近日，EXPERIMENTAL AND THERAPEUTIC MEDICINE?（Exp Ther Med）?雜志發(fā)表了上海第九人民醫(yī)院副院長、心內(nèi)科主任王長謙課題組的研究成果“Identification of mundoserone by zebrafish in vivo screening as a natural product with anti-angiogenic activity”。該研究利用斑馬魚模型篩選了來自Natural Products Collection of MicroSource的具有抗血管生成潛力的天然產(chǎn)物，并首次發(fā)現(xiàn)Mundoserone作為抗血管小分子的治療前景。

該案例涉及的斑馬魚研究工作，全部在南模斑馬魚平臺上完成。

天然產(chǎn)物一直是國外制藥巨頭進行新藥研發(fā)的重要源頭之一。據(jù)?Newman 和 Cragg（2007）統(tǒng)計，在1981 年 1 月至 2006 年 6 月全世界批準上市的 1184 個新化學實體藥物中，有 52% 來源于天然產(chǎn)物或與天然產(chǎn)物有關(guān)。

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利用血管轉(zhuǎn)基因斑馬魚通過活體高通量篩選技術(shù)、斑馬魚獨有的表型分析技術(shù)、以及藥物作用信號通路分析等，南模生物與上海第九人民醫(yī)院副院長、心內(nèi)科主任王長謙課題組合作，成功的從美國天然產(chǎn)物庫中篩選到4個全新的抗血管小分子，而Mundoserone這種小分子便是其中之一。

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• Mundoserone抑制斑馬魚中ISV的形成

利用fli1a:EGFP轉(zhuǎn)基因斑馬魚評估Natural Products Collection of MicroSource中240種天然產(chǎn)物的抗血管生成活性。在受精后24小時，用文庫化合物處理胚胎24小時，然后使用熒光顯微鏡評估節(jié)間血管（ISV）的形態(tài)，然后計數(shù)ISV并計算抑制率。

結(jié)果發(fā)現(xiàn)，Mundoserone抑制了ISV和DLAV的形成（圖1），而且Mundoserone的抗血管生成作用是具有劑量依賴性的（圖2）。在10μM濃度下觀察到顯著的抗血管生成活性，導致ISV抑制率為73.6±1.3％。

Figure 1.?Among the natural products screened in Tg(fli1a: EGFP)y1 zebrafish, mundoserone was identified as an anti?angiogenic compound.?Representative (A?C) bright?field and (D?F) fluorescent images of zebrafish embryos at 24 h post?fertilization treated with 0.1% dimethyl sulfoxide (control), 10 μM mundoserone or 5 μM PTK787 (positive control) for 24 h (magnification, x40). (G?I) Magnification of images (D?F) (magnification, x112.5). Compared with those in the control group, embryos treated with mundoserone presented with a lower number of incomplete ISVs and only occasional sprouts (asterisks) of the DA were observed. (J) Chemical structure of mundoserone. DLAV, dorsal longitudinal anastomotic vessels; ISVs, intersegmental vessels; DA, dorsal aorta; PCV, posterior cardinalvein.

Figure 2.?Mundoserone inhibits the trunk angiogenesis of zebrafish in a dosedependent manner.?Representative (AE)?bright field and (FJ)?fluorescent images of zebrafish embryos at 24?h postfertilization treated with 0.1% dimethyl sulfoxide (control), mundoserone (2.5, 5 or 10?μM) or 5?μM PTK787 (positive control) for 24?h (magnification, x40). (KO)?Magnification of images FJ (magnification, x112.5). Compared with the control, embryos exposed to mundoserone exhibited a lower number of incomplete ISVs and only occasional sprouts (asterisks) of the DA were observed. Quantification of (P)?the number of complete ISVs and (Q) inhibition rate in mundoserone-treated embryos. Values are expressed as the mean ± standard error of the mean (n=10).

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• Mundoserone通過下調(diào)SLIT3/ROBO1和FGFR/PTP-RB以及上調(diào)NOTCH1A來發(fā)揮抗血管生成作用

通過RT-qPCR分析血管生成相關(guān)基因的表達。結(jié)果表明，Mundoserone顯著降低SLIT3，ROBO1，ROBO2，F(xiàn)GFR2，F(xiàn)GFR3和PTP-RB的表達，但增加了NOTCH1A的表達（圖3）。COX2, SLIT2, ROBO4, DLL4, HEY2以及EFNB2的表達沒有變化。由此推測Mundoserone有效的血管生成抑制活性可能是通過下調(diào)SLIT / ROBO1和FGFR / PTP-RB以及NOTCH1A信號傳導的上調(diào)來起作用的（圖4）。

Figure 3.?Expression of angiogenesis?associated genesin zebrafish embryos after treatment with 10 μM mundoserone.?Values are expressed asthe mean ±?standard error of the mean (n=4). *P<0.05 vs. Control. FGFR, fibroblast growth factor receptor; SLIT3, slit guidance ligand 3; ROBO, roundabout guidance receptor; PTP-RB, protein tyrosine phosphatase, receptor type B; DLL, deltalike ligand 4; COX, cyclooxygenase; VEGFR, vascular endothelial growth factor receptor; PIK3R2, phosphoinositide-3-kinase regulatory subunit 2; HEY2, hes related family bHLH transcription factor with YRPW motif 2; EFNB2, ephrin B2.

Figure 4.?Schematic model illustrating the mechanism of action of mundoserone on angiogenesis.FGFR, fibroblast growth factor receptor; SLIT3, slit guidance ligand 3; ROBO, roundabout guidance receptor; PTP?RB, protein tyrosine phosphatase, receptor type B; VE?PTP, vascular endothelial cell?specific phosphotyrosine phosphatase.

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Mundoserone作為抗血管小分子的首次發(fā)現(xiàn)，對于將來開發(fā)治療血管新生相關(guān)的疾?。ㄈ缒[瘤、糖尿病視網(wǎng)膜病、血管瘤、牛皮癬、類風濕性關(guān)節(jié)炎、月經(jīng)失調(diào)等）提供一種全新的治療選擇。