Preliminary Studies On Screening,Fmoc Solid Phase Synthesis And The Anti-Pulmonary Fibrotic Activity Of FGF-2 Targeting Peptide G8

Objective This study is based on the preliminary research of laboratory and used the phage peptide library to screen targeting FGF-2 binding peptides G8.We then constructsed the idiopathic fibrosis animal model to study the inhibitory effect of targeted FGF-2 binding peptide G8 on pulmonary and providing a new approach for the treatment of pulmonary fibrosis.Method(1)The peptides targeting FGF-2 were screened by phage display peptide library.We verifiy the binding specificity of small molecule to FGF-2 by ELISA.(2)The Fmoc solid-phase chemistry was used to synthesize the peptide G8,and optimal the process conditions,including the optimal coupling agent,the optimal reaction time,and used reverse-phase HPLC to separate and purify the small peptides to optimal mobile phase elution gradient.(3)At the cellular level,in order to detect the biological activity of G8.The CCK-8 method was used to detect the toxic effect of G8 on mouse fibroblast L929,and the CCK-8 method was used to detect the inhibitory effect of G8 on FGF-2 induced proliferation of mouse fibroblast L929.The WB test was used to detect the inhibitory effect of this peptide on the expression of phosphorylated fibroblast growth factor receptor 2 (Phosphorylated-FGFR2,p-FGFR2)in human non-small cell lung cancer cell H1299 induced by FGF-2.(4)In vivo,we used the SD rat lung fibrosis by topical application BLM,to determine the role of G8 in pulmonary fibrosis.By measuring the body weight of the modeled rats,lung tissue hematoxylin-eosin staining(HE),Massion staining and immunity Histochemical detection of collagen ?,FGF-2 expression in lung tissue,to study the inhibitory effect of G8 on pulmonary fibrosis.Results(1)The phage peptide library screening targeting FGF-2 binding peptide is 12 peptides,we named it G8,and its theoretical molecular weight is 1357.56.ELISA results show that compared with other selected peptides,G8 can significantly bind to FGF-2 at 40 ?g / ml,with significant specificity,G8 is the target peptide.(2)Fmoc solid phase synthesis G8 used Wang-Resin as the carrier,the purity of the crude peptide synthesized using HBTU/DIE composite condensing agent is 39.552%,and the purity of the crude peptide using HOBt/DIC condensing agent is 54.247%.The crude peptide has higher purity and less heteropeptide.According to the color reaction of ninhydrin,the optimal condensation time of each amino acid is 30 min,60 min and 75 min respectively.Therefore,HOBt/DIC is selected as the optimal composite condensing agent to synthesize G8 with the most suitable condensation time,use the cutting fluid with TFA: EDT: TIS: water = 95: 2:2:1,and cut for 3 h.The molecular weight of the crude peptide obtained by MS detection was 1357.00,and the purity after purification was 95.124%,which reached the ideal purity requirement and can be used in subsequent experiments.(3)In the range of 0?5.12?g/ml,G8 has no toxic effect on L929 cells,and CCK-8 results show that G8 shows inhibitory effect on FGF-2 induced proliferation of L929 cells at the beginning of 50 ng/ml,while G8 significantly inhibited FGF-2 induced phosphorylation of FGFR2 in H1299 cells,indicating that solid-phase synthesized G8 has good biological activity against FGF-2.(4)In vivo,to construct SD rat lung fibrosis model induced by bleomycin,the results showed that G8 has a higher survival rate,a better lung structure,and significantly reduced collagen and FGF-2.The results show that G8 has a significant inhibitory effect on pulmonary fibrosis.FGF-2 is involved in the development of pulmonary fibrosis.Conclusion Successfully screened a small molecule peptide G8 targeting FGF-2 with a purity of more than 95%,which can specifically bind to FGF-2.G8 has no cytotoxic to L929 cells,G8 significantly inhibited the proliferation of L929 cells induced by FGF-2;G8 is down-regulated FGF-2-induced phosphorylation of FGFR2 in H1299 cells,So G8 had good biological activity against FGF-2.The results of animal models showed that G8 can inhibit pulmonary fibrosis by antagonizing FGF-2 and has potentially good therapeutic effects.