The Role Of IRAK-M In Asthma And The Efficacy And Safety Of Tiotropium In The Treatment Of Severe Persistent Asthma

Part 1:The Effect of IRAK-M on Airway Hyperresponsiveness、Airway Inflammation and Airway Remodeling in The Chronic Asthma MiceObjectiveAsthma is a heterogeneous disease, usually characterized by the persistence of chronic airway inflammation, which can further lead to airway hyperresponsiveness (AHR), mucus hypersecretion and airway remodeling. Interleukin-1 receptor-associated kinase M(IRAK-M) is a kind of serine/threonine kinase, which can combine with IL-1 receptor, regulate TLR signal and lots of diseases. It is found that IRAK-M expressed on macrophagocytes^ bronchial epithelial cells and alveolar epithelial cells. IRAK-M is genetically linked to the early-onset persistent asthma and upregulated in airway epithelium from asthma patients. However, the mechanism mediated by IRAK-M is yet to be understood. This study aims to understand the role of IRAK-M in pathogenesis of asthma using knockout mice.(1)To observe the effect of IRAK-M on airway hyperresponsiveness> airway inflammation and airway remodeling in the chronic asthma using animal model.(2) To analyze the mechanism of IRAK-M in asthma. MethodsThe Specific pathogen free(SPF) C57BL/6 female mice were randomly divided into four groups:the control groupl (WT/NS group)、the control group2 (KO/NS group)、the asthma groupl (WT/OVA group)、the asthma group2 (KO/OVA group).All groups include two parts:sensitization and challenge.The following parameters were measured:(1)the ordinary behavior of the mice; (2)induced by inhaled methacholine, airway resistance was determined by invasive pulmonary impedance method; (3)carrying out the total cell count and cell differentiation of inflammatory cells in bronchoalveolar lavage fluid(BALF); (4)evaluation of lung histopathology using HE staining、PAS staining and Masson staining to observe kinds of inflammatory cell infiltration mucus secretion and collagen deposition; (5) measurement of inflammatory factors in BALF by Enzyme linked immunosorbent assay (ELISA);(6)detecting protein level of IRAK-M and other inflammatory proteins in lung tissue by Western blot; (7) measurement of inflammatory factors in lung tissue by Real-time PCR; (8) measurement of hydroxyproline concerntrations in lung tissue.Using SPSS 20.0 and GraphPad Prism software 6.0 to analyze data and make figure. Result(1)The mice in OVA group eat and exercise as normally, but scratch noses in embarrassment more than the mice in NS group, some mice are irritable.(2)The expression of IRAK-M mRNA and protein level in the WT/OVA group was increased compared with the WT/NS group.(3)Airway responsiveness in the WT/OVA group was significantly increased compared with the WT/NS group; airway responsiveness in the KO/OVA group was significantly attenuated compared with the WT/OVA group.(4)The inflammatory cell infiltration and mucus secretion in lung were more obvious in the WT/OVA group compared with the WT/NS group; the inflammatory cell infiltration and mucus secretion in lung were alleviative in the KO/OVA group compared with the WT/OVA group.(5)The total leukocytes、eosnophils、 lymphocytes、neutrophils、 macrophagocytes of BALF in the WT/OVA group were increased compared with the WT/NS group; the total leukocytes、eosnophils、lymphocytes、neutrophils、 macrophagocytes of BALF in the KO/OVA group are decreased compared with the WT/OVA group.(6)The IL-4 mRNA、MUC5AC mRNA expression in the WT/OVA group was increased compared with the WT/NS group, IFN-γ mRNA decreased; the IL-4 mRNA、MUC5AC mRNA expression in the KO/OVA group was decreased compared with the WT/OVA group. The IL-4 concentration of BALF in the WT/OVA group was increased compared with the WT/NS group, the IFN-γ concentration was decreased compared with the WT/NS group; the IL-4 concentration of BALF in the KO/OVA group was decreased compared with the WT/OVA group, the IFN-γ concentration was increased compared with the WT/OVA group; the IL-13 concentration of BALF in the WT/OVA group was increased compared with the WT/NS group.