| IntroductionThe lung fibrotic reaction is characterized by an exaggerated accumulation of collagen and other extracellular matrix components leading to an irreversible distortion of normal tissue architecture and loss of function.Lung fibrosis includes idiopathic lung fibrosis,pneumoconiosis,fibrosis induced by drug and radioactive ray and so on. Investigations to identify the cytokines involved in lung fibrosis suggest that transforming growth factor-β1(TGF-β1) plays an important role in the occurrence and the development of the lung fibrosis.TGF-β1 is secreted primarily as a latent complex consisting of the mature TGF-β1 and latent-associated peptide(LAP) that is termed latent TGF-β1(L-TGF-β1),after bleomycin induced lung injury.Mature TGF-β1 remains non-covalently associated with LAP at a 1:1 ratio.The non-covalent interactions of TGF-β1 with LAP block TGF-β1 from binding to its receptor,thus inducing it to become biologically inactive.It has been demonstrated that thrombospondin-1(TSP-1) has important effects in the activated process of L-TGF-β1 in humans and animal models of lung fibrosis. TSP-1 is a 450kDa homotrimer glucoprotein comprising 3 subunits.Each subunit contains an amidocyanogen-terminal and a carboxyl-terminal,a protocollagen homology region,a properdin-like domain(Typel repeated sequence),an epidermal growth factor-like(Type2 repeated sequence) and a calcium-binding domain(Type3 repeated sequence).The Typel repeat sequence is made of 3 properdin motifs(TSR1 TSR2,TSR3).TSP-1 specifically combines with CD36,which is a receptor for TSP-1 on alveolar macrophages that depends on a specific sequence CSVTCG,in TSR2 and TSR3 motifs.TSP-1 associates with the alveolar macrophage-derived L-TGF-β1 by KRFK sequence.The TSP-1/L-TGF-β1 complex then associates with the cell surface of the alveolar macrophage by interaction with TSP-1 and CD36.The plasmin generated by the macrophages releases TGF-β1 from the LAP after association of CD36 with the TSP-1/L-TGF-β1 complex.TGF-β1 is then available to react with its receptor to bring about its biological effect.The CSVTCG sequence in TSR2 and TSR3 of TSP-1 combines with its cell receptor CD36,which is the key point during the activation of L-TGF-β1.Therefore, TSP-1 functional fragment contained CSVTCG sequence specifically combined with CD36,and blocked both the association of the TSP-1/L-TGF-β1 complex with CD36 and the antagonistic effect against lung fibrosis.We cloned the gene of a TSR2-3 segment contained CSVTCG sequence,expressed and purified the target protein and successfully obtained a purified TSR2-3 GST fusion protein.We showed inhibitory effects of the TSP-1 functional fragment on the activation of L-TGF-β1 and lung fibrosis.These studies provide an experimental approach that contributes to our understanding of the molecular mechanism of lung fibrosis.Methods1.Construction and identification of pGEX-5X-2-TSR2-3 recombinant plasmidExtract tissue total RNA with TRIZOL method.Amplify TSR2-3 gene segments by RT-PCR.The gene segment pGEX-5X-2 and TSR2-3 were digested with EcolR I and Xol I and connected to pGEX-5X-2-TSR2-3 recombinant plasmid. Transform the DH5αand apply it on the LB culture medium to culture for overnight. Extract pGEX-5X-2-TSR2-3 recombinant plasmid and identify the positive colony with the enzymes.2.The recombination pGEX-5X-2-TSR2-3 plasmid expressing and expression proteins identifying and purifyingE.coli BL21 transformed with the positive recombinant plasmid expressed proteins induced by IPTG whose final concentration was 1.0mmol/L,cultured for 3 hours.Assay the expression products by SDS-PAGE and identify the proteins by Western blot.Apply GST antibody to incubation overnight and show the specific reaction band using ECL chemiluminescence system.Purify the recombination protein using the way of affinity chromatograph by GSTrapFF column.3.Assay of activity of TSR2-3 GST fusion protein and inhibitory effects of TSP-1 functional fragments on activation of L-TGF-pl by immunofluorescenceAlveolar macrophages(AMs) were obtained by bronchoalveolar lavage 7 days after intratracheal bleomycin administration(1)AMs were cultured in the presence of TSR2-3 GST fusion protein,GST protein.The cells were incubated overnight with anti-GST and anti-CD36 antibody and the cells were then incubated with secondary antibodies conjugated to TRITC or conjugated with FITC.Nuclear staining was achieved using TOP3.