title: Angiogenic efficacy of simplified 2-herb formula (NF3) in zebrafish embryos in vivo and rat aortic ring in vitro.
abstract: BACKGROUND
  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.
  
  
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong Special Administrative Region.
authors.forename: Ho Yan Gloria
authors.initials: HY
authors.lastname: Tse
authors.forename: Michelle Nga Yu
authors.initials: MN
authors.lastname: Hui
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Simon Ming Yuen
authors.initials: SM
authors.lastname: Lee
authors.forename: Anskar Yu Hung
authors.initials: AY
authors.lastname: Leung
authors.forename: Shuk Han
authors.initials: SH
authors.lastname: Cheng
mesh_terms: D043925:Angiogenesis Inducing Agents / Q000494:pharmacology* / Q000627:therapeutic use / Q000633:toxicity; D042501:Angiogenic Proteins / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D001011:Aorta / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D017719:Diabetic Foot / Q000188:drug therapy; D004305:Dose-Response Relationship, Drug; D004365:Drugs, Chinese Herbal / Q000494:pharmacology* / Q000627:therapeutic use / Q000633:toxicity; D004625:Embryo, Nonmammalian / Q000098:blood supply / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007928:Lethal Dose 50; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000235:genetics; D008297:Male; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000235:genetics; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D059807:S Phase Cell Cycle Checkpoints / Q000187:drug effects; D015027:Zebrafish / Q000196:embryology / Q000235:genetics
chemical_list: D043925:Angiogenesis Inducing Agents; D042501:Angiogenic Proteins; D004365:Drugs, Chinese Herbal; D051789:Proto-Oncogene Protein c-fli-1; D012333:RNA, Messenger