Polysaccharides from astragali radix restore chemical-induced blood vessel loss in zebrafish

Abstract

Background: Astragali Radix has been used widely for the treatment of cardiovascular and cerebrovascular diseases, and to enhance endurance and stamina in traditional Chinese medicine (TCM) for over 2000 years. The polysaccharide constituents of Astragali Radix (ARP) are considered as one of the major constituents contributing to the multiple pharmacological effects of this medicinal plant. The purpose of the study is to evaluate the vascular regenerative activities of ARPs in a chemically-induced blood vessel loss model in zebrafish.

Methods: Blood vessel loss was induced in both Tg(fli-1a:EGFP)y1 and Tg(fli-1a:nEGFP)y7 embryos by administration of 300 nM VEGFR tyrosine kinase inhibitor II (VRI) for 3 h at 24 hpf (hour post-fertilization). Then, the blood vessel damaged zebrafish were treated with ARPs for 21 h and 45 h after VRI withdrawal. Morphological changes in intersegmental vessels (ISVs) of zebrafish larvae were observed under the fluorescence microscope and measured quantitatively. The rescue effect of ARPs in the zebrafish models was validated by measuring the relative mRNA expressions of Kdrl, Kdr and Flt-1 using real-time PCR.

Results: Two polysaccharide fractions, P4 (50000 D < molecular weight & diameter < 0.1 μm) and P5 (molecular diameter > 0.1 μm), isolated from Astragali Radix by ultrafiltration, produced a significant and dose-dependent recovery in VRI-induced blood vessel loss in zebrafish. Furthermore, the down-regulation of Flk-1 and Flt-1 mRNA expression induced by VRI was reversed by treatment with P4.

Conclusion: The present study demonstrates that P4 isolated from Astragali Radix reduces VRI-induced blood vessel loss in zebrafish. These findings support the hypothesis that polysaccharides are one of the active constituents in Astragali Radix, contributing to its beneficial effect on treatment of diseases associated with a deficiency in angiogenesis.

Figure 1

The effect of ARPs on VRI-induced blood vessel loss in Tg(fli-1a:EGFP)y1 zebrafish (result of morphological observation). White arrows indicate DLAV, ISV and DA of zebrafish. Scale bar = 500 μm. (A-A') Control group: 24 hpf embryos were treated with 0.1% DMSO for 24 h and 48 h. 24 hpf embryos were treated with VRI (300 nM) at for 3 h. After that, the VRI was washed out and replaced with 0.1% DMSO (v/v) embryo medium (B-B') or 300 μg/ml P1 (C-C'), 100 μg/ml P2 (D-D'), 300 μg/ml P3 (E-E'), 10 μg/ml (F-F'), 30 μg/ml (G-G') and 100 μg/ml (H-H') P4, 30 μg/ml (I-I'), 100 μg/ml (J-J') and 300 μg/ml (K-K') P5 for 24 h.

Figure 2

Effects of ARPs on VRI-induced blood vessel loss in Tg(fli-1a:EGFP)y1 zebrafish (result of statistical analysis). Percentage recovery (the total length of the selected ISVs in the treatment group over the total length of the selected ISVs in the vehicle control group) of each ISV of Tg(fli-1a:EGFP)y1 zebrafish were calculated. Data are plotted as the mean ± SD, (n = 3), * P < 0.05, # P < 0.001 vs the VRI-only treatment group.

Figure 3

The effect of ARPs on VRI-induced blood vessel loss in Tg(fli-1a:nEGFP)y7 zebrafish (result of morphological observation). White arrows indicate DLAV, ISV and DA of zebrafish. Scale bar = 500 μm. (A-A') Control: embryos treated with 0.1% DMSO at 24 hpf for 24 h and 48 h. 24 hpf embryos treated with VRI (300 nM) for 3 h. After that, the VRI was washed out and replaced with 0.1% DMSO (v/v) embryo medium (B-B') or 300 μg/ml P1 (C-C'), 100 μg/ml P2 (D-D'), 300 μg/ml P3 (E-E'), 10 μg/ml (F-F'), 30 μg/ml (G-G') and 100 μg/ml (H-H') P4, 30 μg/ml (I-I'), 100 μg/ml (J-J') and 300 μg/ml (K-K') P5 for 24 h.

Figure 4

Effects of ARPs on VRI-induced blood vessel loss in Tg(fli-1a:nEGFP)y7 zebrafish (result of statistical analysis). Numbers of endothelial cells in ISVs of Tg(fli-1a:nEGFP)y7 zebrafish embryos were assessed by direct counting of the total number of green light points within the twenty selected ISVs. Each green light point represents one endothelial cell (GFP+). Data are plotted as the mean ± SD, (n = 3), * P < 0.05, # P < 0.001 vs the VRI-only treatment group.

Figure 5

Gene expression of P4 treated zebrafish. Data are expressed as the mean ± SD, (n = 3), * P < 0.05, # P < 0.001 vs the VRI-only treatment group.