Expression And Function Of Galectin-9 And Tim-3 In Lungs Of Mice With Asthma

BackgroundAsthma is an immunologic dissonance disease, Namely T lymphocytes controlling the inflammation, Thl/Th2 reaction imbalance and Th2 type immune response increasing exceptionally.Thl type immune response weakening plays an important role in the pathogenesis of asthma and this balance maladjustment is believed to be the key mechanism of Asthma incidence.The application of Glucocorticoid has improved the symptoms in patients with asthma and the quality of life,which becomes an classical drug for treating bronchial asthma. However, long-term application,especially in a large dose,has obvious toxic effect.There may be glucocorticoids insensitivity or resistance for a few severe asthma patients. Therefore, people have been finding a more effective treatment of asthma to seek for a new breakthrough, In allergic diseases the quantity of half lactose lectin-9 (Galectin-9) and T cells immunoglobulin mucin molecules-3 (Tim-3) at the transcription level and protein level have markedly increased, whose change is directly related to the quantity of Th1/Th2 cytokines expression.Their change is closely related to allergic inflammatory process.Therefore, this study take the Galectin-9 and Tim-3 expressed specifically on the surface of Thl cells as target target molecules of immunoregulation,.Through the immune regulation,they make Thl/Th2 reaction imbalance recover, hoping to provide a theoretical and experimental basis for current asthma mechanism research and clinical treatment.ObjectiveTo explore the expression of Galectin-9 and Tim-3 in lungs of mice with asthma and the effect of Rosiglitazone (PPAR-y agonist) on their expression.Materials and Methods1 Animal Grouping and Asthma Model Manufacture Method45 BALB/c female mice of clean grade (bought from experimental animal center of zhengzhou university),aged 5-8 weeks,weighed 18-22g.According to random number table method,this 45 mice is divided into Asthma Model Group, normal control group and ROS Group.Every group has 15 mice. Mice in Asthma Model Group are sensitized by injecting 0.2 ml of 1% OVA/Al (OH) 3 suspension liquid intraperitoneally and subcutaneously on postnatal day 1,7 and 14 respectively.On the 21st day, we start using 2% OVA aerosol inhalation for stimulating,30min/time, one time per day for two weeks.On PND 28 the mice in ROS Group are given PPAR-y agonist-rosiglitazone by gastric lavage intervention (10mg/kg) 30 minutes before being stimulated by 2% OVA aerosol inhalation;the other dispose is same to Asthma Model Group.The mice in the Control Group are sensitized and inhaled by equivalent normal saline;the other dispose is same to Asthma Model Group. Egg albumin and rosiglitazone liquid are compounded when needed every time.2 Collecting the sampleSerum collecting:In 24 hours after the last stimulation, under aseptic conditions take blood 0.8 ml through eyeball artery, then put into EP tube, centrifuge(3000rpm) for 5min at 4 degrees centigrade; fetch the supernatant fluid divided into several tubes being preserved at minus 80 degrees centigrade for ELISA analysis. Lung tissue samples:All the mice were anesthetized by pentobarbital sodium (45mg per kg weight) after taking blood,then open the chest immediately to expose the lungs to observe general changes of them.Take the left lung tissue and quickly put it in liquid nitrogen freeze-stored for PT-PCR test. Then intubated into the pulmonary artery through the right ventricle, using normal sodium to lavage quickly,. taken the middle lobe of right lung into 4% methanal to fix them, then imbed in paraffin within 24 hours. Then made 5μm thickness slice, and stain them with hematoxylin eosin.Under optical microscope observe lung tissue, various bronchus and infiltrating situation of perivascular inflammatory cells.3 Detect the level of IL-4 and IFN-y in the serum with enzyme-linked immunosorbent adsorption test (ELISA)Do experiment process according to reagent instructions strictly.On adding stop solution immediately and blending, measure Absorbency OD450 values by automatic microplate. OD value is y-coordinate and standard substance is abscissa.Draw standard curve and determine its concentration according to OD value of samples.4 RT-PCRUsing the trizol method to get the total RNA from the frozen right lung, then reverse transcript RNA into cDNA. Add a specific primer and do the polymerase chain reaction to amplify the objective fragment The agarose gel electrophoresis is conducted to display the DNA. The gel analysis system Band Scan 5.0 can be used to detect the gray scale of galectin-9 Tim-3 and GADPH. Regard the ratio of Galectin-9 and Tim-3 and GAPDH in reference as representive of mice.5 Statistics analysisUsing SPSS16.0 statistical package to analyze, measurement data were demonstrated by mean±standard deviation, using one-way ANOVA to compare the mean of each group, linear correlation analysis was used to assess the relation of variables, p<0.05 is the significance level.Results1 Pathologic Changes The light microscopy of HE-stained lungs tissues of mice reveals:In Control Group,the lung tissue has a complete structure and regular tracheal morphology;the mucous epithelium is complete, bronchial epithelium and alveolar diaphragmatic and perivascular has not seen the inflammatory cells infiltrating, bronchioles smooth muscle that does not appear proliferation, bronchial lumen secretions less, the alveolar morphological rules. Asthma mice lung biopsy visible in lung bronchial submucosal and perivascular infiltration more EOS submucosal hyperemia oedema, mucus glands, the airway epithelial hyperplasia of several fracture and epithelial cell falls off, interstitial lung and bronchus concretions within the inflammatory cell infiltrates and secretion, slightly bronchial wall thickening and basement membrane mild thickening and irregular. ROS mice lung tissue inflammatory cells infiltrating etc pathological changes is asthma group ease but not completely disappear.2 Results of ELISAThe concent intensity of IL-4 (564.48±76.17) in Asthma Group increases compared with that in Control Group (347.55±44.31) and ROS Group (483.81±68.36).While compared with Control Group (599.21±105.31) and ROS group (398.67±120.67),the concent intensity of IFN -γin blood in Asthma Group (371.56±131.49)decreases.Group differences are all significant statistically (p< 0.05).3 Results of RT-PCRThe expression of Galectin-9 and Tim-3 mRNA in asthma group (0.614±0.092, 0.542±0.084) are significantly high compared with control group (0.305±0.051, 0.296±0.054) and ROS intervention group (0.401±0.059,0.466±0.091) (p<0.05).4 Linear Correlation AnalysisThe expression of Galectin-9 and Tim-3 mRNA has a positive correlation with IL-4 (r=0.691 r=0.568 p<0.01) and a negative correlation with IFN-γ(r=-0.507 r=-0.403 p<0.01) ConclusionsGalectin-9 and Tim-3mRNA involved in the process of airway inflammation in asthma, the treatment of rosiglitazone reduced lung inflammation in asthmatic mice,which was due to the decreased expression of Galectin-9 and Tim-3mRNA.