Inhibitory Effect And Mechanism Of A Novel Peptide On Ocular Fibrosis

Purpose: Fibrosis occurs in a variety of tissues and organs,resulting in structural damage and functional impairment.In the eye,fibrotic disease mainly includes proliferative vitreoretinopathy(PVR),scar formation after glaucoma filtration surgery,corneal scar which are easy to cause blindness.However,there is currently no effective means to prevent or treat such diseases.Ophthalmic drugs need to have characteristics such as small molecular weight and low immunogenicity.Therefore,peptide drugs derived from endogenous protein display certain advantages for eye administration.In this study,we screened a novel biological peptide BP2 from the key active site of human endogenous protein bone morphogenetic protein-7(BMP-7),which has natural anti-fibrosis property.We investigated its safety,anti-fibrosis efficacy and mechanism in vivo and in vitro.Methods:(1)Peptides screening: Three different segments of peptide,BP1,BP2 and BP3 were screened from BMP-7 protein by bioinformatics method.The proliferation of human RPE-19 cells under normal condition or with growth factor induction were detected by MTS method in vitro.Scratch detection was used to prove effect of BP2 on RPE-19 cell migration.(2)Effect of BP2 on PVR in vivo and in vitro.In vitro,Transwell method,optical observation,collagen traction test,and immunofluorescence detection were adopted to test cell migration,transforming growth factor-?(TGF-?)induced changes in cell morphology,contraction,and key proteins expression under epithelial mesenchymal transformation(EMT).In addition,through establishment of a rabbit animal model of PVR and intraocular injection of BP2,the intraocular proliferative membrane and retinal detachment of PVR was examined by ultrasound,ophthalmoscope,fundus photography,histopathology,HE and MASSON staining.(3)BP2 intervention after glaucoma filtration surgery to prevent scar formation: in vitro,cultured human Tenon s Fibroblast(HTF)was detected after BP2 intervention by MTS method,flow cytometry,collagen contraction and immunofluorescence.In vivo,a rabbit model of glaucoma filtering surgery was established,changes of filtering bleb pathology and intraocular pressure monitoring were observed and HE and MASSON staining was examined after BP2 or mitomycin C(MMC)intervention.(4)Other scar models: we tested effect of BP2 on the proliferation of human corneal fibroblasts(HCF),the change of HCF mechanical migration,the level of inflammatory factors and the change of key proteins during EMT transformation of HCF.Besides,inhibitory effect was observed by establishing the rabbit skin scar model in vitro and examination of peptides on skin scarring by subcutaneous injection of BP2.(5)Safety of peptide in vivo: The safety of BP2 by intraocular injection,subconjunctival injection and ocular surface administration was evaluated by clinical observation,histopathology,intraocular pressure,electrophysiology and microstructure observation.(6)Mechanism of inhibition of fibrosis by peptides: The possible mechanisms of BP2 inhibition of fibrosis were investigated by Western blotting to detect the expression of important proteins in EMT and phosphorylation of Smad signaling,which is one of the critical pathways in fibrosis.Results:(1)Three peptides BP1,BP2 and BP3 were screened from the original protein BMP-7,and BP2 showed the best biological activity in vitro not only in safety but also in inhibition of abnormal proliferation and mechanical migration of RPE cells.(2)BP2 effectively inhibited fibrosis in vitro and in vivo in PVR model and RPE cells.The expression of ?-smooth muscle actin(?-SMA)in EMT cells was significantly lower than that in the control group(p <0.05)and zonula occludens-1(ZO-1),respectively(p <0.05).In vivo,intraocular injection of BP2 could reduce the formation of proliferative membrane and retinal detachment in the rabbit PVR model.(3)BP2 inhibited HTF cell myofibroblast transformation and glaucoma bleb scar formation.(P <0.05).After BP2 intervention,the apoptosis was significantly lower than that of MMC.HTF contraction and expression of cytoskeleton protein,?-SMA and collagen were significantly inhibited after TGF-? stimulation.In vivo,BP2 can maintain the shape of filter bleb.Although the inhibition effect against the scar was not superior to MMC,but the side effects were much less than the MMC group.(4)Peptide could inhibit the mechanical migration of corneal cells in vitro,release of inflammatory factors,and ?-SMA expression(p <0.05).BP2 reduced the rabbit ear skin scar model scar severity.(5)BP2 had no obvious toxicity on the microstructure,electrophysiological function and intraocular pressure of rabbits.(6)BP2 inhibited fibrosis by inhibiting Smad2,Smad3 phosphorylation and expression of important proteins in EMT.The expression of ?-SMA,ZO-1,E-cadherin,and fibronectin changes after TGF-? induction in RPE,HTF and HCF cells but was attenuated by BP2 in Western detection(P <0.05),so were the phosphorylation of Smad2 and Smad3(p <0.05).Conclusion: Bioinformatics-based novel bio-peptide BP2 can safely and effectively inhibit ocular fibrosis in vivo and in vivo,including PVR,glaucoma filtering scar,corneal scar and skin scar.The anti-fibrosis mechanism was probably through reducing EMT transformation and collagen expression by inhibiting Smad2 / 3 pathway.BP2 is a novel polypeptide with a natural anti-fibrotic property and has the potential for clinical transformation.