Bee-derived antibacterial peptide, defensin-1, promotes wound re-epithelialisation in vitro and in vivo (original) (raw)

Royal jelly (RJ) has successfully been used as a remedy in wound healing. RJ has multiple effects, including antibacterial, anti-inflammatory and immunomodulatory activities, in various cell types. However, no component(s) (other than antibacterial) have been identified in RJ-accelerated wound healing. In this study, we demonstrate that keratinocytes are responsible for the elevated production of matrix metalloproteinase-9 (MMP-9) after incubation with a water extract of RJ. Furthermore, the keratinocyte migration and wound closure rates were significantly increased in the presence of RJ extract. MMP-9 production was reduced significantly following proteinase K treatment but remained stable after heat treatment, indicating that active component(s) have a proteinous character. To identify the component responsible for inducing MMP-9 production, RJ extract was fractionated using C18 RP-HPLC. In fractions exhibiting stimulatory activity, we immunochemically detected the bee-derived antibacterial peptide, defensin-1. Defensin-1 was cloned, and recombinant peptide was produced in a baculoviral expression system. Defensin-1 stimulated MMP-9 secretion from keratinocytes and increased keratinocyte migration and wound closure in vitro. In addition, defensin-1 promoted re-epithelisation and wound closure in uninfected excision wounds. These data indisputably demonstrate that defensin-1, a regular but concentration variable factor found in honey and RJ, contributes to cutaneous wound closure by enhancing keratinocyte migration and MMP-9 secretion. Keratinocytes, a major cellular component of the epidermis, are responsible for restoring the epidermis after injury through a process termed epithelialisation. The migration, proliferation, and differentiation of fibroblasts and keratinocytes, as well as interactions between these cells are critical for effective re-epithelialisation and wound healing. Immature keratinocytes produce matrix metalloproteinases (MMPs), including MMP-9 and MMP-2, and plasmin, which enables their dissociation from the basement membrane and facilitates their migration. MMP-9 (gelatinase B) is a zinc-dependent endopeptidase that is involved in the proteolytic degradation of extracellular matrix proteins, such as type III and IV collagens and elastin. MMP-9 plays an important role in normal wound healing, particularly related to extracellular matrix (ECM) remodelling and re-epithelialisation. Wound healing is impaired when MMP-9 is inhibited 1, 2. Historically, honeybee products, such as honey and royal jelly (RJ), have been used to treat a broad spectrum of injuries. RJ is part of the diet of honeybee larvae and is secreted from the hypopharyngeal and mandibular glands of worker honey bees 3. RJ has been used since ancient times to facilitate wound healing. RJ acts as an antimicrobial 4-8 and antioxidative agent 9 and as an immunomodulator with anti-inflammatory properties 10, 11. The topical application of RJ to treat human diabetic foot ulcers 12-14 provides compelling evidence that RJ can accelerate wound healing. Furthermore, RJ promotes wound healing in an animal model of uninfected wound 15. However, the mechanisms of action, other than antibacterial effects, associated with the effects of RJ