Antagonisms And Mechanisms Of Bone Morphogenetic Protein-7 On Silica-induced Pulmonary Fibrosis Through Modulating The Balance Of Smad Signaling Pathway

Silicosis is a pulmonary fibrosis disease caused by long-term inhale of silica dust,which is pathologically characterized by diffuse pulmonary fibrosis.The cause of silicosis is clear,but the exact pathogenesis is complex and has not been fully elucidated.The epithelial-mesenchymal transition(EMT)is a prevailing phenomenon that plays crucial roles in organic fibrosis.As a strong pro-fibrogenic cytokine,transforming growth factor-β(TGF-β)can induce the alveolar epithelial cell transition to fibroblasts via EMT and fibroblast-like behavior.Bone morphogenetic protein-7(BMP-7),belonging to the TGF-β superfamily,has the roles of remodelling airway,maintaining epithelial phenotype,inhibition of EMT and resistance to apoptosis.In recent years,BMP-7 is known as an antagonist against TGF-β-dependent fibrogenic activity in several animal and cell models involving fibrosis of a variety of organs such as kidney,liver,heart and colon.However,the relationship between BMP-7 and silica-induced fibrosis was seldom investigatedThis study aimed to:(1)explore the possibilities and mechanisms of silica induced EMT and fibrosis in RLE-6TN cells;(2)investigate how the anti-fibrosis effect of BMP-7 on silica-induced pulmonary fibrosis via regulating the balance of Smad signaling pathwayPart ⅠObjective:To elucidate whether the canonical Smad-dependent pathway took part in the silica induced EMT in RLE-6TN cells in vitroMethods:RLE-6TN and RAW264.7(RAW)cells were recoveried and activated morphologically,and third passages of the cells were used in vitro experiments.RAW medium were added 0,25,50,100μg/mL silica solution so as to collect supernatant after 24h,and the does-dependent experiments maintained for 48h according to the differently concentration of silica.The expressions of epithelial-mesenchymal,collagens and signal transducer proteins were detected by Western blot.Culture medium was kept for measurement of Hydroxyproline(Hyp)contentResult:(1)silica can damage epithelial cells and the level of SP-C was significantly decreased in the presence of supernatant in a dose-dependent manner;(2)Silica can induce RLE-6TN cells morphological changes,turned into a spindle-like mesenchymal phenotype,and lost their cell-cell contact.The expression of E-cadherin(E-cad),a characteristic epithelial phenotypic marker,gradually reduced in a dose-dependent manner.On the contrary,as a mesenchymal marker,Vimentin(Vim)and Fibronectin(FN)level gradually increased;(3)Silica can induce RLE-6TN cells fibrotic changes,the level of collagen Ⅰ,Ⅲ and Hyp were significantly increased;(4)Silica led to the activation of TGF-β/Smad pathway and inhibition of BMP-7/Smad pathway.Meanwhile,the mentioned above changes present a dose-dependent manner with a fibroblast-like behavior and migratory ability increasingPart ⅡObjective:To elucidate whether the balance of TGF-β/BMP/Smad signaling pathway regulated EMT and fibrosis on RLE-6TN cells induced by silica in vitroMethods:RLE-6TN and RAW264.7(RAW)cells were recoveried and activated morphologically,and third passages of the cells were used in vitro experiments.After the does-dependent experiments for choosing the optimal concentration of silica,the different stimulate factors were added and maintained for 48h according to the differently treated groups:(1)DMEM only,as a negative control(Control group);(2)DMSO or ddH2O,as vehicle control(Vehicle group);(3)RAW supernatant(50lug/mL silica)only,as a positive control(Silica group);(4)incubated together with additional RAW supernatant(50μg/mL silica)and SB-431542(5μM)or LDN-193189(InM)(Low-dose inhibitor group);(5)incubated together with additional RAW supernatant(50μg/mL silica)and SB-431542(10μM)or LDN-193189(5nM)(Medium-dose inhibitor group);(6)incubated together with additional RAW supernatant(50μg/mL silica)and SB-431542(20μM)or