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ABSTRACT: 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.
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.
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.
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.
Vascular cell. 02/2012; 4(1):2.
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ABSTRACT: Astragaloside IV (AS-IV) is a natural product isolated from the Chinese medical herb, Radix Astragali, which has been reported to be a potential candidate for treating diseases associated with abnormal angiogenesis; however, the effect of AS-IV on angiogenesis and its underlying mechanisms are yet to be fully elucidated. In the present study, we investigated the angiogenic effect of AS-IV in vitro using human umbilical vein endothelial cells (HUVECs), and in vivo using zebrafish. AS-IV was found to stimulate the proliferation and migration of HUVECs in an XTT assay and a wound healing migration assay, respectively. Moreover, AS-IV stimulated the invasive ability of HUVECs and significantly increased the mean tube length of HUVECs in Matrigel. AS-IV induced an angiogenic response in HUVECs and enhanced mRNA expression of vascular endothelial growth factor (VEGF) and a VEGF receptor known as kinase‑domain region/fetal liver kinase-1/VEGF receptor 2 (KDR/Flk-1/VEGFR2), as well as activation of Akt as demonstrated by quantitative real-time PCR and Western blot analysis, respectively. The AS-IV-induced proliferation of HUVECs was capable of being suppressed by a KDR inhibitor (SU5416) and an Akt inhibitor (SH-6). AS-IV also rescued blood vessel loss in Tg (fli-1:EGFP) zebrafish. Altogether, our results suggest that AS-IV exerts potential pro-angiogenic effects in vitro and in vivo, and that its pro-angiogenic activity probably involves both VEGF- and Akt-dependent signaling pathways.
Molecular Medicine Reports 12/2011; 5(3):805-11. · 0.42 Impact Factor
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ABSTRACT: Although zebrafish has become a popular animal model for drug discovery and screening, drug metabolism in zebrafish remains largely unknown. In this study, we probed the metabolic capability of zebrafish larvae with calycosin, one of the major isoflavone constituents of Radix Astragali that was previously demonstrated to be angiogenic in the zebrafish model. The metabolism of calycosin and accumulation of its metabolites in zebrafish larvae were determined using an LC-MS/MS method. Calycosin showed a slow but steady decrease from the culture medium as well as a steady accumulation in zebrafish larvae. Calycosin underwent major conjugation and minor oxidation in zebrafish larvae. A total of ten calycosin metabolites formed from glucuronidation, glucosylation, sulfation, oxidation or a combination of two of these metabolisms were identified, most of which were reported for the first time. Most metabolites increased steadily in the larvae over 24-h experimental period. The dominant phase II conjugation of calycosin in zebrafish larvae matched well with existing knowledge of isoflavone metabolism in mammalians. The findings shed a light in certain degree of similarity of phase II drug metabolism between zebrafish larvae and mammals and warrant further investigation on feasibility of adopting the zebrafish larvae as a whole-organism model for examining drug metabolism.
Xenobiotica 10/2011; 42(3):294-303. · 1.79 Impact Factor
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Jing Yan Tang,
Shang Li,
Zhen Hua Li,
Zai Jun Zhang, Guang Hu,
Lorita Chi Veng Cheang,
Deepa Alex,
Maggie Pui Man Hoi,
Yiu Wa Kwan,
Shun Wan Chan,
George Pak Heng Leung,
Simon Ming Yuen Lee
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ABSTRACT: Angiogenesis plays an important role in a wide range of physiological processes, and many diseases are associated with the dysregulation of angiogenesis. Radix Astragali is a Chinese medicinal herb commonly used for treating cardiovascular disorders and has been shown to possess angiogenic effect in previous studies but its active constituent and underlying mechanism remain unclear. The present study investigates the angiogenic effects of calycosin, a major isoflavonoid isolated from Radix Astragali, in vitro and in vivo.
