Over Expression Of α-Smooth Muscle Actin And Type Ⅰ Collagen Induced By FIZZ1/RELM-α In OVA-Induced Asthma Animal Model

Objectives:Asthma is a chronic airway disease with inflammation characterized by airway hyperresponsiveness(AHR)and pathological changes features with the dysfunction of the smooth muscle and the deposition of extracellular matrix.Several lines of evidence suggested that not only severe asthma but also mild and moderate asthma were sometimes difficult to control by routine treatments,due to the early stage of airway remodeling in lung.The early stage airway remodeling in asthma thus might predispose persons with asthma to asthma exacerbations and even death from airway obstruction caused by smooth muscle contraction,airway edema,and mucus plugging.There were evidences from studies demonstrating that the occurrences of early stage airway remodeling in asthma were associated with the pulmonary myofibroblast differentiation,which was known to be a critical modulator ofα-smooth muscle actin(α-SMA).Furthermore,bronchial biopsies of asthmatic patients showed thickening of the subepithelial layer due to the deposition of fibrillar collagen,such as elastin,collagenⅠ,and collagenⅢ.Thus,α-SMA and typeⅠcollage were regards as the main parameters in asthma airway remodeling.So the early stage airway remodeling was a hallmark of the clinical symptom of asthma, which could cause irreversible airflow limitation.Furthermore,Liu et al has provided some clues as to the potential role of certain genes in pathogenesis of airway remodeling.FIZZ1 was highly induced in lung fibrosis,and high FIZZ1-expressing AECⅡsignificantly stimulated both collagen typeⅠandα-SMA expression in fibroblasts.Therefore,both of these endpoints are reflective of fibroblast activation and differentiation to myofibroblasts,which were thought to share the resemblance pathogenesis of airway remodeling.We used pEGFP-FIZZ1 to transfect lung fibroblasts independent of TGF-β1 in order to provide more indirect evidences to elucidate the function of FIZZ1 in airway remodeling process.The myofibroblasts were major producers of collagenous matrix molecules which correlate with subepithelial collagen deposition andα-SMA.Cumulated evidences showed that the appearance ofα-SMA was correlated with the extent of airflow obstruction and likely contributed to airway wall thickening,at the same time collagen deposition caused the structural alterations in the airway,finally leading to airway collapse and airflow limitation.These findings provided valuable information in further understanding of FIZZ1 signal transduction pathways,which may ultimately lead to recognize more clearly mechanism of airway remodeling in asthma.Taken together, these results indicated a novel and critical function of FIZZ1 on the pathogenesis of airway remodeling.Methods:70 rats were divided into 2 groups:saline group 35 and OVA group 35. Pulmonary function tests were performed to evaluate the airway responsiveness;Isolation and culture the AECⅡ;The concentration of FIZZ1mRNA in AECⅡwere measured by the RT-PCR;Theα-SMA expression level were measured by immunohistochemistry;The lung fibroblast(LF)were transfected by pEGFP-FIZZ1 independent of TGF-β1;The expression of typeⅠcollagen were determined by ELISA.Theα-SMA in the LF expression was measured by western blotting.Results:1.The Rat Asthma ModelStudies in the OVA-induced model had demonstrated that a subset of overaction as a major features of asthma successful model.When compared with rats in saline group,the rats from OVA group showed dysphoria,more activity,upper limb raise, urinary and fecal incontinence and so on.2.Pulmonary function testsWhen stimulation of ACH with concentrations(0.025 mg/kg),the mean Re and Cldyn of two groups have no difference(P=0.379,P=0.843).After stimulation of ACH with accelerated concentrations(0.05-0.2mg/kg mg/kg),the mean expiratory resistance(Re)of OVA group was higher than that of saline group(P＜0.05).After stimulation of ACH with accelerated concentrations(0.05-0.2mg/kg mg/kg),the mean Cldyn of OVA group was attenuate than that of saline group(P＜0.05).3.Pato-manifestation by HE in Lung TissuesStudies in the OVA-induced model had demonstrated that a subset of inflammatory reaction as a major histopathological features of asthma.When compared with rats in saline group,the lung tissue from OVA treated rats showed wall thickening,inflammatory cells infiltration,subepithelial fibrosis,mucous metaplasia,and myocyte hyperplasia and hypertrophy.At the same time,we observed no obviously inflammatory reactions in saline group. 4.Immunohistochemical Staining ofα-SMA in Lung TissuesAs expected,positiveα-SMA staining was highly expressed in peribronchial lung sections isolated from the OVA group,and restricted to the muscular layer of the airway in the intrapulmonary bronchus.Neither the terminal bronchioles,nor the bronchial epithelia showed positive staining in saline group(P=1.49×10^-5).5.FIZZ1 mRNA Expression in AECⅡThe AECⅡisolated from lung had typical cube shape features under microscope. Previously studies demonstrated that FIZZ1 was specifically expressed in AECⅡof allergic pulmonary inflammation.When normalized toβ-actin,level of FIZZ1 mRNA was upregulated in AECⅡ,in contrast to very low level in control AECⅡ(P=2.71×10^-8).6.The relationship between the expression of FIZZ1 mRNA andα-SMAThe FIZZ1 mRNA expression has positive correlation with the peribronchial ofα-SMAin lung(r=0.95,P=3.24×10^-6).7.Expression of Collagen TypeⅠin Lung FibroblastBecause AECⅡwere shown to be a major source for FIZZ1 in OVA-induced asthma,their effect on certain fibroblast functions related to airway remodeling could be used to explore the potential role of this molecule in airway remodeling.In vitro,we tested the level of collagen typeⅠin lung fibroblast transfected with the pEGFP-FIZZ1 plasmid independent of TGF-β1 by ELISA.These results showed that FIZZ1 significantly activates rat lung fibroblasts with respect to typeⅠ collagen expression(P=3.64×10^-4).8.Expression ofα-SMA Protein in Lung FibroblastsThere were evidences showing that airway remodeling in asthma were associated with theα-SMA.In vitro,we tested the level ofα-SMA protein in lung fibroblasts transfected with the pEGFP-FIZZ1 plasmid independent of TGF-β1 by western blotting.α-SMA protein level in lung fibroblasts transfected with empty vector plasmid did not show any change.These results showed that FIZZ1 significantly activates rat lung fibroblasts with respect toα-SMA expression(P=0.0065).Conclusion:1.FIZZ1 expressed higher in AECⅡand induced the emergance ofα-SMA in peribronchial lung sections.2.FIZZ1 induced myofibroblast differentiation to releaseα-SMA and typeⅠcollagen independent of TGF-β1,and FIZZ1 plays a critical function in the pathogenesis process of airway remodeling.