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Effects of Periploca forrestii Schltr on wound healing by Src meditated Mek/Erk and PI3K/Akt signals

伤口愈合 蛋白激酶B 体内 MTT法 传统医学 肉芽组织 药理学 免疫印迹 成纤维细胞 MAPK/ERK通路 化学 PI3K/AKT/mTOR通路 医学 体外 分子生物学 生物 免疫学 磷酸化 生物化学 信号转导 生物技术 基因
作者
Tsu-Wei Chou,Lei Chen,Jian Xu,Zijian Xie,Yunhui Xu,Pan Jiang,Bingjing Duan,Xiaoxian Huang,Feng Feng,Wenyuan Liu
出处
期刊:Journal of Ethnopharmacology [Elsevier]
卷期号:237: 116-127 被引量:14
标识
DOI:10.1016/j.jep.2019.03.046
摘要

Periploca forrestii Schltr. (PF) is a traditional folk medicine in China that has been used widely for treating rheumatoid arthritis and traumatic injuries for a long history. Previously, we have roughly demonstrated that the ethanol extract of PF possessed in vitro wound healing potential, and more in depth research deserves to be conducted. The present study is aiming to fully evaluate the wound healing activity of PF in vitro and in vivo, clarify the mechanism of actions and the primary constituents responsible for wound healing. The total extract of Periploca forrestii Schltr. (EPF) and its fraction (65% ethanol fraction, EPFE65) were obtained and evaluated on in vitro wound healing properties using mouse dermal fibroblasts (L929). Cell proliferation was tested by MTT and EdU assay, confirmed by cell cycle analysis, cell migration was evaluated by scratch and transwell assay and collagen production was also determined. Then EPFE65 was tested on in vivo wound healing activity using the excision rat models. The wounded skin of rats was topically applied with 0.1% EPFE65 once daily for 6 days with hydrogel as the carrier and the recombinant bovine basic fibroblast growth factor hydrogel (rbFGF) as positive control. Histopathology of the wounded skin on day 6 and day 12 was studied via hematoxylin and eosin (HE) staining. The expression of phosphorylation of Src, Akt and Erk1/2 was determined after the treatment with EPFE65 by western blot. In order to figure out whether the activation of Src, Akt and Erk1/2 was directly in conjunction with wound healing process promoted by EPFE65, cell proliferation and migration were tested in the presence of three inhibitors of Src, Akt and Erk1/2. Finally, the chemical composition of the effective fraction EPFE65 was analyzed by HPLC-Q-TOF-MS/MS. In vitro experiments suggested that EPFE65 was comparable to EPF that had potent effect on promoting L929 fibroblasts proliferation, migration and increasing collagen production. 0.1% EPFE65 hydrogel also exhibited significant effect on promoting wound healing in rats. The wound closure was significantly faster in EPFE65 and positive rbFGF group than that in negative control group since the third day post wounding (p < 0.05). Specifically, on day10–12, the wounds in EPFE65 and rbFGF group were almost healed as the wound areas diminished into 13.3–5.3% and 7.7–4.0%, while the wound in control group was still apparent with 36.8–22.1% wound area. HE staining demonstrated that EPFE65 and rbFGF group could advance re-epithelialization in the early days and promote the transition of granulation tissue into complete dermis tissue with more skin appendages resembling those of normal skin in the last days. Western blot results suggested that the active fraction EPFE65 could increase the phosphorylation of Src, Akt and Erk1/2 in both dose-dependent and time-dependent manner, whereas Akt and Erk1/2 phosphorylation caused by EPFE65 could be abolished by Src inhibition. Inhibition experiments confirmed that the activation of Src, Akt and Erk1/2 were involved in cell proliferation and migration. All of these demonstrated that EPFE65 promoted wound healing at least in part via Src mediated Mek/Erk and PI3K/Akt signaling pathways. Analysis of chemical composition of EPFE65 revealed that cardiac glycosides were major components in EPFE65, among which periplocin showed effectiveness on promoting fibroblasts proliferation indicating that cardiac glycosides in EPFE65 maybe the active compounds responsible for wound healing. The present study confirmed that EPFE65, ethanol extract of Periploca forrestii Schltr. could accelerate wound healing in vitro and in vivo through Src meditated Mek/Erk and PI3K/Akt signaling pathways.

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