Effect Of Formoterol And Budesonide On Calcium Handling Protein In Asthma Rats

Background: Asthma is a kind of inflammatory disease characterized by variable airflow obstruction, airway chronic inflammation and the airway hyper-responsiveness. Asthma is one of the most common diseases in the world and has become a heavy global burden. The mechanism of the onset of asthma is complicated, but all of them have close relationship with the constructional and functional disorder of the airway smooth muscle. Ca²⁺ plays an essential role in the contraction of the smooth muscle, for the relaxation of the smooth muscle due to the declination of [Ca²⁺]i in the cytoplasm. The pumping back activity of Ca²⁺ by sarco-endoplasmic Ca²⁺-ATPase (SERCA) is the main channel [Ca²⁺]i decreases. The airway smooth muscle mainly expresses the SERCA2b isoform, whose activity is mainly regulated by Phospholambam (PLB). Researches from both domestic and abroad showed that the altered expression of genes and proteins of both SERCA and PLB could harm the contraction of the myocardial cells. Further research proved the application of β-blockers could increase the expression of genes and proteins of SERCA, and at the same time decrease the genes and proteins expression of PLB, and consequently partially recovered the normal contraction ability of myocardial cells. So, can we hypotheses that during the onset of asthma, the alternation of the genes and proteins expression of SERCA and PLB play the same role as in the myocardialcells? Whether the mechanisms of asthma therapeutic drugs β₂ receptor agonist and glucocorticosteroid induce the alternation of the genes and proteins expression of SERCA and PLB also needs to be validated.Aims: We initially sensitized and consequently repeated expose the rats with the antigen to form the asthma model of rats, observing the alternation of the genes and proteins expression of SERCA and PLB. At the same time, we treated the model rats with selective inhibitor of SERCA, cyclopiazonic acid, to confirm the role it played in the asthma mechanism. And we also treated the model rats with glucocorticosteroid using the same method, and finally discuss the role of SERCA and PLB in the mechanism of asthma.Material and methods: 48 clean level male SD rats were randomly divided into 6 groups with 8 rats per group. The 6 groups were the formoterol group, the budesonide group, the formoterol plus budesonide group, the cyclopiazonic acid group, the asthma model group and the control group. The asthma model was established by ovalbumin sensitization followed by repeated allergen exposure, and before each time of allergen exposure, rats were exposed to aerosolise of the corresponding drugs. 24 hours after the last aerosolization, lung function tests were conducted and lung resistance and lung dynamic compliance were computed. After the rats were executed, alveolar wash was conducted and total white blood cells and the proportion of eosinophile granulocyte in the bronchoalveolar lavage fluid were counted. Tracheal smooth muscles were separated and total RNA was extracted using TRIZOL, the expression of SERCA and PLB mRNA in the tracheal smooth muscle were determined by RT-PCR. After the extraction of total protein using the protein lysate, the protein expressions of SERCA and PLB in the tracheal smooth muscle were determined by Western blot. Statistical data was obtained using SPSS13.0 software package. Results:1. The establishment of asthma model of rats: After the repeated exposureto the antigen, the pulmonary function was tested of the model group rats.Compared to the control group, the pulmonary resistance of the model group wassignificantly elevated (P<0.01) and the lung dynamic compliance was decreased (P<0.01). Inflammation caused by eosinophile granular leukocytes infiltration was present in the airway, the count of the eosinophile granular leukocytes in the BALF was significantly elevated compared to the control group (P<0.01). The pathological section also revealed the hypertrophy of the smooth muscle and the increase of the inflammatory cells such as mensenchymal eosinophile granular leukocytes. According to the characteristics asserted above, we can conclude that the establishment of the asthma model is succeeded.2. The effect of drugs: â‘  Compared to the asthma model group, the asthma rats treated with formoterol and budesonide had significantly improved lung dynamic compliance (P<0.01) and decreased pulmonary resistance (P<0.01). Though the combined therapy group also got improved pulmonary function, but got no significant difference with the monotherapy groups. The pulmonary function of the cyclopiazonic acid group was no difference with the model group. The count of the BALF revealed the budesonide group (P<0.01) and the combined therapy group (P<0.01) had less total cell count than the model group. The eosinophile granular leukocytes of the formoterol group (P<0.01), the budesonide group(P<0.01), the combined therapy group(P<0.01) and the cyclopiazonic acid group (P<0.01) were all decreased compared to the model group. And consequently, formoterol and budesonide were effective drugs for asthma rats. â‘¡ The protein expressions of SERCA and PLB: The gene and protein expressions of SERCA of the model group were significantly elevated compared to the control group (P<0.01). The expression of SERCA mRNA of the budesonide group, the combined therapy group and the cyclopiazonic acid group were all lowered (P<0.05), The protein expressions of SERCA was also lowered, but had no statistical significance. The gene and protein expressions of PLB of the model group were significantly decreased compared to the control group (P<0.01). The gene and protein expressions of PLB of the budesonide group, the combined therapy group were consequently decreased (P<0.05); Though the gene and protein expressions of PLB of the formoterol group and the cyclopiazonic acidgroup were all lowered, but had no statistical significance.Conclution: 1. The gene and protein expressions of SERCA and PLB in the asthma rats group were significantly elevated, which indicate that gene and protein of SERCA and PLB had taken part in the mechanism of the onset of asthma. 2. Long termβ₂ receptor agonist formoterol had no effect to the gene and protein expressions of SERCA and PLB in the rats airway smooth muscle. 3. Inhaled glucocorticosteroid budesonide had significant inhibited effect to the gene expressions of SERCA and PLB in the rat's airway smooth muscle, which indicate the therapeutic effect of glucocorticosteroid may be correlated to the alternation of SERCA and PLB gene expression.