近日，EXPERIMENTAL AND THERAPEUTIC MEDICINE （Exp Ther Med） 杂志发表了上海第九人民医院副院长、心内科主任王长谦课题组的研究成果“Identification of mundoserone by zebrafish in vivo screening as a natural product with anti-angiogenic activity”。该研究利用斑马鱼模型筛选了来自Natural Products Collection of MicroSource的具有抗血管生成潜力的天然产物，并首次发现Mundoserone作为抗血管小分子的治疗前景。

该案例涉及的斑马鱼研究工作，全部在南模斑马鱼平台上完成。

天然产物一直是国外制药巨头进行新药研发的重要源头之一。据 Newman 和 Cragg（2007）统计，在1981 年 1 月至 2006 年 6 月全世界批准上市的 1184 个新化学实体药物中，有 52% 来源于天然产物或与天然产物有关。

利用血管转基因斑马鱼通过活体高通量筛选技术、斑马鱼独有的表型分析技术、以及药物作用信号通路分析等，南模生物与上海第九人民医院副院长、心内科主任王长谦课题组合作，成功的从美国天然产物库中筛选到4个全新的抗血管小分子，而Mundoserone这种小分子便是其中之一。

• Mundoserone抑制斑马鱼中ISV的形成

利用fli1a:EGFP转基因斑马鱼评估Natural Products Collection of MicroSource中240种天然产物的抗血管生成活性。在受精后24小时，用文库化合物处理胚胎24小时，然后使用荧光显微镜评估节间血管（ISV）的形态，然后计数ISV并计算抑制率。

结果发现，Mundoserone抑制了ISV和DLAV的形成（图1），而且Mundoserone的抗血管生成作用是具有剂量依赖性的（图2）。在10μM浓度下观察到显着的抗血管生成活性，导致ISV抑制率为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).

• Mundoserone通过下调SLIT3/ROBO1和FGFR/PTP-RB以及上调NOTCH1A来发挥抗血管生成作用

通过RT-qPCR分析血管生成相关基因的表达。结果表明，Mundoserone显著降低SLIT3，ROBO1，ROBO2，FGFR2，FGFR3和PTP-RB的表达，但增加了NOTCH1A的表达（图3）。COX2, SLIT2, ROBO4, DLL4, HEY2以及EFNB2的表达没有变化。由此推测Mundoserone有效的血管生成抑制活性可能是通过下调SLIT / ROBO1和FGFR / PTP-RB以及NOTCH1A信号传导的上调来起作用的（图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.

Mundoserone作为抗血管小分子的首次发现，对于将来开发治疗血管新生相关的疾病（如肿瘤、糖尿病视网膜病、血管瘤、牛皮癣、类风湿性关节炎、月经失调等）提供一种全新的治疗选择。