Tg(fli1:EGFP) and Tg(fli1:nEGFP) transgenic zebrafish embryos were treated with different concentrations of calycosin (10, 30, 100 microM) from 72 hpf to 96 hpf prior morphological observation and angiogenesis phenotypes assessment. Zebrafish embryos were exposed to calycosin (10, 100 microM) from 72 hpf to 78 hpf before gene-expression analysis. The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis were studied using 72 hpf Tg(fli1:EGFP) and Tg(fli1:nEGFP) zebrafish embryos. The pro-angiogenic effects of calycosin were compared with raloxifene and tamoxifen in 72 hpf Tg(fli1:EGFP) zebrafish embryos. The binding affinities of calycosin to estrogen receptors (ERs) were evaluated by cell-free and cell-based estrogen receptor binding assays. Human umbilical vein endothelial cell cultures (HUVEC) were pretreated with different concentrations of calycosin (3, 10, 30, 100 microM) for 48 h then tested for cell viability and tube formation. The role of MAPK signaling in calycosin-induced angiogenesis was evaluated using western blotting.
Calycosin was shown to induce angiogenesis in human umbilical vein endothelial cell cultures (HUVEC) in vitro and zebrafish embryos in vivo via the up-regulation of vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 mRNA expression. It was demonstrated that calycosin acted similar to other selective estrogen receptor modulators (SERMs), such as raloxifene and tamoxifen, by displaying selective potency and affinity to estrogen receptors ERalpha and ERbeta. Our results further indicated that calycosin promotes angiogenesis via activation of MAPK with the involvement of ERK1/2 and ER. Together, this study revealed, for the first time, that calycosin acts as a selective estrogen receptor modulator (SERM) to promote angiogenesis, at least in part through VEGF-VEGFR2 and MAPK signaling pathways.
PLoS ONE 01/2010; 5(7):e11822. · 4.09 Impact Factor
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Yi Zhang, Guang Hu,
Hui-Chao Lin,
Si-Jia Hong,
Yan-Hui Deng,
Jing-Yan Tang,
Sai Wang Seto,
Yiu-Wa Kwan,
Mary Miu-Yee Waye,
Yi-Tao Wang,
Simon Ming-Yuen Lee
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ABSTRACT: Angiogenesis plays an important role in a wide range of physiological processes and many diseases are associated with dysregulation of angiogenesis. Radix Astragali, commonly used in traditional Chinese medicine, is a potential candidate for treating such diseases. However, the biological effects of Radix Astragali on angiogenesis and its underlying mechanisms are yet to be elucidated fully. This study describes the angiogenic effects of Radix Astragali extract (RAE) on human umbilical vein endothelial cells (HUVEC) in vitro. It was shown that RAE treatment stimulated HUVEC to proliferate. A significant increase in migration was observed in RAE-treated HUVEC using the wound healing migration assay. In addition, a significant increase in the number of branching points was observed during endothelial cell capillary formation after RAE treatment. It was shown that RAE enhances vascular endothelial growth factor (VEGF) mRNA expression, and that a specific blocker of VEGF receptor 2 (KDR/Flk) inhibited the RAE-induced HUVEC proliferation. In addition, a decrease in the RAE-induced HUVEC proliferation was observed after treatment with inhibitors of phosphatidylinositol 3-kinase (PI3K), Akt and endothelial nitric oxide synthase (eNOS). Taken together, these data suggest that RAE is a potent stimulator of angiogenesis and that its pro-angiogenic effects involve the VEGF-KDR/Flk and PI3K-Akt-eNOS pathways.
