WAm/Pbm were analysed.4The expression of STAT3and p-STAT3in chronic asthmatic mice and the changes after the intervention with AG490(JAK pathway inhibitor)The protein expression of p-STAT3in lung tissue were detected by western blotting and immunohistochemical methods.The expression of STAT3were observed by fluorescence quantitative PCR. After using AG490,we observed the expression changes of STAT3and p-STAT3, the changes in the airway inflammation and airway remodelling in mice.5The airway remodeling changes and the index of IL-21,IL-21R and p-STAT3after the intervention of dexhmesoneTo analyse the airway wall thickness (WAt/Pbm) and smooth muscle thickness (WAm/Pbm) in dexamethasone intervention group by using IPP6.0image analysis software. Immunohistochemical method and western blotting method were used to detect the protein expression of IL-21, IL-21R and p-STAT3in lung tissue of dexamethasone group. The expression of IL-21mRNA and IL-21RmRNA in the lung tissue were detected by fluorescent quantitative PCR method.6Statistical processingAll data were expressed as means±SD. All statistical analysis was operated with SPSS16.0statistic software.Difference among two independent groups was analyzed by t-test. Differences among one-way designed groups were analyzed by one-way ANOVA followed by Bonferroni test. When homogeneity of variance can not achieve, multiple comparisons were analyzed by Dunnett’s T3test. Correlations were analyzed by Pearson test. A value of P<0.05was considered statistically significant. Results1.44mice were all alive, after OVA sensitized/challenged,the mouse model of chronic asthma was successfully established.Control group mice had no obvious abnormal reaction after atomization, asthma group of mice appeared symptoms such as dysphoria, shortness of breath, abdominal muscle twitching, mainland incontinence, and could be heard the wheezing sound with a stethoscope auscultation. Dexamethasone group and AG490group had similar but significantly gentle symptoms as asthmatic group. The lung tissue by HE staining seen microscopically conformed to the pathological features of asthma.Around bronchioles and vessels,there were a large number of inflammatory cells infiltration in asthmatic mice.Airway wall and lung tissue visible was priority to eosinophilic granulocyte and lymphocyte of inflammatory cell infiltration, bronchial wall thickening and luminal stenosis, bronchial lumen mucous plug.2.The results showed that IL-21and IL-21R were all expressed in lung tissue of asthma group and control group by immunohistochemical method.Compare with the control group, the expression of IL-21and IL-21R enhanced in asthma group (P<0.05), and the result was consistent with the means of western blot.It also showed that the mRNA expression of IL-21and IL-21R in asthma group were much more than control group (P<0.05). The index of WAm/Pbm and WAt/Pbm about airway remodeling in asthma mice were higher than the control group (P<0.05).3.In group of asthma, AG490and dexamethasone, the total number of cells, neutrophils, acidophilic granulocyte and lymphocyte count in alveolar lavage fluid were significantly increased compared with normal control group (P<0.01). Compared with the asthma group, the total number of cells, neutrophils, acidophilic granulocyte and lymphocyte count in dexamethasone treatment group were significantly reduced (P<0.01), the index in AG490group also fell comaring with asthmatic group (P<0.05).Compared AG490group with dexamethasone group, total number of cells, eosinophils, lymphocytes and neutrophils in alveolar lavage fluid in dexamethasone group were lower than AG490group (P<0.05).4.Immunohistochemistry and western blot analysis showed that the control group almost without the protein expression of P-STAT3.In contrast to control group, increased protein expression of P-STAT3wer detected in lung tissue (P<0.05).The expression of P-STAT3protein in lung tissue of AG490group were weaker than in the asthma group (P<0.05).Compared to the control group Fluorescence quantitative PCR showed that the expression of STAT3mRNA in asthma group and AG490group increased (P<0.05), between the two groups, there were no significant differences(P>0.05).5.The index of WAm/Pbm and WAt/Pbm about airway remodelling in dexamethasone group was higher than the control group (P<0.05), lower than the asthma group (P<0.05). The protein expression of IL-21, IL-21R and p-STAT3in the lung tissue of dexamethasone group declined compared with the asthma group by using western blot method (P<0.05).The expression of IL-21in AG490group fell compared with the asthma group (P<0.05).No statistical significance were observed between the AG490group and dexamethasone group (P>0.05). For the protein expression of p-STAT3,there was no statistical significance between AG490group and dexamethasone group (P>0.05). Compared to control group,the level of STAT3mRNA in lung tissue of AG490group increased(P<0.05). However, there was no statistical significance between asthma group and AG490group (P>0.05).6.Correlation analysis:The protein expression of IL-21, IL-21R, p-STAT3were detected by using western blot method, the results showed that these indicators and the total number of cells, eosinophil count in the mice alveolar lavage fluid were positively correlated (r>0.5, P<0.01), the protein expression of IL-21, IL-21R,p-STAT3and the index of WAm/Pbm, WAt/Pbm were positively correlated (r>0.5, P<0.01).ConclusionsIL-21mainly transmit signal through the JAK-STAT pathway, the experiment firstly used different methods to observe the expression of IL-21and its receptor in the lung tissue in control group and asthma group. The expression changes of STAT3and activation of STAT3in lung tissue of mice were observed. After the intervention of JAK pathway inhibitors AG490and dexamethasone, the index changes were observed. Draw the following conclusions:1.IL-21and its receptor express in the lung tissue of mice. Compare to the control group, the expression in asthmatic group is increased.IL-21is an important proinflammatory factor in the incidence of asthma. IL-21that combines with it’s receptor involve in the onset of chronic asthma and participate in the process of airway remodeling.2.STAT3and activation of STAT3involve in the pathogenesis of asthma, may affect the airway remodeling.3.After the intervention of AG490in asthma model, the expression of p-STAT3is reduced, degree of airway inflammation and airway remodeling is reduced, its curative effect are roughly equal to dexamethasone.4.IL-21involve in the onset of asthma disease by activating STAT3signaling pathway, dexamethasone downgrade the expression of IL-21and its receptors, inhibit the activation of STAT3in the lung tissue, relieve the airway inflammation and airway remodeling, IL-21/STAT3signal transduction pathway may be one of the targets of dexamethasone in treatment of asthma. |
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