Structure And Function Of Wound Healing Peptide In Skin Secretions Of Rana Plateau Abdomen(Nanorana Ventripunctata)

Objective:Cathelicidins are a host defense molecule peculiar to vertebrates.They play important biological functions in vivo,such as anti-microbial,anti-inflammatory,anti-oxidation,wound healing and angiogenesis.Many cathelicidins have been found in aquatic and terrestrial vertebrates,and their structures and functions have been studied in detail,especially the human cathelicidin molecule(LL-37).Amphibians are the transitional type between aquatic and terrestrial vertebrates.At present,about 7937 species of amphibians are recorded in the amphibian database(http://www.amphibiaweb.org/).However,little research has been done on the structure and function of cathelicidins in amphibians.At present,only cathelicidin(tylotoin)with wound repair activity has been isolated and purified from Tylototriton verrucoosus Anderson,but cathelicidin isolated from Anura has rarely been reported.In this study,nanorana ventripunctata,an amphibian tailless frog with strong ultraviolet radiation and long sunshine time,was used to screen skin repair related molecules in the body of the frog,to study its structure and possible functions.Methods:After the skin secretions of frog were collected,sephadex g-50 chromatography column and rp-hplc were used to isolate and purify the skin secretions,and the peaks with the activity of promoting cell migration were collected to determine the primary structure.At the same time,RNA was extracted from the skin of the frog,cDNA library was constructed,and polypeptides were synthesized.Then the synthetic samples and the purified samples were functionally identified.Minimal inhibitory concentration method(MIC)was used to detect the antimicrobial activity of the samples(15 strains,including 7 gram-positive,6 gram-negative and 2 fungi).The proliferation of HaCaT cells and HSF cells was detected by MTT assay.Migration of HaCaT cells was detected by cell scratch assay.The secretion levels of McP-1,TGF-1,TNF-and VEGF in mouse abdominal macrophage RAW264.7 were detected by ELISA.Expression levels of MAPK family proteins were detected by Western blot.Full-thickness skin injury model in mice was used to detect the activity of promoting wound repair.HE staining was used to detect epidermal regeneration and collagen distribution at the wound site.The number of myofibroblasts was detected by immunohistochemistry.Results:We isolated and purified a polypeptide with wound healing activity from the skin secretion of Rana plateau abdomen(Nanorana ventripunctata).The amino acid sequence of the N-terminal to C-terminal mature peptide was ARGKKECKDDRLLMKRGSFSYV determined by Edman degradation method.The result of molecular cloning showed that its precursor was composed of 146 amino acids.Blast sequence alignment showed that the polypeptide belonged to the cathelicidins family,so it was named cathelicidin-NV.The precursor peptide of cathelicidin-NV is composed of 146 amino acids.The relative molecular weight of MALDI-TOF was 2845.9 Da,while its theoretical molecular weight was 2847.34 Da,with a difference of only 1.47 Da.The inhibition ring test showed that cathelicidin-NV had no antimicrobial activity;MTT assay showed that cathelicidin-NV could promote the proliferation of HaCaT cells and HSF cells.Scratch test showed that cathelicidin-NV could promote the migration of HaCaT cells.ELISA results showed that cathelicidin-NV could promote the secretion of TGF-beta 1,TNF-alpha,VEGF and MCP-1 cytokines in RAW264.7 cells.Western blot analysis showed that cathelicidin-NV could up-regulate the expression of P-P38 and P-ERK.Compared with negative control,cathelicidin-NV can effectively promote wound healing in mouse trauma model.HE staining showed that cathelicidin-NV could accelerate epidermal regeneration and promote collagen production.Immunohistochemical results showed that cathelicidin-NV could promote the transformation of fibroblasts into myofibroblasts.Conclusion:A new wound healing peptide was isolated and purified from the skin secretion of Rana plateau abdomen.The amino acid sequence of the mature polypeptide was ARGKKECKDDRCRLLMKRGSFSYV.BLAST sequence alignment revealed that the polypeptide belonged to cathelicidin family.The results showed that cathelicidin-NV from the skin of Rana plateau abdomen had no antimicrobial activity,cytotoxicity and hemolytic activity to mammalian cells.Cathelicidin-NV can directly promote the proliferation of HSF cells in the injured area,thus accelerating the formation of granulation tissue.2.Cathelicidin-NV can directly promote the proliferation and migration of HaCaT cells in epidermis,thus accelerating the growth of epidermis in injured areas.3.Cathelicidin-NV promotes the secretion of monocyte chemokine-1,thus recruiting macrophages to aggregate and secreting a large number of TGF-beta 1,TNF-alpha,and VEGF.These cytokines activate proliferation-related signaling pathways.4.Cathelicidin-NV promotes wound healing by activating the MAPK proliferation signaling pathway.Cathelicidin-NV,compared with skin repair drugs currently used in clinic,can rapidly promote wound healing and show excellent therapeutic effect.It has the characteristics of small molecular weight,easy synthesis and low cost,and can be used as a reference template for future drug development.