基于斑马鱼模型和分子对接技术的心可舒片促血管生成活性成分研究

Study on angiogenesis promoting active constituents of Xinkeshu Tablets based on zebrafish model and molecular docking technology

目的基于斑马鱼模型和分子对接技术研究心可舒片的促血管生成活性成分。方法以人脐静脉内皮细胞HUVECs为模型,采用CCK-8与EdU-488细胞增殖检测试剂盒检测细胞活性与增殖能力,采用划痕实验与Transwell实验评价细胞迁移能力,采用成管实验检测各组细胞成管腔能力,评价心可舒片对HUVECs的促血管生成活性。以斑马鱼为模型,采用血管内皮细胞生长因子受体抑制剂PTK787建立斑马鱼节间血管损伤模型,根据各组节间血管长度,评价心可舒片对斑马鱼的促血管生成活性。心可舒片29个成分与表皮生长因子受体(epidermal growth factor receptor,EGFR)、血管内皮生长因子受体1(vascular endothelial growth factor receptor 1,VEGFR1)、蛋白激酶B(protein kinase B,AKT)和VEGFR2的7个晶体结构进行分子对接,通过打分值与对接能筛选潜在活性成分,采用斑马鱼模型评价潜在活性成分的促血管生成活性。结果心可舒片可显著提高细胞增殖能力(P<0.001),提高细胞迁移能力及成管能力(P<0.05、0.001),恢复PTK787造成的斑马鱼节间血管损伤(P<0.01、0.001);通过分子对接共得到12个心可舒片潜在活性成分,斑马鱼验证结果表明丹酚酸A、丹酚酸B、丹酚酸D、葛根素、3′-甲氧基葛根素、3′-羟基葛根素、大豆苷、紫草酸、迷迭香酸和考迈斯托醇可显著恢复PTK787诱导的斑马鱼节间血管损伤(P<0.05、0.01、0.001)。结论采用分子对接技术与斑马鱼模型从心可舒片中筛选得到10个具有促血管生成作用的活性成分。

Objective To study the potential angiogenesis promoting components of Xinkeshu Tablets(心可舒片,XKS)based on zebrafish model and molecular docking technology.Methods Human umbilical vein endothelial cells(HUVECs)were used as model.The cell viability and proliferation ability of each group was detected by CCK-8 and EdU cell proliferation kit.The cell migration ability of each group was evaluated by wound-healing assay and Transwell test.The tube formation ability of each group was detected by tube formation assay.The angiogenesis promoting activity of XKS on HUVECs was evaluated.Zebrafish was used as model.The model of intersegular vascular injury in zebrafish was established by using Vatalanib dihydrochloride(PTK787),and angiogenesis promoting activity of XKS on zebrafish was evaluated according to the length of intersegular vascular in each group.A total of 29 constituents of XKS were obtained.And seven crystal structures of epidermal growth factor receptor(EGFR),vascular endothelial growth factor receptor 1(VEGFR1),protein kinase B(AKT)and VEGFR-2 were employed as molecular docking targets.The potential active components were picked out on the basis of docking score and docking energy.The angiogenesis promoting activity of potential active components was evaluated by zebrafish.Results The cell proliferation ability in XKS groups was significantly increased(P<0.001).The cell migration and tube forming ability were significantly enhanced(P<0.05,0.001).The PTK787-induced intersegular vascular injury in XKS groups was significantly rescued(P<0.01,0.001).According to the molecular docking results,12 potential active components of XKS were obtained.In the zebrafish validation experiments,the PTK787-induced intersegular vascular injury was significantly rescued in salvianolic acid A,salvianolic acid B,salvianolic acid D,puerarin,3′-methoxy puerarin,3′-hydroxy puerarin,daidzin,lithospermic acid,rosmarinic acid and coumestrol groups(P<0.05,0.01,0.001).Conclusion Ten angiogenesis promoting components were screened and verified from XKS by molecular docking technology and zebrafish model.The present study provided reference for clarifying effective substances of XKS in the treatment of cardiovascular diseases.