Effects Of Inhaled Budesonide On Airway Inflammation And Remodeling Of The Asthmatic Rat

Objective: Bronchial asthma is recognized as one of the world's four major chronic medical profession. The leading cause of death was listed as the most. Now, with people's lives change, the incidence rate increase every year. The clinical manifestations is sudden onset recurrent wheezing, difficulty breathing, chest tightness and coughing. The pathological basis of bronchial asthma is airway inflammation and airway remodeling. In this study, through the establishment of asthma model, we observe inhaled budesonide in asthmatic rat the LI-25/ IL-12 ratio change and explore the relationship between the immune imbalance and the expression of NF-κB dynamic in asthma. Observation of inhaled budesonide airway NF-κB, MMP-9 expression in asthmatic rat, we explore relationship between airway remodeling and NF-κB.Methods:1 Animals and drugsWe selected 30 wistar mouse, provided by the Experimental Animal Center of Hebei Province (experimental animal quality certification number: 90301 );Budesonid produced by AstraZeneca Pharmaceuticals; Immunohistochemistry kit, one anti-cox2 rabbit anti-IgG polyclonal antibody, purchased in boshide company, SP9001 kit and DAB reagent purchased in Bio-Technology ,Beijing T-shirt. Enzyme immunoassay kit purchased in the United States RB; other experimental drugs provide by immunity teaching of the Hebei Medical University.2 Model preparation and experimental groupsthe law use random numbers and 30 male Wistar rats were randomly divided into three groups:normal control group,asthma model group, budesonide-treated group,10 in each group.Preparation of asthma model group, two-step:sub-sensitized and challenged. In addition to the normal control group, the other two groups were injectioned antigen solution 1ml (including egg protein, 100mg, aluminum hydroxide powder 100mg) by intraperitoneal sensitized on 1 day, 8 day, 15 day three times. and normal control group with equal of saline to sensitize; the 21 days, the rats were placed not completely closed self-made plexiglass box, asthma model group and the budesonide treatment group were Sucked in 1% ovalbumin (OVA) solution by the air compression pump spray Fog excitation,once a day,each20 minutes,continuously 7 days.The budesonide treatment group after excitation are used to budesonide inhalation treatment ,method 0.5mg/ml adding to 1ml saline. Budesonide compressed into the inhalation nebulizer, bid. Bronchial asthma group inhalation the saline after excitation. normal control group were stimulated with saline for the treatment. This last 7 days.3 The preparation of tissue samplesIn each groups, in the Last 24 hours after induced asthma, intraperitoneal injection of anesthesia, the right femoral artery get blood samples. the serum -70℃preservation, prepared to do enzyme-linked immunosorbent . ligation of the left lung, the right lung was slowly poured into 4% paraformaldehyde for the right lung inflated. then ligating of the right bronchus and placing the fixedan.taking right middle lobe organization prepared to do immunohistochemistry.4 Target detection4.1 IL-12 and IL-25 detectionApplication of ELISA to detect serum IL-12 and IL-25 levels, their manual steps carry out the reagen.4.2 TO detect NF-kB and MMP-9 expression in the lung tissue with immunohistochemistrySP9001 Universal two-step method using to detect NF-kBand MMP-9 expression in the lung tissue, light microscopic observation, the positive part of brownish yellow. Set-positive rate, parallel statistically.Results: 1 Identification of rat model of asthmaThe model group rats after inhalation of OVA appear restless, scratch facial fur and other allergic symptoms, then bowed down and not move , shortness of breath, difficulty breathing ( the notable abdominal breathing), and some rats were audible and louddly expiratory wheezing, nose and mouth out of mucus,severe cases,irregular breathing could occur.Repeatedly stimulated, fur dull, lassitude and unresponsiveness.2 Enzyme-link ed immunosorbent assay results2.1 The serum IL-25 levelsThe serum IL-25 concentration (40.71±3.87) pg/ml in asthma group, The serum IL-25 concentration (30.43±1.48) pg/ml in hormone therapy group, The serum IL-25 concentration (25.96±1.74) pg/ml in control group ,three groups have more statistically significant (P <0.05). And asthma group serum IL-25 levels(40.71±3.87) higher than the hormone therapy group (30.43±1.48) pg/ml, their have more significant differences. Asthma group serum IL-25 levels (40.71±3.87) significantly higher (25.96±1.74) pg /ml than the control group, their have more significant difference (P <0.05), The hormone therapy group and the control group was no significant difference. (P> 0.05).2.2 The serum IL-12 levelsThe serum IL-12 concentration (25.91±3.20) pg/ml in asthma group, The serum IL-12 concentration (29.42.