Periplocin promotes wound healing through the activation of Src/ERK and PI3K/Akt pathways mediated by Na/K-ATPase

Periploca forrestii(PF) is mainly utilized for treatment of arthritis and traumatic injury historically. We had previously demonstrated that a fraction rich in cardiotonic steroids isolated from PF had the potential to facilitate wound healing. However, the exact material basis and mechanism of action responsible for wound healing is still unclear. Periplocin(PP) is the highest level of cardiotonic steroid included in PF. The present study aims to evaluate the efficacy of periplocin on wound healing systematically in vitro and in vivo.The L929 proliferation was determined by both MTT and EdU assay. Cell migration was tested by both scratch and transwell assay. The total amount of soluble collagen was assessed using a Sircol Collagen Assay Kit. The wound healing activity was evaluated in vivo using the excision rat models. Histopathology of the wounded skin on day 9 was studied via hematoxylin and eosin staining (HE) for general morphological observations and masson's trichrome staining for collagen deposition, respectively. The alteration in Src/ERK and PI3K/Akt pathways mediated by Na/K-ATPase was determined by western blot after the treatment with periplocin. The interaction between Na/K-ATPase and Src was tested by immunoprecipitation and immunostaining analysis.The results revealed that periplocin could significantly boost proliferation, migration and stimulate collagen production in fibroblast L929 cells, which is dependent on activation of Src/ERK and PI3K/Akt pathways mediated by Na/K-ATPase, and thus promoting wound healing. Indeed, inhibition of Na/K-ATPase/Src complex receptor by Src specific inhibitor or knocking down the Na/K-ATPase expression would abolish the subsequent activation of Src/ERK and PI3K/Akt pathways and attenuate periplocin-induced beneficial effects on wound healing. Additionally, the wound healing activity is also confirmed in a rat excisional wound model as evidenced by increased rate of wound closure, reepithelization, formation of granulation tissue and collagen accumulation.Collectively, we lay the rationale for traditional usage for traumatic injury, suggesting that periplocin and periploca forrestii is a promising candidate for management of chronic wounds.