LDN-193189(10nM)(High-dose inhibitor group).In the inhibitor experiments,different concentrations of inhibitors were added in the cell cultures lh before adding RAW supernatant(50lug/mL silica).The expressions of fibrosis markers,as well assignal transducer proteins,were detected by Immunofluorescence and Western blot.Culture medium was kept for measurement of Hyp content.In addition,MTT assay was used to explore the toxic effects of SB-431542 and LDN-193189Result:(1)Western blot results showed that pre-treating with SB-431542 could attenuate the decrease of phosphorylated Smadl/5(P-Smadl/5)and the increase of phosphorylated Smad2/3(P-Smad2/3)and fibrosis-related markers(collagen Ⅰ,collagen Ⅲ,and FN).On the contrary,using LDN-193189 could promote the secretion of Hyp and fibrosis-related marker’s expression by increasing P-Smad2/3 and decreasing P-Smadl/5.After SB-431542 blocked TGF-β/Smad signal pathway,P-Smadl/5 expression was significantly increased.Meanwhile,LDN-193189 blocked BMP/Smad signal pathway,P-Smad2/3 expression was significantly increased Meanwhile,the mentioned above changes present a dose-dependent manner;(2)Immunofluorescence results demonstrated that SB-431542(20μM)could suppress the increase of collagen Ⅰ in comparison to silica group and LDN-193189(10nM)could promote the increase of collagen I.Part ⅢObjective:To investigate the anti-fibrotic effects and possible mechanisms of BMP-7 on silica induced fibrosis in RLE-6TN cellsMethods:RLE-6TN and RAW264.7(RAW)cells were recoveried and activated morphologically,and third passages of the cells were used in vitro experiments Firstly,we established BMP-7-over-expressing(BMP-7(+)/RLE-6TN)and BMP-7-silent(BMP-7(-)/RLE-6TN)cell lines by lentivirus-mediated gene transduction on RLE-6TN,then followed by RAW supernatant(50μg/mL)for 48h Secondly,the cells were incubated with exogenous recombinant human bone morphogenetic protein-7(rhBMP-7)for 48h,each containing one of the following conditions:(1)DMEM only,as a negative control(Control group);(2)RAW supernatant(50μg/mL)only,as a positive control(Silica group);(3)incubated together with additional RAW supernatant(50μg/mL)and BMP-7(800ng/mL)(BMP-7 group).Thirdly,the cells were incubated with neutralizing antibody of BMP-7,each containing one of the following conditions:(1)DMEM only,as a negative control(Control group);(2)RAW supernatant(50μg/mL)only,as a positive control(Silica group);(3)incubated together with RAW supernatant(50μg/mL)and BMP-7 neutralizing antibody(10μg/mL)(BMP-7 neutralizing group).Using qPCR and Western blot or Immunofluorescence,the mRNA and protein levels of BMP-7,fibronectin(FN),collagen Ⅰ and collagen Ⅲ were detected.The Smad signaling pathway proteins,including P-Smadl/5 and P-Smad2/3 were detected by using Western blot.Culture medium was kept for measurement of Hyp contentResult:(1)Silencing of BMP-7 could promote the secretion of Hyp and fibrosis-related mRNA and the protein’s expression by increasing P-Smad2/3 and decreasing P-Smadl/5;(2)Over-expressing BMP-7 could attenuate the decrease of P-Smadl/5 and the increase of P-Smad2/3 and fibrosis-related markers(collagen Ⅰ,collagen Ⅲ,and FN)induced by silica;(3)rhBMP-7 can protect RLE-6TN cells partly from silica-induced damage,which might be associated with partial with the block TGF-β/Smad signaling and the recovery BMP/Smad signaling;(4)Neutralizing BMP-7 can aggravate the fibrosis induced by silicaConclusions:(1)Silica could induce epithelial injury,EMT and mesenchymal changes on RLE-6TN cells in vitro,Smad signal pathway might be related to this process;(2)The TGF-β and BMP signaling balance was important to the development of silica induced EMT and mesenchymal changes on RLE-6TN cells;(3)BMP-7 exerts an inhibitory effect on the silica induced mesenchymal changes on RLE-6TN cells via a blockade of TGF-β signaling and a restoration of BMP signaling BMP-7 can stimulate the development of new therapeutic agents in silica induced fibrosis.