±2.43) pg/ml in hormone therapy. serum IL-12 concentration (33.71±2.69) pg/ml in control group, three groups have more statistically significant (P <0.05). And asthma group serum IL-12 levels (25.91±3.20) pg/ml lowwer than the hormone therapy group,Their have a statistically significant difference (P <0.05), asthma group serum IL-12 levels (25.91±3.20) pg / ml significantly lower than the control group, their have significant difference (P <0.05),the hormone therapy and the control group were statistically significant (P <0.05).2.3 Serum IL-25 / IL-12 ratio of changeCompared with the normal group, asthma group serum IL-25 / IL-12 ratio was significantly higher, the difference was significant (P <0.01); After hormone treatment, serum IL-25 / IL-12 ratio, compared with asthma group, ratio decreased; compared with the control group increased, the difference also was significant (P <0.01).3 Identification of biopsy results3.1 Light microscopyAsthmatic rat bronchioles had spasm,contraction, lumen containing secretions,bronchioles, the small blood vessels around the apparent infiltration of inflammatory cells,including eosinophils, lymphocyte predominance and neutrophils;alveolar interval widened, fibrosis, a large number of lymphocytes, macrophages infiltration. Treated rats had no significant bronchiole spasm, contraction, non-intraluminal secretions, wall significantly reduced infiltration of inflammatory cells ,without interval widened.3.2 Immunohistochemistry resultsNF-κB and MMP-9 in the asthmiatic group, hormone therapy group and normal control rats were positive expression in lung tissue and were mainly expressed in the nucleus, a small amount of expression in the cytoplasm.3.2.1 Lung tissue NF-κB expressionAsthmatic group, the lung tissue expression of NF-κB was significantly higher compared with the normal group, the difference was significant (P <0.01); After hormone treatment, the rat lung tissue expression of NF-κB compared with Asthmatic group lower , when compared with the normal group increased, the differences were significant (P <0.01)3.2.2 Lung tissue MMP-9 expressionAsthmatic group,The lung tissue expression of MMP-9 was significantly higher compared with the normal group,the difference was significant (P <0.01); After hormone treatment, the rat lung tissue expression of MMP-9 compared with the Asthmatic group reduced,compared with the control group increased, the differences were significant (P <0.01).4 The correlation between the various indicatorsApplication of Spearman Rank Correlation analysis showed that:IL-25 and IL-12 were negative correlation (r =- 0.754, P <0.01), IL-25 and NF-κB were positive correlation (r = 0.812, P < 0.01), IL-12 and NF-κB were negative correlation (r =- 0.747, P <0.01), MMP-9 and NF-κB were positive correlation (r = 0.797, P <0.01).Conclusion:1 The immune balance of Th1/Th2 was broken,and it migrant to Th2 direction.Th2 dominant reaction play an important role in the pathogenesis of asthma.2 The percentage of NF-κB in asthma mice was notable higher than that of the control group.It hint that plays an important role in the process of the airway inflammation and remodeling of asthma.3 In asthma group.the positive percentage of NF-κB and the level of IL-25 increase signicantly,but the level of IL-12 decreased, the ratio of IL-25/IL-12 increase, correlation analysis showed: NF-κB and IL-25 were positively correlated:NF-κB and IL-12 were negative correlation.4 Asthma, NF-κB activation and MMP-9 expression in bronchopulmonary tissues were significantly higher than control group.Bronchial mucosal hyperplasia hypertrophy significantly, indicating that both participate in the process of airway remodeling,correlation analysis showed a positive correlation between the two.5 Budesonide as the effective drug treatment of asthma can inhibit NF-κB activity,so that it is IL-25 levels decrease, IL-12 levels increased, and reduce airway inflammation;Budesonide also can reduce the level of MMP-9 and improve airway remodeling.Can be used as first choice for treatment of asthma and provide a theoretical basis for the treatment of asthma.