王不留行黄酮苷激活bFGFR及下游通路促进创伤愈合机制研究
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摘要
目的考察王不留行黄酮苷促创伤愈合作用,并探讨其作用机制。方法构建SD大鼠皮肤开放性创伤模型,创伤部位分别涂抹0.02 g空白软膏剂基质(模型组)、0.02 g含0.1%王不留行黄酮苷的软膏剂(王不留行黄酮苷组)、0.02 g美宝润湿烧伤膏(阳性药)。观察创伤愈合速率,并取创伤部位皮肤制作石蜡切片,通过HE染色进行组织病理学评估,通过免疫组化染色观察增殖细胞核抗原(PCNA)、p-碱性成纤维细胞生长因子受体(p-b FGFR)、p-血管内皮细胞生长因子受体(p-VEGFR)、CD31、p-Akt、p-Erk表达,Western blotting法分析Erk和Akt蛋白的磷酸化水平、b FGFR磷酸化水平。结果与模型组比较,创伤后3、6、9 d,王不留行黄酮苷组显著促进开放性创伤愈合(P<0.05、0.01);随着时间的延长,与模型组比较,王不留行黄酮苷组中成纤维细胞和内皮细胞大量增殖,炎症细胞增殖减少,微血管密度显著增加(P<0.01);免疫组化及Western blotting结果显示,与模型组比较,王不留行黄酮苷组内皮细胞膜受体中b FGFR的磷酸化程度明显升高(P<0.05、0.01),p-VEGFR磷酸化程度无明显变化,PI3K/Akt与MAPK/Erk信号通路的节点蛋白Akt和Erk磷酸化程度均明显升高(P<0.05)。结论王不留行黄酮苷促进开放性创伤愈合,机制可能与激活b FGFR及其下游MAPK/Erk和PI3K/Akt信号通路相关。
Objective To investigate the effect of vaccarin on wound healing and to explore the mechanism of vaccarin promoting wound healing. Methods The open wound model of SD rat skin was constructed, and the wound sites were smeared with 0.02 g blank cream matrix(model group), 0.02 g containing 0.1% vaccarin(vaccarin group) or 0.02 g MEBO wetting burn ointment(positive drug). The rate of wound healing was observed and paraffin sections were made from the skin of the wound. Histopathological evaluation was performed by HE staining. Proliferating cell nuclear antigen(PCNA), p-Akt, p-Erk P-basic fibroblast growth factor receptor(p-bFGFR), p-vascular endothelial cell growth factor receptor(p-VEGFR), and CD31 were observed by immunohistochemical staining. Western blotting method were used to analyze the phosphorylation level of Erk, Akt, and bFGFR protein. Results Compared with model group, 3, 6, and 9 d after trauma, vaccarin significantly promoted the healing of open wound healing(P < 0.05, 0.01). Compared with model group, the fibroblasts and endothelial cells in vaccarin group were proliferating, the proliferation of the inflammatory cells decreased and the microvessel density increased significantly(P < 0.01); The results of immunohistochemistry and Western blotting showed that the degree of phosphorylation of bFGFR in the endothelial cell membrane receptor of vaccarin group was significantly higher than that in model group(P < 0.05, 0.01), and the degree of p-VEGFR phosphorylation was not significantly changed, and the degree of Akt and Erk phosphorylation of PI3 K/Akt and MAPK/Erk signaling pathway were significantly increased(P < 0.05). Conclusion Vaccarin could promote open wound healing, and may be related to activation of bFGFR and its downstream MAPK/Erk and PI3 K/Akt signaling pathways.
Objective To investigate the effect of vaccarin on wound healing and to explore the mechanism of vaccarin promoting wound healing. Methods The open wound model of SD rat skin was constructed, and the wound sites were smeared with 0.02 g blank cream matrix(model group), 0.02 g containing 0.1% vaccarin(vaccarin group) or 0.02 g MEBO wetting burn ointment(positive drug). The rate of wound healing was observed and paraffin sections were made from the skin of the wound. Histopathological evaluation was performed by HE staining. Proliferating cell nuclear antigen(PCNA), p-Akt, p-Erk P-basic fibroblast growth factor receptor(p-bFGFR), p-vascular endothelial cell growth factor receptor(p-VEGFR), and CD31 were observed by immunohistochemical staining. Western blotting method were used to analyze the phosphorylation level of Erk, Akt, and bFGFR protein. Results Compared with model group, 3, 6, and 9 d after trauma, vaccarin significantly promoted the healing of open wound healing(P < 0.05, 0.01). Compared with model group, the fibroblasts and endothelial cells in vaccarin group were proliferating, the proliferation of the inflammatory cells decreased and the microvessel density increased significantly(P < 0.01); The results of immunohistochemistry and Western blotting showed that the degree of phosphorylation of bFGFR in the endothelial cell membrane receptor of vaccarin group was significantly higher than that in model group(P < 0.05, 0.01), and the degree of p-VEGFR phosphorylation was not significantly changed, and the degree of Akt and Erk phosphorylation of PI3 K/Akt and MAPK/Erk signaling pathway were significantly increased(P < 0.05). Conclusion Vaccarin could promote open wound healing, and may be related to activation of bFGFR and its downstream MAPK/Erk and PI3 K/Akt signaling pathways.
