Effect And Mechanism Of Active Peptide RL-QN15 On Diabetic Foot Ulcer Repair

Objectives: Wound management of diabetic foot ulcers(DFUs)is a complex and challenging task,and existing strategies fail to meet clinical needs.Therefore,it is important to develop novel drug candidates and discover new therapeutic targets.However,reports on peptides as molecular probes for resolving issues related to DFUs remain rare.In this study,RL-QN15,a skin-derived active peptide from Rana limnocharis,was used as an exogenous molecular probe to explore its possible regulatory mechanism of "cell signaling pathway-micro RNA-cell behavior eventdiabetic foot ulcer wound healing axis",providing new ideas for clinical treatment of diabetic foot ulcer and exploring potential drug targets.Methods: First,we simulated a high glucose environment in vitro,and explored the ability of RL-QN15 to promote the wound repair activity,migration and proliferation of Ha Ca T cells in a high glucose environment by using cell scratch,cell proliferation and Transwell migration experiments.Secondly,we established a rat model of DFUs to explore the role of RL-QN15 in promoting wound healing in vivo.Next,RNA sequencing,in vitro transfection,quantitative real-time polymerase chain reaction,western blotting,dual luciferase reporter gene detection were performed to explore the potential mechanism underlying the promoting effects of RL-QN15 on DFU repair.Subsequently,changes in keratinocyte migration and proliferation were analyzed by cell proliferation assay,cell scratch assay,and Transwell migration assay.Results: Peptide RL-QN15 can significantly promote the scratch repair activity,migration and proliferation of Ha Ca T cells in a high-glucose environment and accelerated wound healing in a DFU rat model.Histopathological results showed that RL-QN15 promoted re-epithelialization and angiogenesis at the wound site.Based on results from RNA sequencing,we found a new miRNA(miR-4482-3p)related to the promotion of wound healing.The bioactivity of miR-4482-3p was verified by inhibiting and overexpressing miR-4482-3p.Inhibition of miR-4482-3p enhanced the migration and proliferation ability of Ha Ca T cells as well as the expression of vascular endothelial growth factor B(VEGFB),The opposite trend was found for keratinocytes treated with the miR-4482-3p mimic.RL-QN15 also promoted the migration and proliferation ability of Ha Ca T cells and VEGFB expression was mediated via inhibition of miR-4482-3p expression by the p38 MAPK and smad3 signaling pathways.The dual luciferase reporter assay also showed binding between hsa-miR-4482-3p and VEGFB.Conclusions: RL-QN15 is an effective intervention for the treatment of DFUs,with the underlying mechanism related to the inhibition of miR-4482-3p expression via the p38 MAPK and smad3 signaling pathways,ultimately promoting re-epithelialization,angiogenesis,and wound healing.This study provides a theoretical basis for the clinical application of RL-QN15 as a molecular probe in promoting DFUs wound healing.