The Study Of RGD Peptide Hydrogel Anti-scarring Through Extracellular Matrix Mechanical Signal YAP

Background Reducing intraocular pressure is currently the only clinically proven effective treatment that can reduce the risk of glaucoma progression.The purpose of glaucoma filtering surgery is to form a functional filtering channel to ensure the smooth discharge of aqueous humor.The success of surgery mainly depends on the degree and time of conjunctival fibrosis during postoperative wound healing.If the Tenon ’s capsule fibroblasts at the site of conjunctival injury continue to transform into myofibroblasts,the extracellular matrix(ECM)will deposit and harden to produce scars,resulting in the disappearance of filtering blebs and the failure of surgery.Yes-associated protein(YAP)is the key mechanical signal of fibroblast activation and fibrosis through mechanical force pathway.Fibroblasts can regulate YAP and express pro-fibrotic protein through mechanical signal and transforming growth factor-β(TGF-β)in fibrotic ECM,thus further causing fibroblast activation and ECM protein secretion and aggravating scarring.Objective To explore whether the new biomaterial RGD peptide hydrogel can reduce the activation of Tenon ’s capsule fibroblasts and secrete ECM-related proteins by inhibiting the mechanical signal YAP.Methods In vitro experiments:(1)By commercially synthesizing 0.5%,1% and 2%concentrations of self-assembled peptide RGD polypeptide hydrogel,the internal microstructure was observed by transmission electron microscopy(TEM),and the mechanical properties were detected by rheometer.(2)Successfully cultured human embryonic Tenon’s cystic fibroblast HFTFs in vitro,and used TGF-β2 to stimulate HFTFs activation to construct a scarred cell model after glaucoma filtration in vitro.(3)According to different treatment factors,HFTF cells were divided into:(1)normal control group: no special treatment;(2)TGF-β2 group:TGF-β2 stimulation;(3)Experimental group: After TGF-β2 stimulation,three concentrations of RGD polypeptide hydrogel were given: 0.5% concentration,1% concentration,and 2%concentration;(4)Positive control group: After TGF-β2 stimulation,verteprofin is given.(4)The expression of YAP,α-SMA,FN,Col I,CTGF proteins and the relative transcription levels of genes in each group of cells were detected by western blot experiment(WB)and real-time PCR(q-PCR),and the expression and localization of cytoskeletal proteins F-actin and YAP were detected by cellular immunofluorescence staining.In vivo experiments:(1)20 7-weekold male SD rats were evenly divided into model group and experimental group: after constructing the mouse eye injury model,three concentrations of RGD polypeptide hydrogel of 0.5%,1% and 2% were injected under the conjunctiva,and the lesion site sections were stained by Masson staining one week later to observe collagen fiber morphology,and the average optical density of YAP and α-SMA was detected as their expression after immunohistochemical staining,and finally the results were statistically analyzed.Results In vitro experiments:(1)TEM results showed that RGD peptide hydrogels with three concentrations could self-assemble into nanofiber network structure,and the pore structure was relatively simple.However,with the increase of concentration,the crosslinking of hydrogels was more intensive.Rheological results showed that the three concentrations of RGD peptide hydrogels were G ’ ≥ G ’,which conformed to the gel kinetic characteristics and showed dynamic viscoelasticity under alternating stress.Young ’s modulus increased with the increase of peptide concentration,which was about 0.067 k Pa,0.15 k Pa and 2.17 k Pa,respectively.The matrix stiffness of 1 % RGD peptide hydrogel was more consistent with the stiffness of connective tissue.(2)The results of WB and q-PCR experiments showed that the protein expression levels and gene relative transcription levels of YAP,α-SMA,FN,Col I and CTGF were significantly increased after TGF-β2-induced activation of HFTF cells.In the experimental group,the protein expression levels and gene relative transcription levels of YAP,α-SMA,FN,Col I and CTGF were significantly decreased compared with the TGF-β2 group after treatment with different concentrations of RGD peptide hydrogel in the postoperative scar cell model,and the difference was statistically significant(P < 0.05).The inhibitory effect of RGD peptide hydrogel with 1 % concentration was the best,which was equivalent to that of YAP inhibitor verteporfin group,and the difference was not statistically significant.(3)The results of immunofluorescence showed that HFTFs were activated by TGF-β2,and the morphology of HFTFs was star-shaped and extended to the periphery.The fluorescence signal of F-actin was enhanced,and YAP was highly expressed in the nucleus.After adding different concentrations of hydrogel,the cell pseudopods were recovered,the F-actin fluorescence signal was weakened,and YAP was gradually expressed in the cytoplasm.The 1 % concentration group could significantly induce YAP translocation to the cytoplasm.Verteporfin can inhibit the high expression of YAP in the cytoplasm and reduce the production of filamentous stress fibers.In vivo experiments:(1)Masson staining: Compared with the simple model group,the proliferation area of collagen fibers in the 0.5 % and 2 % concentration groups was small,the color was light,and the arrangement between collagen was slightly disordered.The collagen fibers in the 1 % concentration group were slightly darker,the collagens were arranged neatly and continuously,and the area of new collagen fibers was the smallest,which was statistically significant compared with the model group(P < 0.05).(2)Immunohistochemical staining :Compared with the model group,the expression of YAP and α-SMA in the three concentration RGD peptide hydrogel groups decreased to varying degrees,and the 1 % concentration group was the lowest(P < 0.05).Conclusion(1)RGD peptide hydrogels with concentrations of 0.5 %,1 % and 2 % can self-assemble to form nanofiber network structure,and have certain mechanical properties,which can simulate ECM with different hardness.(2)TGF-β2 can induce the activation of HFTF cells and the synthesis of ECM proteins.Different concentrations of RGD peptide hydrogels can inhibit the expression of activation phenotype protein α-SMA of HFTF cells,reduce the expression of mechanical signal YAP and its accumulation in the nucleus,inhibit the transcriptional expression of downstream target gene CTGF,and down-regulate the expression of ECM protein FN and Col I.Among them,1 % concentration of RGD peptide hydrogel has the best inhibitory effect.(3)0.5 %,1 %,2 % concentration of RGD peptide hydrogel had antiscarring effect in mouse eye injury model,which could reduce the regeneration and deposition of collagen fibers,as well as the expression of YAP and α-SMA,but 1 % concentration had the best effect.