引文
[1]中国药典[S].一部.2015.
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[8]Gould L,Abadir P,Brem H,et al.Chronic wound repair and healing in older adults:current status and future research[J].J Am Geriatr Soc,2015,63(3):427-438.
[9]He H,Xia D L,Chen Y P,et al.Evaluation of a two-stage antibacterial hydrogel dressing for healing in an infected diabetic wound[J].J Biomed Mater Res B Appl Biomater,2017,105(7):1808-1817.
[10]Zhu X,Sun Y,Mu X,et al.Phospholipase Cepsilon deficiency delays the early stage of cutaneous wound healing and attenuates scar formation in mice[J].Biochem Biophys Res Commun,2017,484(1):144-151.
[11]Chen X,Peng L H,Li N,et al.The healing and anti-scar effects of astragaloside IV on the wound repair in vitro and in vivo[J].J Ethnopharmacol,2012,139(3):721-727.
[12]Weidner N.Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors[J].Breast Cancer Res Treat,1995,36(2):169-180.
[13]Dai X,Liu D,Liu M,et al.Anti-metastatic efficacy of Traditional Chinese Medicine(TCM)ginsenoside conjugated to a VEFGR-3 antibody on human gastric cancer in an orthotopic mouse model[J].Anticancer Res,2017,37(3):979-986.
[14]Pranjol M Z I,Gutowski N J,Hannemann M,et al.Cathepsin D non-proteolytically induces proliferation and migration in human omental microvascular endothelial cells via activation of the ERK1/2 and PI3K/AKT pathways[J].Biochim Biophys Acta,2017,1865(1):25-33.
[15]Takeuchi K,Yanai R,Kumase F,et al.EGF-likedomain-7 is required for VEGF-induced Akt/ERK activation and vascular tube formation in an ex vivo angiogenesis assay[J].PLo S One,2014,9(3):91849.
[16]Li B,Qiu T,Zhang P,et al.IKVAV regulates ERK1/2 and Akt signalling pathways in BMMSC population growth and proliferation[J].Cell Prolif,2014,47(2):133-145.
[2]孟贺,陈玉平,秦文杰,等.王不留行中黄酮苷的分离与鉴定[J].中草药,2011,42(5):874-876.
[3]付起凤,薛娟,张万鹏,等.正交法优化王不留行中王不留行黄酮苷的超声提取工艺[J].哈尔滨医药,2016,1:69-70,73.
[4]Qiu Y,Qiu L,Cui J,et al.Bacterial cellulose and bacterial cellulose-vaccarin membranes for wound healing[J].Mater Sci Eng C Mater Biol Appl,2016,59:303-309.
[5]Xie F,Feng L,Cai W,et al.Vaccarin promotes endothelial cell proliferation in association with neovascularization in vitro and in vivo[J].Mol Med Rep,2015,12:1131-1136.
[6]Li J,Chen J,Kirsener R.Pathophysiology of acute Wound healing[J].Clin Dermatol,2007,25:9-18.
[7]Chen X,Peng L H,Li N,et al.The healing and anti-scar effects of astragaloside IV on the wound repair in vitro and in vivo[J].J Ethnopharmacol,2012,139:721-727.
[8]Gould L,Abadir P,Brem H,et al.Chronic wound repair and healing in older adults:current status and future research[J].J Am Geriatr Soc,2015,63(3):427-438.
[9]He H,Xia D L,Chen Y P,et al.Evaluation of a two-stage antibacterial hydrogel dressing for healing in an infected diabetic wound[J].J Biomed Mater Res B Appl Biomater,2017,105(7):1808-1817.
[10]Zhu X,Sun Y,Mu X,et al.Phospholipase Cepsilon deficiency delays the early stage of cutaneous wound healing and attenuates scar formation in mice[J].Biochem Biophys Res Commun,2017,484(1):144-151.
[11]Chen X,Peng L H,Li N,et al.The healing and anti-scar effects of astragaloside IV on the wound repair in vitro and in vivo[J].J Ethnopharmacol,2012,139(3):721-727.
[12]Weidner N.Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors[J].Breast Cancer Res Treat,1995,36(2):169-180.
[13]Dai X,Liu D,Liu M,et al.Anti-metastatic efficacy of Traditional Chinese Medicine(TCM)ginsenoside conjugated to a VEFGR-3 antibody on human gastric cancer in an orthotopic mouse model[J].Anticancer Res,2017,37(3):979-986.
[14]Pranjol M Z I,Gutowski N J,Hannemann M,et al.Cathepsin D non-proteolytically induces proliferation and migration in human omental microvascular endothelial cells via activation of the ERK1/2 and PI3K/AKT pathways[J].Biochim Biophys Acta,2017,1865(1):25-33.
[15]Takeuchi K,Yanai R,Kumase F,et al.EGF-likedomain-7 is required for VEGF-induced Akt/ERK activation and vascular tube formation in an ex vivo angiogenesis assay[J].PLo S One,2014,9(3):91849.
[16]Li B,Qiu T,Zhang P,et al.IKVAV regulates ERK1/2 and Akt signalling pathways in BMMSC population growth and proliferation[J].Cell Prolif,2014,47(2):133-145.