The Designer Antimicrobial Peptide A-hBD-2 Facilitates Skin Wound Healing By Stimulating Keratinocyte Migration And Proliferation

Part I: Design of New Human ? Defensins 2 and Detection of Its Various PropertiesObjective: Antimicrobial peptides(AMPs)has gained wide attention for its broad-spectrum antimicrobial activity.However,its application in clinical was limited by its high cytotoxicity and poor structure stability.By changing the structure of hBD2 could effectively improve its properties.Methods: By changing the GIGDP of human ? defensins 2(hBD2)with APLAM,we designed a new hBD2 which was called A-hBD-2.The online website tools were used to predict the primary structure and secondary structure of A-hBD-2.Then its cytotoxicity to Ha Ca T cells,salt resistance and antibacterial to Staphylococcus aureus were detected.Results: The peptide hydrophobicity and charge were increased in the primary structure of A-hBD-2.Besides,an alpha helix was found in the secondary structure of A-hBD-2.The cytotoxicity of A-hBD-2 to Ha Ca T cells were significantly decreased when compared with hBD2.In addition,its ability of antibacterial to Staphylococcus aureus and salt resistance were significantly increased when compared with hBD2.Conclusions: A-hBD-2 has advantages of lower cytotoxicity,higher structure stability and stronger antibacterial properties.Part ? The Effect of A-hBD-2 on Ha Ca T Cells and Its MechanismsObjective: To assess the effect of A-hBD-2 on Ha Ca T cells and its underlying mechanisms.Methods: CCK8 and WB was used to detected the proliferation of Ha Ca T cells after cells were incubated with PBS,hBD2 and A-hBD-2.The effect of A-hBD-2 on Ha Ca T migration was observed by Transwell assay.The underlying mechanism of A-hBD-2 on the proliferation and migration of Ha Ca T cells was investigated.Then,we observed the effect of A-hBD-2 on the migration of Ca2+.In addition,the PCR and ELISA kit was used to assess the effect of A-hBD-2 to inflammatory cytokines(IL-6,IL-10 and TNF-?)and chemokines(MCP-1).Results: A-hBD-2 promotes proliferation and migration of Ha Ca T cells via EGFR and STAT3 signaling pathway.In addition,A-hBD-2 elicited mobilization of intracellular Ca2+ and stimulated keratinocytes to produce pro-and anti-inflammatory cytokines and chemokines via PLC signaling pathway.Conclusions: A-hBD-2 has the ability of promotes Ha Ca T cells proliferation,migration and produces inflammatory cytokines.Part ? The Effect of A-hBD-2 on Wound HealingObjective: To assess the effect of A-hBD-2 on wound healing of rat.Methods: After one full-thickness dorsal wound was created on each rat,the rats were randomly divided into three groups: control group(PBS),hBD2 group and A-hBD-2 group.Then the PBS,hBD2 or A-hBD-2 were dropped into wound site each day.The wound was monitored by taking photographs on days 0,3,5,7 and 10.Then,wound healing rate was calculated and epidermal thickness was measured through HE staining.Results: A-hBD-2 has the ability of promotes wound healing and increases the thickness of epidermal.Conclusions: Our data provide evidence that A-hBD-2 could promotes wound healing,suggesting that A-hBD-2 may be a promising candidate therapy for wound healing.In addition,our data provides theoretical basis for clinical trials.