基于斑马鱼模型及分子对接技术研究丹参促进血管生成的关键活性成分

Crucial proangiogenic components in Salvia miltiorrhiza based on zebrafish model and molecular docking technique

目的建立新方法探索丹参中潜在的促进血管生成的活性成分。方法以绿色荧光蛋白标记血管的转基因斑马鱼为实验动物,以血管抑制剂PTK787损伤造模建立血管生成活性评价模型,评价不同产地的丹参提取物对血管生成的促进作用。采用分子对接技术筛选丹参促血管生成的潜在活性成分,结合分子对接筛选结果与斑马鱼活性实验结果,确认关键的活性成分。结果与模型组比较,不同产地的丹参提取物均能明显促进斑马鱼节间血管的生成(P<0.01)。通过分子对接筛选发现丹酚酸B与VHL-缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)复合物的对接结合能最低,并初步揭示可能的作用位点。利用斑马鱼模型实验发现,丹酚酸B具有明显的促进血管生成作用(P<0.01)。结论建立了一种基于斑马鱼模型的“虚拟筛选-活性评价”的中药药效物质辨识新方法,将计算化学数据挖掘技术与斑马鱼活性实验结合,发现了丹酚酸B是丹参中促血管生成的关键活性成分之一,为冠心病的防治及后续的新药研发提供科学依据。

Objective To establish a new method to explore the potential angiogenesis-promoting active components in Danshen(Salvia miltiorrhiza).Methods Transgenic zebrafish with green fluorescent protein-labeled blood vessels was used as experimental animal,and PTK787,a vascular inhibitor,was used to establish the angiogenesis inhibition model.And this kind of experimental model was used to evaluate the angiogenesis-promoting effect of S.miltiorrhiza extracts of different origins.The molecular docking technique was used to screen the potential active components of S.miltiorrhiza for pro-angiogenesis.The results of molecular docking screening were combined with the results of zebrafish activity assay to identify the key active ingredients.Results Compared with model group,S.miltiorrhiza extracts from different origins significantly promoted intersegmental vessels development in zebrafish(P<0.01).The lowest docking binding energy of salvianolic acid B with VHL-hypoxia inducible factor-1αcomplex was found by molecular docking screening,and the possible sites of action were initially revealed.The experiments using zebrafish model revealed that salvianolic acid B had a significant angiogenesis-promoting effect(P<0.01).Conclusion A new method of“virtual screening-bioactivity evaluation”based on zebrafish model was established for identification of pharmacological substances in traditional Chinese medicine.Combining computational chemistry data mining technology with zebrafish activity experiments,salvianolic acid B was discovered as one of key pro-angiogenic active ingredients in S.miltiorrhiza.The research provides a scientific basis for the prevention and treatment of coronary heart disease and subsequent development of new drugs.