- 作者：Cheuk-Lun Liu ; Hin-Fai Kwok ; Ling Cheng ; Chun-Hay Ko ; Chun-Wai Wong ; Tina Wai Fong Ho ; Ping-Chung Leung ; Kwok-Pui Fung ; Clara Bik-San Lau
- 关键词：Bioassay-guided fractionation ; Rehmannia glutinosa ; zebrafish ; Angiogenesis ; PCR array
- 刊名：Journal of Ethnopharmacology
- 出版年：10 January, 2014
- 年：2014
- 卷：151
- 期：1
- 页码：565-575
- 全文大小：2785 K
Ethnopharmacological importance
The root of Rehmannia glutinosa (Rehmanniae Radix (RR)) is clinically used as a wound-healing agent in traditional Chinese medicine. Angiogenesis acts crucially in the pathogenesis of chronic wound healing. The present study investigated the angiogenesis effect and its underlying mechanism of RR through zebrafish sprout angiogenesis guided-fractionation.Materials and methods
The in vivo angiogenesis effect was studied by analyzing the number of ectopic sprouts formed upon sub-intestinal vessel of transgenic TG(fli1:EGFP)y1/+(AB) zebrafish embryos by fluorescence microscopy. Quantitative real-time PCR gene expression of the zebrafish embryos was further performed using a panel of 30 angiogenesis-associated genes designed for zebrafish sprout angiogenesis. Classical in vitro angiogenesis assays using human microvascular endothelial cells (HMEC-1) was accompanied.
Results
We demonstrated that among all RR sub-fractions tested, C1-1 treated-zebrafish embryos possessed the most potent angiogenesis activities (from 190 to 780 ng/ml, p<0.001) in sprout formation in the zebrafish model. Quantitative gene expression of the treated embryos demonstrated significant up-regulation in MMP-9 (p<0.05), ANGPT1 (p<0.05), EGFR (p<0.05), EPHB4 (p<0.01), and significant down-regulation in Ephrin B2 (p<0.05), Flt-1 (p<0.05) and Ets-1 (p<0.05). C1-1 treatment could also significantly (p<0.001-0.05) stimulate HMEC-1 cell migration in scratch assay. Significant increase (p<0.05) in mean tubule length was observed in the C1-1-treated HMEC-1 cells in the tubule formation assay.
Conclusions
Our zebrafish sprout angiogenesis model-guided fractionation revealed that C1-1 possessed the most potent angiogenesis effect in RR. The design of the panel with 30 tailor-made angiogenesis-associated genes exhibited in zebrafish gene expression analysis showed that C1-1 could trigger differential expression of various angiogenesis-associated genes, such as VEGFR3 and MMP9, which played key role in angiogenesis. The pro-angiogenic activity of C1-1 was further confirmed in the translated study in motogenic and tubule-inducing effect using HMEC-1.