Induction of Angiogenesis in Zebrafish Embryos and Proliferation of Endothelial Cells by an Active Fraction Isolated from the Root of Astragalus membranaceus using Bioassay-guided Fractionation.
The objective of the study was to identify the active fraction(s) from AR aqueous extract responsible for promoting angiogenesis using bioassay-guided fractionation. The angiogenic activity was screened by monitoring the increase of sprout number in sub-intestinal vessel (SIV) of the transgenic zebrafish embryos after they were treated with 0.06-0.25 mg/ml of AR aqueous extract or its fraction(s) for 96 h. Furthermore, the angiogenic effect was evaluated in treated zebrafish embryos by measuring the gene expression of angiogenic markers (VEGFA, KDR, and Flt-1) using real-time polymerase chain reaction (RT-PCR), and in human microvascular endothelial cell (HMEC-1) by measuring cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, (3)H-thymidine uptake assay, and cell cycle analysis. A major active fraction (P1-1-1), which was identified as glycoproteins, was found to significantly stimulate sprout formation (2.03 ± 0.27) at 0.125 mg/ml (P < 0.001) and up-regulate the gene expression of VEGFA, KDR, and Flt-1 by 2.6-fold to 8.2-fold. Additionally, 0.031-0.125 mg/ml of P1-1-1 was demonstrated to significantly stimulate cell proliferation by increasing cell viability (from 180% to 205%), (3)H-thymidine incorporation (from 126% to 133%) during DNA synthesis, and the shift of cell population to S phase of cell cycle. A major AR active fraction consisting of glycoproteins was identified, and shown to promote angiogenesis in zebrafish embryos and proliferation of endothelial cells in vitro.
Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. ; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Patrick Kwok-Kin
PK
Lai
Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. ; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Judy Yuet-Wa
JY
Chan
Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. ; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Hin-Fai
HF
Kwok
Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. ; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Ling
L
Cheng
Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. ; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Hua
H
Yu
Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. ; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Ching-Po
CP
Lau
Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. ; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Ping-Chung
PC
Leung
Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. ; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. ; School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Kwok-Pui
KP
Fung
Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. ; State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Clara Bik-San
CB
Lau
Astragali Radix; Glycoproteins; Traditional Chinese Medicine; Wound healing