| BackgroundAsthma is a common heterogeneous disease characterized by airway inflammation and airway remodeling.Airway remodeling is the main reason for the rapid decrease of lung function and the limitation of the fixation airflow in asthmatic patients.Meanwhile,5 to 10%of severe asthmatics are significantly correlated with airway remodeling.Currently,the cornerstone of asthma therapies is corticosteroids,long-acting ?2 agonists and leukotriene antagonists.These therapies may be effective in alleviating inflammation,but are not effective in preventing and reversing airway remodeling.Early prevention of airway remodeling has the potential to reduce disease severity,to improve control and to prevent disease progression.So,it is urgent to detect the mechanism of airway remodeling in order to find more useful therapies thattarget molecules associated with structural changes,such as epithelial thickening and subepithelial fibrosis.Epithelial mesenchymal transition(EMT)is a novel clinical therapeutic target in many chronic airway diseases related with tissue remodeling.EMT is characterized by a series of changes,during which epithelial cells lose epithelial features,such as tight and adherent junctions as well as epithelial markers,and acquire mesenchymal properties.Airway remodeling is thought to intensify the degree of subepithelial fibrosis via EMT.Follistatin-like 1(FSTL1)is a secreted extracellular glycoprotein.Prior studies have identified that FSTL1 is required for lung organogenesis and development.Sonomi Maruyama et al.found that FSTL1 is essential for the acute repair of the infracted myocardium and Fstll activates fibroblast migration and proliferation via ERKIl2 signaling.The induction of collagen I and fibronectin protein expression in ischemic tissue was significantly attenuated in Fstl1-cfKO compared to wide type(WT)mice in the ischemic heart tissue.Otherwise,FSTL1 induces lung fibrosis.Fstl1+/-mice and blockage of FSTL1 with a neutralizing antibody have an attenuated fibrotic phenotype after bleomycin-induced lung injury.Disco inter-acting protein 2 homolog A(DIP2A)can be a receptor of FSTL1 and FSTL1 may induce the airway remodeling through binding DIP2A in asthma.Autophagy,a catabolic intracellular pathway,plays a critical role in maintaining cellular homeostasis by degrading misfolded or dysfunctional proteins and defective or excessive organelles.Autophagy involves in many processes that have different effects on disease and could prevent or promote disease progression.Previous studies have suggested autophagy could affect the pathogenesis of asthma.Poon et al.found that epithelial cells and fibroblasts isolated from bronchial biopsy tissue taken from asthmatic patients had a greater number of autophagosomes compared to healthy subjects.These studies also demonstrated that autophagy is associated with reduced FEV1 in asthmatic patients.In addition,the single nucleotide polymorphism(SNP)of autophagy-related gene(ATG)is related to asthma.These results indicated that FSTL1 and autophagy may be involved in multiple steps of the asthma airway remodeling process,potentially providing novel mechanisms for the development of new therapeutic strategies for asthma.ObjectivesTo explore the mechanism of FSTL1 and autophagy on airway remodeling in asthma and to find the effective targets as new therapeutic strategies for asthma airway remodeling.Methods1.The expression of FSTL1 and autophagic markers in airways from asthma1.1 PatientsBronchoscopy was performed in seven patients with asthma at Qilu Hospital,Shandong University(Jinan,Shandong,China).Control specimens(n =7)were obtained from Qilu Hospital Cadaver Donating Center.Control subjects did not have asthma,according to their history.The experiments were approved by local ethics committee,and written informed consent was obtained from these patients.1.2 Hematoxylin&eosin(H&E)staining was used to detect the basic pathology of airway from asthma patients1.3 Masson's trichrome staining was used to detect the expression of collagen of airways,1.4 Immunohistochemical staining was used to detect the expression of FSTL1?beclinl?the marker of EMT(E-Cadherin?N-Cadherin?vimenin)?the marker of remodeling(fibronectin??