Angiogenic efficacy of simplified 2-herb formula (NF3) in zebrafish embryos in vivo and rat aortic ring in vitro

Tse, H.Y., Hui, M.N., Li, L., Lee, S.M., Leung, A.Y., and Cheng, S.H.
Journal of ethnopharmacology   139(2): 447-53 (Journal)
Registered Authors
Cheng, Shuk Han, Leung, Anskar
diabetic foot ulcer, radix astragali, radix rehmanniae, angiogenesis, VEGF, FGF, MAPK, wound healing, zebrafish embryos
MeSH Terms
  • Angiogenesis Inducing Agents/pharmacology*
  • Angiogenesis Inducing Agents/therapeutic use
  • Angiogenesis Inducing Agents/toxicity
  • Angiogenic Proteins/genetics
  • Animals
  • Animals, Genetically Modified
  • Aorta/drug effects*
  • Cell Proliferation/drug effects
  • Diabetic Foot/drug therapy
  • Dose-Response Relationship, Drug
  • Drugs, Chinese Herbal/pharmacology*
  • Drugs, Chinese Herbal/therapeutic use
  • Drugs, Chinese Herbal/toxicity
  • Embryo, Nonmammalian/blood supply
  • Embryo, Nonmammalian/drug effects
  • Endothelial Cells/drug effects*
  • Endothelial Cells/metabolism
  • G2 Phase Cell Cycle Checkpoints/drug effects
  • Gene Expression Regulation/drug effects
  • Humans
  • Lethal Dose 50
  • MAP Kinase Signaling System/drug effects
  • MAP Kinase Signaling System/genetics
  • Male
  • Neovascularization, Physiologic/drug effects*
  • Neovascularization, Physiologic/genetics
  • Proto-Oncogene Protein c-fli-1/genetics
  • RNA, Messenger/metabolism
  • Rats
  • Rats, Sprague-Dawley
  • Real-Time Polymerase Chain Reaction
  • S Phase Cell Cycle Checkpoints/drug effects
  • Zebrafish/embryology
  • Zebrafish/genetics
22138660 Full text @ J. Ethnopharmacol.

Ethnopharmacological relevance

Diabetic foot ulceration results in high risk of lower extremity amputation, and represents a significant health care expenditure worldwide. Radix Astragali (RA) and Radix Rehmanniae (RR) are widely used Chinese medicinal herbs in treating diabetes, and have shown positive effects in enhancing wound healing in diabetic foot ulcer animal model.

Materials and methods

The angiogenic efficacy of NF3, a simplified 2-herb formula consisting of RA and RR in 2:1 ratio, was investigated. Median lethal concentration (LC50) and median effective concentration (EC50) were determined by treating zebrafish embryos with different concentrations of NF3 from 20 hpf to 72 hpf. The angiogenic activity of NF3 was examined in zebrafish embryos in vivo and by rat aortic ring assay in vitro. Cell cycle analysis of endothelial cells induced by NF3 was analyzed by flow cytometry using transgenic zebrafish Tg(fli1:EGFP). Real-time PCR was used to analyze mRNA expression profiles of selected genes involved in VEGF, FGF and MAPK pathways.


NF3 enhanced blood vessel formation as indicated by extra growth of intersegmental vessels in zebrafish embryos, and increased microvessels formation in rat aortic ring. NF3 also enhanced endothelial cells proliferation as shown by increased percentage of cells accumulating in S phase and G2/M phase of the cell cycle. NF3 exposure significantly induced up-regulation of VEGF-A, Flk-1, fgf1 and bRaf expression in zebrafish embryos.


Our results demonstrated that NF3 was effective in promoting angiogenesis in zebrafish embryos and by rat aortic ring assay, which provided scientific basis to support the use of NF3 as potential therapeutics in treating diabetic foot ulceration.

Genes / Markers
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes