| Bronchial asthma (bronchial asthma)are the most common childhood chronicrespiratory diseases, which main features include airway hyperresponsiveness,airflow limitation and lung function reduction. The pathogenesis of asthma iscomplex, which associated with immune, environment and gene. The pathogenesis ofasthma remains unclear now, but the imbalance between Th1/Th2subsets andfunctional imbalance still is currently accepted immunological pathogenesis.Signaling pathway of non-receptor tyrosine kinase/signal transducer and activator oftranscription is an important intracellular signal transduction pathway. IL-4, IL-13cytokines and other inflammatory mediators may play a biological role mediated bythe road. Signal transduction and activator of transcription (STAT) proteins familywas a group of cytoplasmic proteins, after phosphorylation and poly modificationenter the nucleus, combined with a specific response element, they can activate targetgenes and regulate the related gene transcription. STAT6is a member of the STATfamily, which plays an important role in the development of asthma. STAT6is a Th2cell-specific transcription factor, which activated IL-4, IL-13and other cytokines topromote the proliferation of Th2cell differentiation and lead to hyperthyroidism.Orosomucoid like3(ORMDL3) has been strongly linked with asthma in geneassociated studies in2007. Its encodes which consists of153amino acids on thetransmembrane protein localized in the endoplasmic reticulum (endoplasmic reticulum, ER). Although ORMDL3is asthma susceptibility genes, but itsrelationship with airway remodeling is still not clear. In this study, we establishedairway remodeling model and apply it to budesonide intervention therapy, thenobserve the airway remodeling improvement, the expression of STAT6protein andmRNA, ORMDL3mRNA, to investigate the possible mechanisms involved inasthma.ObjectiveUse ovalbumin stimulate and challenge the mice to establish airway remodelingmodels, application budesonide suspension atomization intervention. Observed thecases of thickness of airway, inflammatory cells and subepithelial fibrosis. Measurethe expression STAT6mRNA and protein, ORMDL3mRNA in the lung, and theeffects of budesonide intervention on its expression.Materials and Methods1Experimental mice groups and asthma model preparation30BALB/c mice feed them one week to adapt to the environment, and thenrandomly divided into control, asthma and Budesonide intervention groups. The micewere sensitized and challenged with ovalbumin to establish asthma model. The BUDintervention group received inhalation of Budesonide（1mg)30minutes before eachOVA challenge. The control group use isodose normal saline supersede OVA mixture.2Lung tissue samples collectedEach group of mice were within24h after the end of the last challenge, deaththem and open the chest, then fully exposed the lungs. The left lung stored at-80℃refrigerator for ELISA and RT-PCR detection, and then external fixate the right lungtissue by the4%formaldehyde solution, and embedded in paraffin, cut consecutivelyinto3μm thickness slice for hematoxylin-eosin (HE) staining, Masson three staining(Masson) and immunohistochemical staining. 3HE staining and Masson stainingAfter dewaxing sections were stained with HE staining and Masson staining, andunder the microscope to observe the change of airway wall structural, the skin fiberdeposition and infiltration of inflammatory cells infiltration condition. Measured thebronchial wall thickness which the same level.4Immunohistochemical stainingUse the SP method for immunohistochemisty staining. Dewaxing, dehydration,antigen retrieval, endogenous peroxidase closed, dropping an anti,4℃overnight,dropping two anti, three anti, DAB coloration, hematoxylin stain again, hydrochloricacid and alcohol differentiation, concentration gradient ethanol dehydration,transparency, mounted. Observed the express of STAT6protein under a microscope athigh magnification (10×40) vision. Choose to take five high-power fields for eachspecimen. Use medical image analysis system to measure protein-positive area, theresult is represented by optical density per unit area (IOD).5IL-13levels of lung tissue were detected by ELISAGrind the lung tissue which stored at-80℃into homogenates, according to kitinstructions measure the levels of IL-13in lung tissue.6RT-PCRTake the lung tissue (100mg) from the-80℃refrigerator,extracting the totalRNA from lung tissue by Trizol method. Add the reverse transcription reagents andreverse-transcribed them into cDNA. Designed primers and amplified gene fragment.The amplification product was subjected to electrophoresis on a1.5%agarose gel,and by the gel electrophoresis imaging system to determine of the target geneSTAT6,ORMDL3and their ratio of the grey value of GAPDH as the mRNAexpression levels.7Statistical analysisApplication SPSS17.0statistical package for analysis of the experimental data. use mean±standard deviation(X S) to express the measurement data, Thedifferences between groups was analized by ANOVA, pairwise comparisons usingLSD-t method, correlation using pearson correlation analysis,=0.05as the lever ofthe test.Results1The pathomorphology of each group of miceIn ordinary optical microscope at high magnification（10×40）observation.Control group:the airway wall thickness and the arranged of the epithelial cells werenormal,and a small amount of inflammatory cell infiltration. Asthma group:the airwaystructural disorder, which include bronchial wall thickening, goblet cell metaplasia,mucus secretion, collagen fibers and a large number of inflammatory cell infiltration.BUD group: the airway structural disorder cases better than the asthma group, and theinflammatory cells less than the asthma group. According to the results of ANOVA: F=109.43, P <0.05, three groups of mice airway wall thickness difference wasstatistically significant. Further pairwise comparison, the airway wall thickness of theasthma group(160.50±21.38μm) was higher than the BUD group(84.50±15.45μm)and the control group(48.70±9.84μm), and the BUD group is higher than the controlgroup, the difference was statistically significant.2Immunohistochemical results of mice in each groupSTAT6protein were expressed in lung tissue of the three groups. Its mainlydistributed in the cytoplasm of epithelial cells and inflammatory cells, showing brownparticles. According to the results of ANOVA: F=66.118, P<0.05, the difference ofthe express of STAT6protein in the three groups were statistically significant. Furtherpairwise comparison, the STAT6protein of the asthma group (123.40±19.60) washigher than the BUD group(80.00±10.43) and the control group(49.30±11.59), andthe BUD group is higher than the control group, the difference was statisticallysignificant. 3IL-13levels in lung tissue of mice in each groupIL-13were expressed in lung tissue of the three groups. According to the resultsof ANOVA: F=416.988, P <0.05, the difference of the express of IL-13in the threegroups were statistically significant. Further pairwise comparison, the expression ofIL-13in the asthma group (56.00±2.6ng/mgpro) was higher than the BUD group(37.20±2.7ng/mgpro) and the control group(26.40±1.5ng/mgpro), and the BUD groupis higher than the control group, the difference was statistically significant.4RT-PCR resultsThe results of RT-PCR was that STAT6mRNA and ORMDL3mRNA wereexpressed in the three groups. The two bands in the asthma group were strongestbrightness, followed by the BUD group, the control group is the weakest. Accordingto the results of ANOVA: F=92.472, P<0.05and F=290.457, P<0.05, the levels ofSTAT6mRNA and ORMDL3mRNA in the three groups of mice lung tissue werestatistically significant differences. Further pairwise comparison, the STAT6mRNAand ORMDL3mRNA of the asthma group[(0.78±0.08) and (0.340±0.032)] werehigher than the BUD group [(0.52±0.05) and (0.194±0.026)] and control group[(0.38±0.07) and (0.075±0.013)], and the BUD group is higher than the control group,the difference was statistically significant.5Correlation AnalysisPearson correlation analysis shows that the expression of STAT6mRNA in theasthma group was positively correlated with ORMDL3mRNA（r=0.676, P=0.032）,and the expression of STAT6mRNA in asthma group was positively correlated withthe airway wall thickness（r=0.738，P=0.015）, the expression of ORMDL3mRNAin asthma group was positively correlated with the airway wall thickness（r=0.668，P=0.035). Conclusions1. STAT6and ORMDL3may be involved in the airway remodeling of mice.2. Budesonide can downregulate the expression levels of STAT6mRNA and proteinand ORMDL3mRNA in the lung to reduce airway inflammation and airwayremodeling in asthmatic mice. |
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