-SMA).1.5 Immunofluorescence staining was used for co-expression of FSTL1 and E-Cadherin in airway epithelial cell.1.6 Transmission electron microscopy was used for detect the autophagosomes in asthmatic bronchial epithelium.2.FSTL1 promotes EMT through binding DIP2A to activate autophagy in epithelial cells2.1 The human bronchial epithelial cell line(16HBE)was purchased from American Type Culture Collection(Manassas,VA).Cells were cultured in high-glucose Dulbecco's modified Eagle's medium complete medium supplemented with.10%fetal bovine serum(FBS).All cells were grown at 37? in humidified air with 5%C02.Cells were transfected with DIP2A siRNA(siDIP2A)and ATG5 siRNA(siATG5)and siRNA negative control(siRNA-NC)for 48 h using Lipofectamine 3000 according to the manufacturer's protocol.2.2 16HBE cells were treated with PBS,FSTL1(FSTL1 100 ng/ml),LY-294002(LY-294002 10 uM)and FSTL1+LY-294002(FSTL1 100 ng/ml+LY-294002 10 uM)respectively.2.3 The effect of FSTL1 treatment and blocking DIP2A on bronchial EMT.Western blot analysis was used to detect the expression of E-Cadherin,N-Cadherin,vimentin,collagen I and a-SMA after the cells were treated with the FSTL1 and si-DIP2A.Cell migration assay was used to detect the migration ability of these cells after they were treated with the FSTL1 and si-DIP2A.2.4 The effect of LY-294002 on bronchial epithelia autophagy and EMT.Transmission electron microscopy was used to detect the autophagosomes in 16HBEs.GFP-LC3 punctuate counts,GFP-LC3-expressing vectors were obtained from Addgene.Lentiviral supernatant was prepared according to the manufacturer's instructions and provided by GenePharma.Lentiviral infection was carried out accordingly.Cells with ?5 puncta were considered as GFP-LC3 puncta-positive cells.The percentage of GFP-LC3 puncta-positive cells was quantified by counting 200 GFP-LC3 stable cells.Western blot analysis was used to detect the expression of E-Cadherin,N-Cadherin,vimentin,collagen I and a-SMA.Cell migration assay was used to detect the migration ability of these cells.Collagen gel contraction assay was used to detect the contraction ability of these cells after receiveing different treatment.2.5 The effect of Knocking out the ATG5 gene on FSTL1-induced EMT.Western blot analysis was used to detect the expression of E-Cadherin,N-Cadherin,vimentin,collagen I and a-SMA after knocking out ATG5 by using si-RNA.3.The effect of blocking FSTL1 and autophagy on airway EMT and remodeling in mouse3.1 Animals.Eight-week-old female wild-type C57BL/6 mice were obtained from the Center of Experimental Animals of Shandong University.Fstllflox/+ mice were generous gifts from Xiang Gao(Nanjing University,Nanjing,China)and Xu Zhang(Institute of Neuroscience,Shanghai Institute for Biological Sciences,Chinese Academy of Sciences,Shanghai,China).Fstl1+/-mice were generated by the mating of Fstl1flox/+ mice and cmv-Cre mice.Mice were maintained in a standard laboratory animal facility for 1 week before the experiments.All the mouse experiments were approved by the Institutional Animal Care and Use Committee of Shandong University.3.2 Animal model.? ovalbumin(OVA)model,Mice were sensitized by intraperitoneal(ip)injection with 100 ug of ovalbumin and 2 mg of aluminum hydroxide(in 200 ul of phosphate-buffered saline(PBS)on days 0 and 14 and followed by intranasal administration of 200 ug of OVA in 20 ul of PBS on days 21,22,and 25.From day 28,mice continued challenges with intranasal OVA twice/wk for 4 wk.Non-OVA-challenged mice were sensitized and challenged with PBS only.? FSTL1 and LY-294002 treatment.Mice were challenged with 10 ug of FSTL1 in 50 ul of PBS intranasally daily for 15 days.LY-294002-treated mice were treated once daily with 7.5 mg/kg LY-294002 intraperitoneally 2 h before the FSTL1 challenge.Control groups received sterile PBS at all time points.3.3 The animal experimental group.CON-WT,OVA-WT,OVA-Fstl1flox/+,OVA-Fstl1+/-FSTL1-WT,FSTL1+LY-294002-WT.3.4 Assessment of AHR.The mice were anesthetized by chloral hydrate 24 h after the last challenge.Aerosolized methacholine was administered at a concentration of 0,4,8,12,or 16 mg/ml.Airway resistance was measured using flexiVent(Scireq)with increasing concentrations of methacholine.3.5 H&E and Periodic Acid-Schiff(PAS)staining were used to detect the basic pathology of airway of the mouse from each group.3.6 Enzyme linked immunosorbent assay(ELISA)was used to detect the level of FSTL1 in plasma and BALF from mice.3.7 Transmission electron microscopy was used for detecting the autophagosomes in bronchial epithelium from mouse.3.8 Immunohistochemical staining was used to detect the expression of FSTL1,beclinl,the marker of EMT(E-Cadherin,N-Cadherin,vimenin)and the marker of remodeling(fibronectin??