(7) The collagenous fiber deposition in lung was more serious in the WT/OVA group compared with the WT/NS group; the collagenous fiber deposition in lung was alleviative in the KO/OVA group compared with the WT/OVA group. The hydroxyproline level of lung in the WT/OVA group was increased compared with the WT/NS group, the hydroxyproline level of lung in the KO/OVA group was decreased compared with the WT/OVA group. The TGF-β1 mRNA expression in the WT/OVA group was increased compared with the WT/NS group; the TGF-β1 mRNA expression in the KO/OVA group was decreased compared with the WT/OVA group. The α-SMA protein expression in the WT/OVA group was increased compared with the WT/NS group, the a-SMA protein expression in the KO/OVA group was decreased compared with the WT/OVA group.(8) The NF-κB p65、p-NF-KB p65 protein expression in the WT/OVA group were increased compared with the WT/NS group, the NF-κB p65Ο p-NF-κB p65 protein expression in the KO/OVA group were decreased compared with the WT/OVA group. Conclusion(1)In the mice model of chronic asthma, knockout of IRAK-M gene can alleviate the airway responsiveness、airway inflammation and airway remodeling, IRAK-M is involved in the pathogenesis of chronic asthma.(2)IRAK-M can regulate inflammation of asthma by activating NF-κB.Part 2:Efficacy and Safety of Tiotropium in The Treatment of Severe Persistent Asthma: Meta-analysisObjectiveTiotropium is a long-acting anticholinergic receptor agonist, which characterized by bronchodilating. Even using glucorticosteroids and long-acting β2 agonist, severe persistent asthma cann’t be controlled well. Using tiotropium as add-on therapy may dilate bronchus and improve airflow limitation.This article aims to evaluate the efficacy and safety of tiotropium in treatment of severe persistent asthma.MethodsReports of randomized controlled trials (RCTs) describing tiotropium for treatment of severe persistent asthma published from January 1946 to February 2015 were searched in Cochrane、ClinicalTrials. gov、PubMed、Ovid Medline、CNKI、CSJD. The data of the included RCTs were extracted and the data quality was evaluated. Meta-analysis was performed with Revman 5.3 software.ResultsFive RCTs including 1433 patients were analyzed. Meta-analysis of the data showed that compared with the placebo group, tiotropium treatment significantly improved the patients’ peak FEV, (Forced expiratory volume in one second) [WMD (Weighted mean difference) 0.13 L,95%CI (Confidence interval, CI) 0.10-0.16 L, P<0.00001], trough FEV, (WMD 0.09 L,95% CI 0.06-0.12 L, P<0.00001), peak FVC(Forced vital capacity) (WMD 0.10 L,95%CI 0.06-0.14 L, P<0.00001). trough FVC (WMD 0.12 L,95%CI 0.08-0.17 L, P<0.00001), morning PEF(Peak expiratory flow) (WMD 9.21 L/min,95%CI 4.2-14.23 L/min, P=0.0003)、 even ing PEF (WMD 22.06 L/min,95%CI 13.05-31.08 L/min, P<0.00001), having statistical difference. The scores of ACQ(asthma-control questionnaire) (WMD 0.01,95% CI-0.07 to 0.09, P=0.86) or AQLQ(asthma quality of life questionnaire) (WMD-0.06,95% CI-0.18 to 0.06, P=0.33) were not affected by tiotropium. No significant difference with adverse events between tiotropium group and placebo group were reported in these included studies.ConclusionOn the basis of using glucorticosteroids and long-acting β2 agonist, inhaling tiotropium for severe persistent asthma can improve FEV1、FVC、PEF, but shows no significant effect on the quality of life of the patients. Tiotropium is well tolerated and can be an add-on therapy for severe persistent asthma.