(2)AMs were cultured in the presence of TSR2-3 GST fusion protein,GST protein,TSP-1 functional peptide and control peptide,anti-CD36 antibody.The cells were incubated overnight with anti-TGF-β1 and anti-CD36 antibody and were then incubated with secondary antibodies conjugated to TRITC or conjugated with FITC.Nuclear staining was achieved using Hoechst.Image analysis was performed using a fluorescence microscope.4.Detection of TGF-β1 secreted by activated AMs by ELISATotal TGF-β1 was determined after acid activation and performed according to the recommendation of the manufacturer.The method for the determination of active TGF-β1 in same sample was modified by omitting acid activation.The wells were coated with capture antibody,followed by incubating overnight at 4℃.The next day,the plates were blocked with block buffer for 1 h.The wells were then added 100μl of samples and standards with incubation for 2 h at room temperature.The detection antibody was then added and the mixture was then incubated for 2h.Streptavidin-HRP was added,following which there was a further incubation for 20 min in the dark.After addition of the substrate solution,there was a further incubation for 20 min.Stop solution was added and the optical density immediately determined using an ELISA reader set to 450 nm.5.Determination of hydroxyproline contentHydroxyproline content was determined,using a hydroxyproline kit,performed according to the recommendations of the manufacturer.The results were calculated as micrograms of hydroxyproline per milligram of wet lung weight with hydroxyproline standards.6.Pathologic examinationSmall pieces of lung tissue were sectioned at 5μm.The tissue sections were stained with hematoxylin and eosin(HE) and van Gieson stains(vG) for collagen fibers.7.Immunohistochemical examination of the collagenâ… andâ…¢All sections were deparaffinized in xylene followed by 100%ethanol and then placed in a freshly prepared methanol-H2O2 solution for 30 minutes to block endogenous peroxidase activity for immunohistochemical examination.After incubation with the rabbit polyclonal anti-collagenâ… andâ…¢antibodies for 2 hours at 37℃,antigen-antibody complexes were detected with a SP kit.Peroxidase activity was revealed using 3,3'-diaminobenzidine-tetrahydrochloride a benzidine peroxide substrate.Sections were counterstained with hematoxylin for 3 minutes,rinsed,and mounted with glycerin gelatin for histological examination.Interpretation of results was as follows:brown particles in the cytoplasm or the cellular membrane were considered positive.The collagenâ… andâ…¢proteins were quantitatively analyzed using an image analysis.Using 10×40 fields,5 fields were randomly selected for every section.The integrated optical density(IOD) average represented the quantitative expressions of collagenâ… andâ…¢.Results1.Screening and assessment of the recombined pGEX-5X-2-TSR2-3 plasmidThe specificity DNA amplification strap was observed at the 390bp,which was coincidence with the prediction.The recombinant plasmid pGEX-5X-2-TSR2-3 was digested by the enzyme EcolR I,Xol I.The enzyme digestion segment was coincidence with the prediction.2.The expression and identification of the recombined TSR2-3 GST fusion proteinThe recombined pGEX-5X-2-TSR2-3 plasmid was expressed in the E.coli.BL21 and the expression product presented a limpid proteinum at the 39kDa,whose molecular weight was coincidence with the prediction.The strap at the corresponding location can combine specifically with the GST antibody by Western blot method.3.Purification of the recombined TSR2-3 GST fusion proteinE.coli.BL21.transformed pGEX-5X-2-TSR2-3 recombined plasmid were destroyed by the hypersound and passed through the GSTrapFF affinity colomn.The single protein band was showed at the 39kDa in the SDS-PAGE gel.4.Cell surface association of TSR2-3 GST fusion protein to CD36 by immunofluorescenceThe much co-localization of CD36(green emission) and GST(red emission) were found on the surface of AMs cultured in the presence of TSR2-3 GST fusion protein, which suggested TSR2-3 GST fusion protein could bind to CD36 and had biological activity. 