Angiogenesis plays an important role in a wide range of physiological processes and many diseases are associated with dysregulation of angiogenesis. Radix Astragali, a commonly used traditional Chinese medicine, also known as Huangqi in Chinese, is a potential candidate for treating angiogenesis dysregulation diseases. Calycosin, a major isoflavonoid isolated from Radix Astragali has been shown to possess angiogenic effect in previous studies but its underlying mechanism remains unclear. The present study investigates the angiogenic effects of calycosin in zebrafish embryo in vivo. Tg(fli1:EGFP) and Tg(fli1:nEGFP)transgenic zebrafish embryos were treated with different concentrations of calycosin (10, 30, 100 μM) from 72 hpf to 96 hpf prior morphological observation and angiogenesis phenotypes assessment, Calycosin was demonstrated to induce phenotypic changes and stimulate endothelial cells growth at subintestinal vessels (SIVs) in zebrafish embryo. Meanwhile, transcriptome profiling by deep sequencing suggested that calycosin modulated expression of genes involved in VEGF, FGF, Delta-Notch and Erbb signalling pathways, and these results were confirmed by qPCR and whole mount in situ hybridization. Importantly, the involvement of VEGF(R) and FGF(R) signaling pathways in calycosin induced angiogenic phenotype in vivo was further confirmed because the effects could be specifically blocked by a VEGF receptor kinase inhibitor and a FGF receptor kinase inhibitor (SU5402), individually.Previous findings suggested that vascular endothelial growth factor and fibroblast growth factor are angiogenic molecules whose combined mitogenic activity and capability to activate PI3K-Akt signaling pathway is more potently synergistic. Interestingly, Mitogen-activated protein (MAP) kinases inhibitor (MEK1/2 Inhibitor) and blockers of PI 3-kinase catalytic activity (Wortmannin or LY294002) also could completely abrogate calycosin-induced angiogenesis actions. Taken together, this study revealed, for the first time, that calycosin is an active compound which can elicit angiogenic effect, involving the interaction between VEGF and FGF signaling pathways. The result should provide scientific clues to underlying mechanism of action of traditional use of Radix Astragali on promotion of “qi”, a Chinese medicine philosophical concept. Key words calycosin, angiogenesis, zebrafish, VEGF/VEGFR, FGF/FGFR, EGF/EGFR, Notch, MAPK, Raf, MEK1/2, PI3-kinase, Src, transcriptome

血管新生在很多生理過程中扮演著重要的角色，並且很多疾病發生與血管 新生的失調有關。黃芪，作為一劑傳統中藥，在治療血管新生失調性疾病有 著潛在的效果。毛蕊異黃酮，作為從黃芪中提取的主要的異黃酮成分，在我 們以前的研究中發現其具有血管新生的效果，但是其中的分子機制依然不清 楚。而我們當前研究既是探索毛蕊異黃酮在體內斑馬魚模型中的血管新生的 效果。將轉基因斑馬魚胚胎 Tg(fli1:EGFP)和 Tg(fli1:nEGFP)在受精 72 小時 後，用 10, 30, 100 μM 濃度的毛蕊異黃酮處理 24 小時後，對其形態的觀察以 及血管新生的形態的評價中發現，毛蕊異黃酮能夠誘導斑馬魚胚胎腸下血管 (SIV)的形態的變化及刺激內皮細胞生長。與此同時，通過深度測序的轉錄學 分析發現，毛蕊異黃酮能夠調節 VEGF, FGF, Delta-Notch 和 Erbb 信號通路中 一些基因的表達,而該一結果，被即時定量 PCR 和原位雜交技術再次證實。 而關鍵的是，血管內皮細胞生長因數(VEGF)受體激酶的抑制劑以及成纖維細 胞生長因數(FGF)受體激酶抑制劑都能特異性的抑制毛蕊異黃酮誘導的體內 血管新生形態的發生，從而進一步證實了毛蕊異黃酮的促進血管新生的作用 是通過 VEGF 和 FGF 信號通路而實現的。以前的研究發現，VEGF 和 FGF 作為血管新生分子都能夠通過有絲分裂活性和啟動 PI3K-AKT 信號通道的能 力而發揮作用。有趣的是，有絲分裂啟動蛋白激酶抑制劑(MEK1/2 抑制 劑)PI3K 酶活性抑制劑(Wortmannin 或者 LY294002) 都能抑制住毛蕊異黃酮 誘導的血管新生的活動。所以，這個研究第一次發現了毛蕊異黃酮是一個促 進血管新生的化合物，並且其促血管新生原理是通過啟動 VEGF 和 FGF 兩條 信號通路實現的。這個結果也能提供一個科學的理解線索，關於中醫哲學上 的黃芪“補氣”的概念。 關鍵字: 毛蕊異黃酮，血管新生，斑馬魚，血管內皮生長因數/受體，成纖維 細胞生長因數/受體，表皮細胞生長因數/受體，有絲分裂啟動蛋白激酶