目的 基于模式生物斑马鱼研究重楼皂苷I的发育毒性、抗血管新生活性，并利用网络药理学探究重楼皂苷I抗血管新生的作用机制。方法 将受精后6 h（6 h post fertilization，6 hpf）的斑马鱼胚胎暴露于不同浓度的重楼皂苷I中96 h，在实验终点确认重楼皂苷I对斑马鱼胚胎的致死曲线，计算20%致死浓度（20% lethal concentration，LC₂₀）。在实验终点，以斑马鱼自主抽动次数、96 hpf心率、斑马鱼肝脏面积、静脉窦-动脉球间距、总胆固醇（total cholesterol，T-CHO）、三酰甘油（triglyceride，TG）、低密度脂蛋白胆固醇（low-density lipoprotein cholesterol，LDL-C）含量等指标，结合吖啶橙、油红O染色观察综合评价重楼皂苷I的发育毒性及其相关靶器官毒性。安全剂量条件下，以斑马鱼肝脏面积和静脉窦-动脉球间距评价重楼皂苷I对相关靶器官的影响，同时以节间血管数评价重楼皂苷I对斑马鱼节间血管生长的影响。基于网络药理学预测重楼皂苷I抗血管新生的作用机制并通过qRT-PCR技术进行验证。结果 重楼皂苷I对斑马鱼胚胎的致死曲线为y＝270x－23.62，LC₂₀为0.16 µg/mL；在亚致死浓度暴露条件下的实验终点，与对照组比较，0.16μg/mL重楼皂苷I能够诱导斑马鱼幼鱼出现卵黄囊吸收延迟，脊柱弯曲，尾巴畸变等明显的毒性特征，且T-CHO、TG、LDL-C含量明显升高（P<0.05、0.01）；吖啶橙染色及油红O染色表明0.16 μg/mL重楼皂苷I能引起斑马鱼肝脏细胞凋亡和脂肪变性。在安全剂量条件下，与对照组比较，0.06、0.09μg/mL重楼皂苷I对主要脏器没有明显影响，但可以抑制节间血管数（P<0.01）。网络药理学预测发现重楼皂苷I可通过调节血管内皮生长因子A（vascular endothelial growth factor A，VEGFA）、哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）、类固醇受体共激活因子（steroid receptor coactivator，SRC）、表皮生长因子受体（epidermal growth factor receptor，EGFR）等关键靶点，调控EGFR酪氨酸激酶抑制剂耐药性等相关信号通路，进而发挥抗血管新生的作用，qRT-PCR实验结果验证了以上靶点。结论 重楼皂苷I过高剂量使用会造成斑马鱼胚胎发育毒性，而在安全剂量条件下，重楼皂苷I通过调节EGFR酪氨酸激酶抑制剂耐药性等相关通路发挥明显的抗血管新生活性，为重楼皂苷I的临床用药安全及开发利用提供了思路，同时也为毒性中药的“有效性-安全性”综合评价提供了参考。

Objective To study the developmental toxicity and anti-angiogenesis of polyphyllin I based on the model organism zebrafish, and explore the anti-angiogenesis mechanism of polyphyllin I by network pharmacology. Methods The zebrafish embryos 6 h post fertilization (6 hpf) were exposed to different concentrations of polyphyllin I for 96 h. The lethal curve of polyphyllin I on zebrafish embryos was confirmed at the end of the experiment, and 20% lethal concentration (LC₂₀) was calculated. At the end point of the experiment, the developmental toxicity of polyphyllin I and its related target organ toxicity were comprehensively evaluated by the number of spontaneous twitches of zebrafish, the heart rate of 96 hpf, the liver area of zebrafish, the distance between venous sinus and arterial bulb, the contents of total cholesterol (T-CHO), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and the observation of acridine orange and oil red O staining. Under the safe dose condition, effect of polyphyllin I on related target organs was evaluated by the liver area of zebrafish and the distance between venous sinus and arterial bulb, and effect of polyphyllin I on growth of zebrafish internode vessels was evaluated by the number of internode vessels. The mechanism of polyphyllin I against angiogenesis was predicted based on network pharmacology and verified by qRT-PCR technology. Results The lethal curve of polyphyllin I on zebrafish embryos was y=270 x-23.62, LC₂₀ was 0.16 µg/mL. The end point of the experiment under the sublethal concentration exposure, 0.16 µg/mL polyphyllin I showed obvious toxic characteristics such as delayed absorption of yolk sac, curvature of spine and tail distortion in larvae zebrafish, and the contents of T-CHO, TG and LDL-C were significantly increased (P < 0.05, 0.01). Acridine orange and oil red O staining showed that 0.16 µg/mL polyphyllin I could induce liver cell apoptosis and steatosis. Compared with control group at safe dose, 0.06, 0.09 µg/mL polyphyllin I had no significant effects on main organs, but could inhibit the number of internode vessels (P < 0.01). Network pharmacology prediction found that polyphyllin I could regulate the resistance of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and other related signaling pathways by regulating vascular endothelial growth factor A (VEGFA), mammalian target of rapamycin (mTOR), steroid receptor coactivator (SRC), EGFR and other key targets, so as to play the role of anti-angiogenesis. The results of qRT-PCR experiment confirmed the above targets. Conclusion Excessive dosage of polyphyllin I can cause developmental toxicity of zebrafish embryos. Under safe dose conditions, polyphyllin I exerts significant anti-angiogenesis activity by regulating EGFR tyrosine kinase inhibitor resistance and other related pathways. This study provided a way of thinking for the safety and development of clinical drug use of polyphyllin I, and also provided a reference for the comprehensive evaluation of "efficacy-safety" of toxic traditional Chinese medicine.

R285.5

国家自然科学基金资助项目（82204753）；国家中药标准化项目（ZYBZH-Y-YN-44）