-SMA).Results1.The expression of FSTL1 and the level of autophagy were upregulated in airways of patients with asthma,with a concomitant airway EMT and remodeling.1.1 There is no statistical difference in age between asthma and control group,the gender composition is same.Airway remodeling,such as mucus metaplasia,loss of epithelia integrity,subepithelial fibrosis,goblet cell hyperplasia was found in the airways of asthmatic patients.1.2 FSTL1 was highly expressed in bronchial epithelium in patients with asthma compared with control subjects,subepithelial fibrosis and decreased expression levels of E-Cadherin and increased expression levels of N-Cadherin,vimentin,and fibronectin were found in asthmatic airway epithelia.In addition,a-SMA was highly expressed in the thickened airway walls obtained from asthmatic samples.1.3 The level of autophagy was upregulated in airways of patients with asthma.Autophagy biomarkers beclin-1 were highly expressed in bronchial epithelium in patients with asthma compared with control subjects and the autophagosomes were detected in asthmatic bronchial epithelium.2.FSTL1 promotes EMT through binding DIP2A to activate autophagy in 16HBEs2.1 FSTL1 promoted EMT in 16HBE cells in vitro.16 HBE cells were treated with variable concentrations FSTL1 recombinant protein for 24h,and the expression of the biomarker of EMT and autophagy was examined.16HBE cells exposed to FSTL1 dose-dependently reduced E-Cadherin expression accompanied by upregulation of N-Cadherin and vimentin.We also detected these markers' expression in different time points.By Western blot analysis,we found that the level of EMT protein markers significantly changed following 100ng/ml FSTL1 stimulation for 24 h.2.2 FSTL1 promoted EMT through binding DIP2A.We blocked the effect of FSTL1 by silencing its receptor-DIP2A with si-DIP2A in 16HBE cells.The expression of E-Cadherin was increased,and N-Cadherin,vimentin,collagen I,and ?-SMA were decreased when DIP2A was silenced.These results demonstrated that FSTL1 could promote EMT and airway remodeling in asthma in vitro.Then,we also observed the effect of FSTL1 on cell migration.Increased cell migration induced by FSTL1 was shown by the transwell assays.2.3 FSTL1 activated the level of autophagy in 16HBEs.TEM analysis clearly demonstrated increased production of autophagosomes after FSTL1 treatment.To further address the role of FSTL1-induced autophagy in16HBE cells,we generated stably expressed GFP-LC3 16HBE cells.The percentage of GFP-LC3-positive puncta increased in FSTLl-treated cells.FSTL1 promoted the expression of beclinl and LC3B II.2.4 Inhibiting autophagy can decrease the FSTL1-induced EMT.LY-294002,an autophagy inhibitor,attenuated the effect of FSTL1 on autophagosome formation as well as EMT and airway remodeling-related protein expression.Additionally,LY-294002 can also attenuate FSTL1-induced cell migration and cell contraction.We further inhibited autophagy in 16HBE cells by knockdown of ATG5,an essential protein involved in the autophagy pathway,via using siRNA and found that si-ATG5 significantly decreased the expression of beclin-1 and LC3B II in FSTL1-treated cells compared with the control group.Taken together,these results indicated that autophagy was required in FSTL1-induced EMT and airway remodeling in 16HBE cells,3.The effect of dficiency of FSTL1 and inhibiting autophagy on airway EMT and remodeling in mouse3.1 The levels of FSTL1 in plasma and BALF were increased in OVA-challenged WT mice.3.2 The levels of autophagy,EMT,and airway remodeling were increased in the OVA-challenged WT mice.3.3 OVA-challenged Fstll+/-mice had a slight degree of autophagy.Concomitantly,the degree of EMT and airway remodeling was significantly attenuated compared with OVA-challenged Fstllflox/+ groups.3.4 LY-294002 treatment attenuated FSTL1-induced autophagy,EMT and airway remodeling in mouse.3.5 Deficiency of FSTL1 and treatment by LY-294002 can protect from methacholine-induced AHR.Conclusion1.The expression of FSTL1 and the level of autophagy were upregulated in airways of patients with asthma,with a concomitant airway EMT and remodeling.2.FSTL1 promoted autophagy and EMT through DIP2A in 16HBE cells in vitro.3.Fstll Knock down and LY-294002 treatment attenuated FSTL1-induced EMT and airway remodeling in mouse... |
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