Effects Of Azithromycin On Th2 Cytokines And Expression Of STAT5, STAT6 In Asthmatic Mice

Background and objective: Asthma is a complex inflammatory disease characterized by mucus hypersecretion, variable airway obstruction, airway hyperresponsiveness (AHR) and airway reconstitution. The mechanism of asthma is complex and many of which are not well understood. Bronchial asthma is the chronic and inflammatory disease that takes place in the airway. The whole process involves many cells such as eosinophil, macrophages, mast cell, T lymphocyte, neutrophils, epithelial cell and cytokines. Eosinophil(EOS)infiltrating in the respiratory tract play a central role in the pathogenesis of asthma. The researches indicate that the imbalance of normal ration and function of Thl/Th2 is the pathogenic mechanism of bronchial asthma. The over activated and proliferative Th2 cells produce Interleukin 4,5(IL-4,IL-5 )is the major reason of asthma. While the activation of signal transducer and activator of transcription (STAT) is the key processes that cytokines produce biologic effects. STAT5,STAT6 are the members of STAT family, IL-4/STAT6 pathway is closely associated with the pathogenesis of asthma. It can regulate Th2 dominant response, promote the differentiation of B lymphocyte and IgE class switch, and induce the airway inflammation and airway hyperresponsiveness. IL-5/STAT5 pathway can induce persistently pluripotential hematopoietic stem cells (HSCs) in bone marrow to differentiate into mature eosinophils and promote eosinophils proliferation which is the major reason of eosinophils increase. Low-dose erythromycin therapy has been accepted as an effective therapy for diffuse panbronchiolitis. Mocrolides (erythromycin, azithromycin, roxithromycin, clarithromycin) which were used in the therapy of asthma showed a particular anti-inflammation effect. But the precise mechanisms in the therapy of asthma remain unclear; several studies have suggested that the beneficial effects are due to nonspecific anti-inflammatory and immunoregulation action. To further study the mechanism of azithromycin(AZM) in treating asthma and find a new way of preventing and curing asthma, the asthmatic mice models were established by Ovalbumin(OVA) and treated with different concentration of azithromycin and the effects of azithromycin on airway inflammation and Th2 cell cytokines IL-4,IL-5 in BALF as well as expression of STAT5,STAT6 in lung tissue were observed.Methods: BALB/C mice were randomly divided into five groups: group A (asthmatic model group, n=8); group B (low dose AZM treated group, n=8); group C (moderate dose AZM treated group, n=8); group D (high dose AZM treated group, n=8); group E (control group, n=8). Mice in group A,B,C,D were respectedly sensitized on days 0,13 by Ovalbumin (OVA) 20ug together with aluminum hydroxide[AL(OH)3] 1mg and challenged from 21-30 by inhalation of 2%OVA to establish a murine model of asthma .On days 15 to 30, the animals in group B,C,D were intraperitoneal treated respectedly with lower dose AZM(50mg/kg per day), moderate dose AZM(100mg/kg per day), higher dose AZM(150mg/kg per day). Group E was sensitized and challenged by 0.9%NaCl. All the animals were killed on day 31. The BALF was collected and the paraffin sections of pulmonary tissues were made. The level of IL-4, IL-5 in BALF was determined by ELISA and the total WBC and EOS were counted. The expression of STAT6, STAT5 in lung was analyzed by immunohistochemistry.Results:1.The total WBC and the numbers of eosinophils in BALF of asthmatic model group (group A) were significantly increased comparing with the control group(p<0.01); After the treatment of AZM , the total cell counts and the numbers of EOS in BALF of the moderate dose AZM treated group and high dose AZM treated group were decreased comparing with the asthmatic model group (p<0.01); But the low dose AZM treated group in the total cell counts and the numbers of EOS were not different comparing with asthmatic model group.(p>0.05)2. The level of IL-4 and IL-5 in BALF of asthmatic model group were significantly increased comparing with the control group (p<0.01). the moderate dose AZM treated group and high dose AZM treated group caused notably decrease in the level of IL-4 and IL-5 in BALF comparing with the asthmatic model group (p<0.01), but the lower does AZM treated group in the level of IL-4 and IL-5 was not different comparing with asthmatic model group.(p>0.05).3. Comparing with control group, the bronchial mucous walls destroyed partly and inside the bronchial lumen were filled with a little mucus as well as inflammatory cells infiltration, including lymphocytes and eosinophils were observed around airway and vessels in asthmatic model group. While the inflammatory reaction in AZM group treated with moderate dose and high dose was relieved. 4. Expression of STAT5, STAT6 was rarely seen in lungs in control group, while the expression of STAT5, STAT6 was obviously increased in asthmatic model group. The expression of STAT5, STAT6 decreased notably after the treatment of AZM with 100mg/kg per day and 150mg/kg per day.5. After the last challenging, in group A, the level of IL-4, IL-5 in BALF is significant positive correlate with the numbers of EOS (r=0.955, p<0.01), (r=0.965, p<0.01).Conclusion:1. Asthmatic animal models with mice had been successfully established by Ovalbumin.2. The basic pathologic change in asthma is characterized with airway chronic inflammation, especially EOS inflammation .The over activated Th2 cytokines (IL-4, IL-5) closely associated with the pathogenesis of asthma. IL-4/STAT6 and IL-5/STAT6 pathways play an important role in the pathogenesis of asthma.3. AZM can suppress the inflammation of airway in asthmatic model. The mechanism may be explained by that AZM could down regulate the expression of STAT6,STAT5 and inhibit the response of Th2, so that it could decrease the airway inflammation.