| Objective: Asthma is a chronic disease characterized by variable airway obstruction, airway hyperresponsiveness(AHR) ,and airway allergic inflammation associated with several inflammatory cells and mediators. The signal transducer and activator of transcription 6 (STAT6) is an obligatory transcription factor in interleukin (IL)-4 and IL-13 signaling, and contributes to the differentiation of naive T cells to the Th2 subtype and immunoglobulin(Ig)E isotype switching in B cells.STAT6 is therefore a key effector in allergic disease and may be central to the pathophysiology of asthma. In this experiment, in order to reveal the role of STAT6 in pathophysiology process of asthma and provide a new target and method for asthma treatment, we observed the STAT6 expression changes in OVA-sensitized mice and in dexamethasone-treated mice, investigated the role of STAT6 in IL-4 secretion of murine spleen lymphocytes by antisense technology in vitro, and then, treated OVA sensitized mice with antisense medicine by intranasal administration. Methods and Results: 1,The expression of STAT6 in lung of OVA-sensitized mice and the effect of dexamethasone treatment. A murine asthma model was induced previously by administering OVA intranasally or dexamethasone, then lungs, were analyzed by histology and the expression of STAT6 mRNA was detected by semi-quantitive RT-PCR. As a result, , an infiltration of inflammatory cells, including eosinophil, lymphocyte, neutrophil around bronchial, vessel and interstitium tissue of lung was observed in untreated OVA-sensitized mice. In contrast, inflammatory cells infiltration was lessened in lung tissue of dexamethasone-treated mice. Compared with control mice , STAT6 mRNA increased ( 2.46±0.24 vs 1.41±0.26 , P<0.01 ) in OVA-sensitized mice. After dexamethasone intervention,STAT6 mRNA level dropped to near the controls(1.80±0.36 vs2.46±0.24,P<0.01)。2,In vitro study for the effect of STAT6 on murine spleen lymphocytes. We synthesized a 20-mer phosphorothioate antisense oligonucleotide (ASODN) of STAT6, and transfected them to murine spleen lymphocytes with cationic liposome (Geneshuttle -20)/oligonucleotide complexes. The uptake and intracellular distribution of FAM-labeled STAT6 antisense oligonucleotide were detected by flow cytometry and fluorescence microscope. ASODN/ Geneshuttle-20 ( W/W ) 2:4 complexes were cocultured with murine spleen lymphocytes, ,the expression of STAT6 was measured by Western-blot and RT-PCR respectively.IL-4 levels in culture supernatants of all groups were detected by ELISA. As a result, Cationic liposome enhanced murine spleen lymphocytes taking antisense oligonucleotides by 67.7%,six times than the controls,and the fluorescence intensity in cytosol and nuclear increased. After STAT6 ASODN/Geneshuttle-20 complexes transfection, The expression of STAT6 protein and mRNA was decreased,and IL-4 levels in culture supernatants were lower than the controls(22.6±5.5 pg/ml vs 33.2±6.1 pg/ml).After 24 hour culture, the levels of STAT6 mRNA and protein decreased to 33.8% vs 58.3%. From then, the levels of STAT6 mRNA and protein increased. 3,In Vivo study for the effect of STAT6 ASODN on OVA-sensitized mice OVA-sensitized mice were given STAT6 ASODN or STAT6 ASODN and dexamethasone complex by intranasal administration ,then we observed the lung tissue pathology and distribution of FAM-labeled STAT6 ASODN in lung, The expression of STAT6 protein and mRNA was detected by immunohistochemistry, Western -blot and RT-PCR respectively. Bronchioalveolar lavage fluid(BALF) was collected for counting eosinophils and analyzing content of IL-4 by ELISA.As a result ,FAM-labeled STAT6 ASODN distributed mainly in lung interstitium and airway epithelium , the expression of STAT6 protein and mRNA was down-regulated. After STAT6 ASODN intervention, the infiltration of inflammatory cells in lung was lessened,the number of eosinophils and the content of IL-4 in BALF decreased(67.8±9.4pg/ml vs 89.5±12.1pg/ml P<0.01).After STAT6 ASODN and dexamethasone complex treatment,...
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