Lei Chen1, Pan Jiang2, Jinsong Li1, Zijian Xie3, Yunhui Xu3, Wei Qu2, Feng Feng4, Wenyuan Liu5. 1. Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China. 2. Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 211198, China. 3. Department of Physiology and Pharmacology, Mail Stop 1008, College of Medicine, University of Toledo, 3000 Arlington Avenue, Toledo, OH 43614, USA. 4. Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 211198, China; Jiangsu Food & Pharmaceutical Science College, 4 Meicheng Donglu, Huaian 223003, China. Electronic address: fengfeng@cpu.edu.cn. 5. Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China. Electronic address: liuwenyuan8506@163.com.
Abstract
BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
BACKGROUND: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.