Structural And Founctional Research Of A Peptide Which Promotes The Healing Of Skin Wound

Objective Skin wound is a common form of skin damage in daily life.How to promote the process of skin wound remains a serious challenge in clinic.With high activity,specificity and stability,bioactive peptides have been considered as potentially therapeutic candidates for wound healing in recent years.The study of structure,function and mechanism is great significance for the research and development of new drugs.Methods We captured a Yunnan Odorrana frog(Odorrana andersonii)in the wild.After cDNA synthesis,illumina MiSeq sequencing and processing of data,a peptide was identified from the skin.Subsequently,scratch test on cells,cell proliferation test and transwell migration experiment were performed to test the wound-healing activity of the peptide in vitro;Animal wound healing and tissue staining analysis were performed in vivo;What is more,the mechanism was further explored by inflammatory cytokines and epidermal growth factor receptor(EGFR)test;In addition,hemolysis test,acute toxicity test,antimicrobial activity test,radical scavenging activity detection and stability analysis were aslo performed.Results The peptide,OA-GL12,identified of amphibian O.andersonii is composed of 12 amino acids and the sequence is 'GLLSGINAEWPC'.Compared with the control,OA-GL12 significantly promoted the healing of HaCaT and HSF cell scratches in both time-and concentration-dependent manners in vitro,which might have little relation with the proliferation but the migration of HaCaT and HSF cells.Meanwhile,when compared with the control,OA-GL12 promoted the healing of full-thickness skin wounds in vivo by the regeneration of epidermis and dermis at the wound site and the contraction of granulation tissue.In addition,compared with the control group,OA-GL12 induced the secretion of TNF in RAW264.7 cells in concentration-dependent manner,which recruited more macrophages to the wound site with relations of TGF-?1.In further mechanism studies,the results indicated that EGFR made a great contribution to the scratch repair activity of OA-GL12.In addition,OA-GL12 showed ABTS+and DPPH free radical scavenging activity but no direct antimicrobial activity,hemolytic or acute toxicity.Conclusion1.A novel peptide,OA-GL12,with a mature sequence of‘GLLSGINAEWPC',was identified from the skin of amphibian O.andersonii.2.Results revealed that OA-GL12 could accelerate wound healing at certain concentrations in vitro and in vivo.3.Further mechanism studies revealed that OA-GL12 improved the healing with the participation of TGF-?1 and EGFR.4.OA-GL12 showed antioxidant activity,but no direct antimicrobial activity,hemolytic or acute toxicity.