Phytotherapy Research 03/2009; 23(9):1205-13. · 2.09 Impact Factor
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ABSTRACT: Angiogenesis plays an important role in a wide range of physiological processes such as wound healing and fetal development. In fact, many diseases are associated with imbalance in the regulation of angiogenesis in which there is either excessive or insufficient blood vessel formation. Panax notoginseng, a blood circulation invigorating herb, is commonly used in traditional Chinese medicine to treat circulation-related diseases. However, the biological effects of saponin extract from Panax notoginseng (PNS) on angiogenesis and the underlying mechanisms are yet to be fully elucidated. This investigation describes the angiogenic effects of PNS on human umbilical vein endothelial cells (HUVECs) in vitro and zebrafish in vivo. The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)5[(phenylamino)carbonyl]2H-tetrazolium hydroxide (XTT) assay and microscopic cell counting demonstrated that the extract was able to stimulate the proliferation of HUVECs. Meanwhile, the numbers of invaded cells and tube branches were significantly increased in PNS treatment groups. PNS was also shown to promote changes in the subintestinal vessels, a feature of angiogenesis, in zebrafish. In addition, by using real-time polymerase chain reaction (PCR), PNS was found to enhance vascular endothelial growth factor (VEGF) and kinase-domain region/fetal liver kinase-1 in mice (KDR/Flk-1) mRNA expression, and the PNS-induced HUVECs proliferation could be abolished by a KDR/Flk-1 inhibitor. Furthermore, the proliferation of HUVECs induced by PNS was significantly attenuated by inhibitors of PI3K-Akt-eNOS. All the results suggest that PNS can promote angiogenesis, and that the proangiogenic effects involve the VEGF-KDR/Flk-1 and PI3K-Akt-eNOS signaling pathways.
Phytotherapy Research 12/2008; 23(5):677-86. · 2.09 Impact Factor
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ABSTRACT: To investigate the effects of the essential oil of Curcuma wenyujin (CWO) on growth inhibition and on the induction of apoptosis in human HepG2 cancer cells.
The cytotoxic effect of drugs on HepG2 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. DNA fragmentation was visualized by agarose gel electrophoresis. Cell cycle and mitochondrial transmembrane potential (Delta psi m) were determined by flow cytometry (FCM). Cytochrome C immunostaining was evaluated by fluorescence microscopy. Caspase-3 enzymatic activity was assayed by the cleavage of Ac-DEVD-R110. Cleaved PARP and active caspase-3 protein levels were measured by FCM using BD(TM) CBA Human Apoptosis Kit.
Treatment with CWO inhibited the growth of HepG2 cells in a dose-dependent manner, and the IC50 of CWO was approximately 70 mug/mL. CWO was found to inhibit the growth of HepG2 cells by inducing a cell cycle arrest at S/G(2). DNA fragmentation was evidently observed at 70 mug/mL after 72 h of treatment. During the process, cytosolic HepG2 cytochrome C staining showed a markedly stronger green fluorescence than in control cells in a dose-dependent fashion, and CWO also caused mitochondrial transmembrane depolarization. Furthermore, the results clearly demonstrated that both, activity of caspase-3 enzyme and protein levels of cleaved PARP, significantly increased in a dose-dependent manner after treatment with CWO.
CWO exhibits an antiproliferative effect in HepG2 cells by inducing apoptosis. This growth inhibition is associated with cell cycle arrest, cytochrome C translocation, caspase 3 activation, Poly-ADP-ribose polymerase (PARP) degradation, and loss of mitochondrial membrane potential. This process involves a mitochondria-caspase dependent apoptosis pathway. As apoptosis is an important anti-cancer therapeutic target, these results suggest a potential of CWO as a chemotherapeutic agent.
World Journal of Gastroenterology 08/2008; 14(27):4309-18. · 2.47 Impact Factor
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ABSTRACT: Furanodiene (C15H20O), a pure compound isolated from Traditional Chinese medicine, Curcuma wenyujin, named Ezhu in Chinese, which structure was determined on the basis of NMR, MS and UV spectrum. In this study, we attempted to characterize in detail the signaling cascades resulted from furanodiene-induced apoptosis in human hepatoma HepG2 cells. Furanodiene inhibited HepG2 cell growth by causing cell cycle arrest at G2/M and inducing apoptosis as evidenced by DNA fragmentation assay. We found that furanodiene induced mitochondrial transmembrane depolarization, release of mitochondrial cytochrome c, activation of caspases-3 and the cleavage of PARP. The furanodiene mediated mitochondria-caspase apoptotic pathway also involved activation of p38 and inhibition of ERK mitogen-activated protein kinase (MAPK) signaling. These results for the first time have identified the biological activity of furanodiene against HepG2 cells and provide rationales for further development of essential oil of Ezhu and its ingredients such as furanodiene on treatment of liver diseases.
Cancer biology & therapy 05/2007; 6(7):1044-50. · 2.64 Impact Factor
