The Synergism And Mechanism Of ICS And LABA In The Treatment Of Asthma

Background and objective:Asthma is a chronic inflammatory disease of the airways characterized by recurrent exacerbations with wheezing, chest tightness, cough and the activation of inflammation cells. Inhaled β2-adrenoceptor agonists (LABA) and inhaled corticosteroids (ICS) are commonly used in the treatment of asthma. In recent years, a combination treatment of LABA and ICS was developed for the treatment of persistent asthma. This study is to investigate the potential synergistic effect of an inhaled β2-adrenoceptor agonists (formoterol) and an inhaled corticosteroid (ciclesonide) in vitro and in vivo, and preliminarily study the synergistic mechanism.Methods:In vivo, establishing mice model of asthma. BALB/c mice was sensitized with100μg ovalbumin adsorbed on a2-mg aluminum hydroxide gel by subcutaneous injection on days0and14. From day21to day28, the mice were challenged with aerosolized ovalbumin (10mg/mL in saline) for30min. A day after the last challenge, treatment mice were administered with drugs by intratracheal instillation. After3h, airway reactivity to aerosolized methacholine (0-50mg/mL) was measured by whole body plethysmography. Then the animals were killed and bronchoalveolar lavage (BAL) was performed, by injecting four times0.3mL of PBS into the airway lumen, for determination of inflammatory cells numbers. In vitro, RBL-2H3cells were used as a model cell line for evaluating the inhibitory effect of des-ciclesonide and formoterol on antigen-induced allergic reactions. The mRNA expressions of IL-4and IL-13were detected by real time RT-PCR. The protein expressions of IL-4and IL-13were measured by ELISA. Western blot was used in the tests of JNK signaling pathway.Results:In vivo,1) In a murine model of OVA-induced asthma, the methacholine-induced increase in Penh was dose-dependently inhibited by ciclesonide and formoterol respectively.2) A synergistic effect on the methacholine-induced increase in Penh was demonstrated for ciclesonide and formoterol given in combination, at the following doses:formoterol/ciclesonide (in ug/kg)7/300,14/300,28/30; formoterol/ciclesonide combinations of7/1000,14/1000,28/1000ug/kg had no a significant synergistic effect on the methacholine-induced increase in Penh.3) In the OVA group, the total cells macrophage, eosinophil and lymphocyte numbers increased significantly; All the inflammatory cell types were dose-dependently inhibited by ciclesonide and formoterol respectively.4) All formoterol/ciclesonide combinations had a synergistic effect on the inhibition of inflammatory cells compared to the single administration of each drug.5) The expression of IL-4mRNA was increased significantly in the OVA group compared with control; The increase of IL-4mRNA was inhibited by formoterol (28,56μg/kg) and ciclesonide (0.3mg/kg) respectively; formoterol/ciclesonide combinations of14/1000,28/1000ug/kg had a synergistic inhibition effect on IL-4mRNA increase. In vitro,1) High concentrations of des-ciclesonide (10-8-10-4M) induced cell toxicity in a concentrtions-dependent manner and formoterol had no toxicity to RBL-2H3cells.2) DNP-BSA (100ng/ml) induced the mRNA expression and protein production of IL-4and IL-13in IgE (200ng/ml)-sensitized RBL-2H3cells.3) The increase in mRNA and protein of IL-4and IL-13was dose-dependently inhibited by des-ciclesonide (0.1-10nM) and formoterol(0.1-10μM) respectively.4) The combinations of des-ciclesonide (0.2nM) and formoterol (1μM) had a synergistic inhibition effect on IL-4mRNA expression and protein production, but no synergy to IL-13.5) Pre-treatment with JNK inhibitor SP600125(10μM) significantly inhibited the IL-4production compared with DNP-BSA treatment alone.6) DNP-BSA increased the phosphorylation of JNK and the level of JNK activation was significantly suppressed by des-ciclesonide (10nM) and formoterol (10μM) respectively.7) Des-ciclesonide (0.2nM) and formoterol (1μM) synergistically inhibited phosphorylation of JNK.Conclusion:1) Ciclesonide and formoterol, two clinically used drugs, act synergistically to relieve the methacholine-induced airway hyperresponsiveness and airway inflammation in allergen sensitized and challenged mice.2) In IgE-sensitized RBL-2H3cells, des-ciclesonide and formoterol had a synergistic inhibition effect on IL-4production induced by DNP-BSA, which is mediated through the signaling JNK phosphorylation.