运用血清药理学初步探讨毛蕊异黄酮苷的血管新生作用

Preliminarily Discussion on the Angiogenesis Effect of Calycosin-7-glucoside by Using Serum Pharmacology

目的首次运用血清药理学方法,并借助体外细胞实验,初步探讨毛蕊异黄酮苷在血管新生方面的作用,为后期深入研究该物质诱导内皮祖细胞(EPCs)参与脑缺血后血管再生修复机制提供重要帮助。方法健康雄性Sprague-Dawley(SD)大鼠随机分为两组:一组大鼠预先实施假手术并作为空白血清供体,采用生理盐水灌胃;另一组大鼠预先实施右侧大脑中动脉阻塞再灌注手术以复制缺血性脑卒中疾病模型,并将造模成功动物作为含药血清供体,术后按照50mg/kg·bw的剂量灌胃毛蕊异黄酮苷溶液。各组动物每天早晚各给药1次,连续3天,末次给药1 h后腹主动脉采血。配置不同血清含量(5%、10%、15%)的培养基并对大鼠骨髓来源的内皮祖细胞进行培养,观察不同含药血清对EPCs形态数量的影响,进而选择适宜的含药血清,借助Transwell小室和Matrigel分别考察内皮祖细胞的迁移能力和形成管腔的能力的变化,并采用聚合酶联免疫反应(ELISA)对血清中血管内皮生长因子(VEGF)、基质细胞衍生因子1(SDF-1)、粒细胞集落刺激因子(G-CSF)、粒细胞巨噬细胞集落刺激因子(GM-CSF)以及促红细胞生成素(EPO)含量进行检测。结果5%和10%含药血清均能促进内皮祖细胞增值且对细胞形态无影响;15%含药血清使细胞形态改变,生长受到显著抑制。10%含药血清能够显著促进内皮祖细胞的迁移作用和形成管腔的能力(P<0.01)。ELISA结果显示,含药血清中内皮祖细胞动员因子(VEGF、SDF-1、G-CSF、GM-CSF、EPO)含量与空白血清中的各参数相比均显著升高(P<0.01)。结论体外实验初步表明,毛蕊异黄酮苷吸收入血后,能够调动多种内皮祖细胞动员因子进入血循环,这些物质对激活内皮祖细胞参与机体脑缺血后的血管再生修复过程起到非常关键的作用。

Objective To preliminarily explore the role of calycosin-o-β-d-glucopyranoside from angiogenesis aspect by using serum pharmacology method and in vitro cell experiments for the first time,which will pave the way for the further study on the mechanism of the monomer compound inducing endothelial progenitor cells to participate in angiogenesis after cerebral ischemia.Methods Healthy male SD rats were randomly divided into two groups:one group previously received sham operation and served as blank serum donor,which was intragastrically administered with normal saline;the other group was treated as the donor of serum containing drug which was preliminary established ischemic stroke model with suture.After surgery,the rats were intragastrically administered calycosin-o-β-dglucopyranoside solution at the dose of 50mg/kg·bw.Each group administrated twice a day in the morning and evening for 3 days.1 hr after the last administration,abdominal aorta blood was collected.Different concentrations of serum(5%,10%,15%)were used to culture rat bone marrow derived EPCs,and the effects of different drug-containing serum on the morphology and number of EPCs were observed.Then,the appropriate drug-containing serum was selected.Transwell chamber and Matrigel were used to investigate the migration ability and tube formation ability of EPCs.ELISA was used to detect the contents of vascular endothelial growth factor(VEGF),stromal cell-derived factor 1(SDF-1),granulocyte colony-stimulating factor(G-CSF),granulocyte macrophage colony-stimulating factor(GM-CSF)and erythropoietin(EPO)in serum.Results 5%and 10%drug-containing serum could promote the proliferation of endothelial progenitor cells and had no effect on cell morphology;15%drug-containing serum changed cell morphology and significantly inhibited cell growth.10%drug-containing serum could significantly promote the migration and tube formation of EPCs(P<0.01).ELISA results showed that the contents of EPCs mobilization factors(VEGF,SDF-1,G-CSF,GM-CSF,EPO)in medicated serum were significantly higher than those in blank serum(P<0.01).Conclusion In vitro experiments demonstrated that calycosin-o-β-d-glucopyranoside could activate the mobilization factors of EPCs into circulatory system,these factors may play an important role of participating in the process of revascularization after cerebral ischemia.