三种斑马鱼血瘀模型在三七抗血栓及抗心肌缺氧能力评价中的应用  

Application of Three Blood Stasis Models in Zebrafish in Evaluation of Anti-thrombosis and Anti-myocardial Hypoxia Activities of Notoginseng Radix et Rhizoma

作　　者：李明利 高波 郭胜亚 罗川 戴明珠 高文雅 边宝林[1] 王宏洁[1] 周严严[1] 张勇 赵海誉[1] LI Ming-li;GAO Bo;GUO Sheng-ya;LUO Chuan;DAI Ming-zhu;GAO Wen-ya;BIAN Bao-lin;WANG Hong-jie;ZHOU Yan-yan;ZHANG Yong;ZHAO Hai-yu(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Anhui China Resources Jinchan Pharmaceutical Co.Ltd.,Huaibei 235000,China;Hunter Biotechnology Co.Ltd.,Hangzhou 310051,China)

机构地区：[1]中国中医科学院中药研究所,北京100700 [2]安徽华润金蟾药业股份有限公司,安徽淮北235000 [3]杭州环特生物科技有限公司,杭州310051

出　　处：《中国实验方剂学杂志》2022年第3期98-108,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：中国中医科学院科技创新工程项目(CI2021A04510);国家“重大新药创制”科技重大专项(2019ZX09201-005-006-002);杭州市钱江特聘专家项目(2020)。

摘　　要：目的:采用不同诱导剂建立3种斑马鱼血瘀模型,优选最适宜评价三七活血能力的模型。方法:花生四烯酸(AA),普纳替尼(Ponatinib),异丙肾上腺素(ISO)分别建立斑马鱼血瘀模型,设置正常组、模型组、阳性药组、不同浓度三七水提物冻干粉组,计算心脏红细胞染色强度、心脏凋亡细胞荧光强度,评价三七的抗血栓作用和抗心肌缺氧能力。基于优选的评价三七活血化瘀活性的血瘀模型—AA,ISO诱导的斑马鱼血瘀模型,比对三七水提取物及70%甲醇提取物之间的活性,并采用UHPLC LTQ-Orbitrap MS/MS技术比对其化学成分差异。结果:AA,Ponatinib诱导后斑马鱼心脏红细胞染色强度显著降低(P<0.01),ISO诱导后心脏凋亡细胞荧光强度显著升高(P<0.01),三七水提物冻干粉可以拮抗AA模型的血栓形成(P<0.01)和ISO模型的心肌凋亡(P<0.05),对Ponatinib模型血栓形成无显著改善作用。三七70%甲醇提取物冻干粉可以抑制ISO血瘀模型的心肌凋亡(P<0.01),作用强于三七水提物冻干粉,其成分差别主要在于人参皂苷Re等部分皂苷类以及氨基酸类、炔醇等成分。结论:AA,Ponatinib,ISO均可建立斑马鱼血瘀模型,其中AA和ISO模型可以用于评价三七水提物冻干粉的活血能力。水提和70%甲醇提取三七冻干粉化学成分差异较大,对ISO血瘀模型,70%甲醇提取三七冻干粉抗心肌缺氧能力更强,皂苷、炔醇类成分可能与其活血化瘀功效密切相关。Objective:To establish blood stasis models in zebrafish using three inducers and select the optimal model for evaluating the activity of Notoginseng Radix et Rhizoma in promoting blood circulation.Method:Arachidonic acid(AA),ponatinib,and isoprenaline(ISO)were used to induce blood stasis models in zebrafish.A normal group,a model group,a positive drug group,and Notoginseng Radix et Rhizoma water extract freeze-dried powder groups at different concentrations were set up.The staining intensity of cardiac erythrocytes and the fluorescence intensity of cardiac apoptotic cells were calculated,the anti-thrombotic effect and anti-myocardial hypoxia activity of Notoginseng Radix et Rhizoma were evaluated.The activities of water extract and 70%methanol extract of Notoginseng Radix et Rhizoma were compared based on the preferred AAand ISO-induced blood stasis models in zebrafish and the difference in the chemical composition was analyzed by UHPLC LTQ-Orbitrap MS/MS.Result:After induction by AA and ponatinib,the staining intensity of cardiac erythrocytes was reduced(P<0.01),and the fluorescence intensity of cardiac apoptotic cells increased after the induction by ISO(P<0.01).The freeze-dried powder of the water extract of Notoginseng Radix et Rhizoma could antagonize the thrombosis in the AA-induced model(P<0.01)and the myocardial apoptosis in the ISOinduced model(P<0.05),while no significant improvement in the thrombosis was observed in the ponatinibinduced model.The freeze-dried powder of 70%methanol extract of Notoginseng Radix et Rhizoma could inhibit myocardial apoptosis in the ISO-induced blood stasis model(P<0.01),and the effect was stronger than that of the freeze-dried powder of Notoginseng Radix et Rhizoma water extract.The difference in chemical composition lay in some saponins(such as ginsenoside Re),amino acids,and acetylenic alcohols.Conclusion:AA,ponatinib,and ISO all can serve as inducers for the blood stasis model in zebrafish.AA-and ISO-induced models can be used to evaluate the activity of freeze-dried po

关 键 词：斑马鱼 三七 血瘀模型 心肌缺氧 血栓 

分 类 号：R2-0[医药卫生—中医学] R22