5.Cell surface association of TGF-β1 to CD36 by immunofluorescenceWhen AMs,obtained 7 days after bleomycin-induced lung injury,were cultured in the presence of TSR2-3 GST fusion protein,TSP-1 functional peptide or anti-CD36 antibody,the presence of CD36(green emission) and TGF-β1(red emission) was markedly reduced and was not detected consistently on the same cells.Furthermore,the co-localization of TGF-β1 and CD36 markedly decreased.These showed that TSR2-3 GST fusion protein,TSP-1 functional peptide or anti-CD36 made the production of TGF-β1 decrease.6.Quantity of TGF-β1 secreted by activated AMs by ELISAThe quantities of total and active TGF-β1 secreted by AMs,activated by bleomycin and cultured in the presence of GST protein or TSP-1 control peptide were higher than those in the presence of TSR2-3 GST fusion protein,TSP-1 functional peptide or anti-CD36 antibody.7.Hydroxyproline contentThere were no significant differences in the hydroxyproline contents of the six groups at 7 days after the instillations.The hydroxyproline contents of the bleomycin groups and both the low-dose and high-dose TSP-1 control peptide groups were significantly higher than those of saline control groups(P＜0.05 or P＜0.01) at 14 and 21 days after the instillation.In addition the hydroxyproline contents of the low-dose and the high-dose TSP-1(447-452) synthetic peptide groups were significantly lower than those of bleomycin groups.8.Light microscopic examinationAnimals of the bleomycin model groups,low-dose and high-dose TSP-1 control peptide groups had patchy areas of inflammation and loosely distributed collagen fibers in the lung at 7 and 14 days after the instillation.There were less inflammation and tiny collagen fibers in the lung of mice of the low-dose and high-dose TSP-1 functional peptide groups.Animals of the bleomycin model groups,low-dose and high-dose TSP-1 control peptide groups had patchy lesions characterized by a progressive increase of the interstitial and alveolar inflammatory cells and alveolar septa thickening in lungas compared to the low-dose and high-dose TSP-1 functional peptide groups at 21 days after the instillation.vG stain was intensely positive for collagen fibers in the bleomycin model groups,low-dose and high-dose TSP-1 control peptide groups. However,vG stain was weakly positive for collagen fibers in the low-dose and high-dose TSP-1 functional peptide groups.9.The expressions of the collagenâ… andâ…¢There were weakly positive reactions for the scattered collagenâ… andâ…¢in the mesenchymal tissue of the saline control group.The expressions of collagenâ… andâ…¢of all groups were not significantly different at 7 days after the instillation.The expressions of collagenâ… andâ…¢of bleomycin model and TSP-1 control peptide and TSP-1 functional peptide groups were greater than those of saline control groups (P＜0.01 or P＜0.05).The expressions of collagenâ… andâ…¢of TSP-1 functional peptide groups were significantly lower than those of bleomycin groups(P＜0.01or P＜0.05).Conclusion1.The mouse TSR2-3 gene segments have been successfully cloned and efficiently expressed in E.coli BL21,and the TSR2-3 GST fusion proteins have been purified by GSTrapFF affinity colomn chromatography.2.TSP-1 functional fragments and the anti-CD36 antibody could inhibit activation of L-TGF-β1 secreted by bleomycin-induced lung injury-derived AMs in vitro.The inhibitory effect of an activation of L-TGF-(β1 may have occurred through a blocking of the binding of TSP-1 to CD36.3.The CSVTCG/TSP-1 functional peptide would be able to substantially reduce hydroxyproline contents and typeâ… andâ…¢collagen accumulation in a mouse model of lung fibrosis and attenuate the severity of lung injury induced by bleomycin,which may have inhibitory effects in the occurrence and the development of the lung fibrosis.
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