重楼皂苷Ⅰ对斑马鱼发育毒性、抗血管新生活性及其机制研究  

Developmental toxicity,anti-angiogenesis activity and mechanism of polyphyllin Ⅰ on zebrafish

作　　者：王璇 陈林珍 林瑞超[1] 陈美琳 范琦琦 李芝奇 赵崇军[1] 李向日[1] WANG Xuan;CHEN Lin-zhen;LIN Rui-chao;CHEN Mei-lin;FAN Qi-qi;LI Zhi-qi;ZHAO Chong-jun;LI Xiang-ri(Beijing Key Laboratory for Quality Evaluation of Chinese Materia Medica/Chinese Medicine Processing Research Center,Beijing University of Chinese Medicine,Beijing 102488,China)

机构地区：[1]北京中医药大学中药品质评价北京市重点实验室/中药炮制研究中心,北京102488

出　　处：《中草药》2023年第14期4548-4555,共8页Chinese Traditional and Herbal Drugs

基　　金：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)。

摘　　要：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0.16μg/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0.16μg/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,m TOR)、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 recepObjective 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

关 键 词：重楼皂苷Ⅰ 斑马鱼 发育毒性 抗血管新生 血管内皮生长因子A 雷帕霉素靶蛋白 类固醇受体共激活因子 表皮生长因子受体 

分 类 号：R285.5[医药卫生—中药学]