| Bronchial asthma is a chronic inflammation of airway, airway inflammatory cells which infiltrate in airway wall, such as eosinophils and lymphocytes, release toxic protein and inflammatory cytokine resulting in airway epithelial injury and activating the curing reaction. The damage and repair alternately led to the airway remodeling eventually. Airway remodeling is characteristic of pathological changes of asthma, It is the considerable patho-foundation of irreversible airflow obstruction and persistent airway hyperresponsiveness. Up to now, the mechanisms of asthmatic airway remodeling is still not completely clear.Epidermal growth factor receptor (EGFR) is a membrane receptor tyrosine kinase and widespread exists in many cells of human body, The ligands including epidermal growth factor (EGF),heparin-binding epidermal growth factor-like growth factor (HB-EGF), transforming growth factor alpha (TGF-α), amphiregulin (AR), etc. HB-EGF which is mitogenic for airway smooth muscle cells, airway epithelial cells, myofibroblasts and vascular smooth muscle cells can promote the differentiation, proliferation and migration. TGF-a up-regulates MUC5AC gene and protein expression in airway epithelial cells and stimulates proliferation of goblet cells, causing airway extraordinary mucus hypersecretion and formation of mucus plugs which is the main reason of airway obstruction and suffocation in asthma and improves morbidity and mortality of asthma. The epidermal growth factor receptor tyrosine kinase inhibitor (AG1478) ameliorates the progression of airway remodeling following prolonged allergen challenge via regulation of EGFR signal pathway and inhibits proliferation of airway epithelial cells,smooth muscle cell and vascular smooth muscle cell and induces apoptosis of them.ObjectiveThrough building the asthma model in mice, to explore the relationship between airway remodeling and expression of EGFR,HB-EGF and TGF-αin asthmatic mice and the effect of AG1478 intervention in asthmatic mice. It provide theoretical basis for clinical treatment.Materials and methods1 Materials and methods32 healthy and male BALB/c mice, aged 6-7 weeks, weighed (15±2) grams, they were randomly divided into 4 groups:control group,asthma group,AG1478 treated group and dexamethasone treated group, every group had 8 rats. On the 1st,8st and 15stday, mice of asthma group,AG1478 treated group and dexamethasone treated group were injected 0.2ml suspension of ovalbumin (OVA) (containing 0.2ml normal saline, 1mg aluminum hydroxide and OVA 20μg) by intraperitoneal to sensitize them, mice of control group were injected lml normal saline. On the 22st day, mice of asthma group,AG1478 treated group and dexamethasone treated group were challenged for 30 minutes by inhaling 1% OVA (once/every other day) in special made terrarium, mice of control group were challenged with normal saline. Mice of AG1478 treated group and dexamethasone treated group were respectively injected AG1478 (15mg/kg) and dexamethasone (10mg/kg) by intraperitoneal an hour before every challenge. The mice appear restlessness, accelerated breathing or immobility and abdominal muscle spasm, indicate that we make the asthma model successfully.2 Collecting the sampleAll the mice were anesthetized by Phenobarbital sodium(45mg for each), then opened the chest, cut off left lung and took into liquid nitrogen for PT-PCR, intubated into pulmonary artery through right ventricle, using normal saline to lavage quickly, taken the middle lobe of right lung into 10% methanal to fix them, then imbed in paraffin within 24 hours. Slicing the samples by two direction:vertical and parallel to the airway, the thickness of each slice was 5μm, then make Masson staining,PAS staining and immunohistochemistry staining.3 Determination of the expression of EGFR,HB-EGF and TGF-α3.1 Immunohistochemistry staining,Masson staining and PAS stainingUsing computer image analysis system, five representative filed of view randomly were choose on each slice under high power lens (×400) and positive area were selected. On immunohistochemistry and Masson staining slice, the density scale were measured. On PAS staining slice, the quantity of goblet cells were calculated. Then the average was the last result.3.2 PT-PCRUsing gel analysis software Band Scan 5.0, A value were compared respectively with EGFR, HB-EGF andβ-actin for semi-quantitative analysis.4 Statistics analysisUsing SPSS16.0 statistical package to analyze, all the result were demonstrated by mean±standard deviation, using one-way ANOVA to compare the mean of each group after the test of homogeneity of variance, linear correlation analysis was used to assess the relation of variables,α=0.05 is the significance level, P value less than 0.05 indicate statistical significance. Result1 Appearance of animal generally observedAfter repeated OVA, the rats of asthma group appeared restlessness, accelerated breathing or immobility, abdominal muscle spasm irritability and restlessness. The rats of AG1478 group and dexamethasone group also appeared similar symptoms of asthma group, but the symptoms were significantly lighter. The rats of normal control group appeared normal.2 Pathological morphology observationThickening of airway wall,hypertrophy of airway smooth muscle,shedding of epithelium cells,proliferation of goblet cells and mucus hypersecretion,subepithelial deposition of collagen and infiltration of inflammatory cells in the lung sample of asthma rats. Compared with asthmatic group, there was mild inflammation reaction and airway remodeling in the lung sample of therapeutic rats. There were not such changes in control group.3 The protein expression of HB-EGF and TGF-αHB-EGF mainly expressed in bronchial mucosal epithelium, TGF-αmainly expressed in bronchial mucosal epithelium, airway smooth muscle cells and vascular smooth muscle cells. The protein expression of HB-EGF and TGF-αin asthmatic group(0.587±0.110 and 0.585±0.078)were significantly higher than normal control group(0.196±0.050 and 0.241±0.062), P＜0.05; Compared with asthmatic group, there was lower expression of HB-EGF and TGF-αin AG1478 group (0.330±0.080 and 0.345±0.072) and dexamethasone group (0.372±0.073 and 0.384±0.054), P＜0.05.4 The mRNA expression of EGFR and HB-EGFRT-PCR was used to detect the mRNA expression of EGFR and HB-EGF. Expression levels (0.720±0.120 and 0.616±0.108) in the epithelial cells of bronchi were significantly higher in asthmatic group than those in control animals (0.229±0.070 and 0.191±0.067), P＜0.05; Compared with asthmatic group, there was lower expression in AG1478 group (0.418±0.093 and 0.340±0.096) and dexamethasone group (0.446±0.010 and 0.391±0.101), P＜0.05.5 The mean area deposition of collagen and the average of goblet cellsAfter repeated allergen challenge, obvious deposition of collagen and proliferation of goblet cells were demonstrated in asthmatic group mice (6.822±0.954 and 17.062±1.328) than those in control animals (2.806±0.466 and 9.388±0.938), P＜0.05; Compared with asthmatic group, there was decreased in collagen deposition and proliferation of goblet cells in AG1478 group (4.007±0.805 and 13.025)and dexamethasone group (4.206±0.832 and 13.412±0.904), P＜0.05.6 Linear correlation analysisThe expression of EGFR was positive correlation with the mean area deposition of collagen and the average of goblet cells in asthma mice(r=0.699 and 0.522, P＜0.01).Conclusion1 EGFR,HB-EGF and TGF-αare tightly correlated with the development of airway remodeling of asthma.2 The epidermal growth factor receptor tyrosine kinase inhibitor (AG1478) ameliorates the progression of airway remodeling following prolonged allergen challenge via regulation of EGFR signal pathway.
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