Fstl1 Is Involved In Bleomycin-induced Pulmonary Fibrosis Via TGF-?1/MAPK Signaling Pathway

Objective: Idiopathic pulmonary fibrosis?IPF?is a chronic,progressive and fatal interstitial lung disease with no outstanding therapeutic effect due to the unclarity of pathogenesis.The pathogenesis of IPF remains unknown,but with the developing study on this disease,iterative alveolar epithelial injury and activated of myofibroblast are now regarded as the direct reason of fibrogenesis.several profibrotic mediators are released during the process of injury and repair of alveolar epithelial cell induced fibroblast differentiation,proliferation,migration and even invasion through autocrine or paracrine way,and this makes extracellular matrix accumulate,hinder normal tissue repair and fibrosis becomes established eventually.Transforming growth factor-?1?TGF-?1?as a profibrotic factor is the central switch in development of IPF.TGF-?1 along with its downstream signaling pathways have influential role on the pathogenesis of IPF making it an attractive therapeutic target.Follistatin-like 1?Fstl1?,initially identified as a TGF-?1-inducible gene,encodes a secreted extracellular glycoprotein.Previous studies of our group have demonstrated that Fstl1 possesses profibrotic effect in lung tissue and fibroblasts from IPF patients,in bleomycin-induced fibrosis mouse model and in Fstl1^+/-mouse model.Fstl1 regulated myofibroblast differentiation and extracellular matrix?ECM?deposition though promoting TGF-?1/Smad2/3 signaling pathway.In addition,neutralizing antibody of Fstl1 alleviated TGF-?1 induced myofibrobalst differentiation and ECM deposition in fibroblast from IPF patients and bleomycin-treated fibrosis in mice in vivo confirming the crucial effect of Fstl1 on lung fibrosis.TGF-?1 regulates its downstream Smad or non-Smad signaling?MAPK or Akt?to facilitate the development of lung fibrosis in various stages.Previous study of our research group has demonstrated that Fstl1 has crutial effect on lung fibrosis though TGF-?1/Smad2/3 signaling,however,whether non-Smad signaling like MAPK is involoved in pulmonary fibrosis remains unreported.Therefore,relative to the canonical TGF-?1/Smad2/3 signaling,the aim of this study was to investigate whether Fstl1 is involved in pulmonary fibrosis via noncanonical MAPK signal pathway,and the likely mechanisms in lung fibrogenesis.Material and Methods: 1.In vivo,Fstl1^+/-and wild type C57BL/6J mice were injected bleomycin into trachea,while saline were injected in control group.Sacrificed them 14 days later,the lung tissue were stained with HE,and determination of hydroxyproline content was measured to verify the establishment of pulmonary fibrosis model.The m RNA and protein level of Fstl1,as well as the change of MAPK signaling phosphorylation were measured by applying q RT-PCR and western blot.Primary pulmonary fibrosis separated from Fstl1^+/-and wild type C57BL/6J mice were observed the difference of MAPK signaling between the two groups.2.In vitro,after stimulated by TGF-?1,lung fibroblast from Fstl1^+/-and wild type mice were measured by western blot and cell immunofluorescence to observe fibroblast differentiation and the effect of Fstl1 deficiency on it.After loss of function and gain of function of Fstl1,activation of MAPK signaling was observed by applying western blot between Fstl1^+/-and wild type group added with ectogenous Fstl1 protein.In MLg,adding inhibitors of ERK,p38 and JNK respectively 1 hour before treated with TGF-?1 and?or?Fstl1 for 24 hours,observing differentiation,proliferation,migration and invasion by western blot,MTT analysis and transwell.Results: After intratracheal injection of bleomycin,the HE stain of lung tissue showed that collagen deposition was aggravating gradually,in the meantime,hydroxyproline content was increasing in 1,7,14 days,turned out the model of pulmonary fibrosis in mice was successfully built.The m RNA and protein level of Fstl1 reveal time-dependent time increase,meanwhile,the MAPK signaling was activated,Fstl1 suppressed MAPK signaling pathway.In cellular experiments,TGF-?1 induced lung fibroblasts differentiate to myofibroblasts and Fstl1 production.TGF-?1 combined with Fstl1 protein facilitated MAPK signaling pathway activation.Not only did Fstl1+ /-attenuate pulmonary fibroblast differentiation,but also inhibited MAPK signaling.In MLg,inhibitors of p38,JNK suppressed MLg differentiation,proliferation,migration and invasion,while ERK inhibitor has no prominent negative effect on MLg profibrotic functions.Conclusions: These results suggest that Fstl1 is a profibrotic factor in pulmonary fibrosis,and has effect on lung fibroblast differentiation,proliferation,migration and invasion through p38,JNK signalings,which may influence development of pulmonary fibrosis.