基于斑马鱼模型和网络药理学的甘草酸拮抗乌头碱心脏毒性作用与机制研究

Study of glycyrrhizic acid ameliorates cardiotoxicity of aconitine and potential mechanism based on zebrafish model and network pharmacology

目的利用斑马鱼模型结合网络药理学,研究甘草酸拮抗乌头碱心脏毒性及作用机制,探讨附子-甘草配伍减毒的现代科学内涵。方法选用受精后48 h(48 hpf)的健康野生型AB系斑马鱼,脱膜后随机移入6孔板中,每孔30枚。对照组加入新鲜养鱼水;模型组和各药物处理组均给予乌头碱溶液,终浓度均为10μmol/L;各药物组同时分别给予低、中、高浓度(10、50、100μmol/L)的甘草酸、甘草素、甘草苷和低、中、高浓度(1、5、10μmol/L)的异甘草素,各组斑马鱼处理后放入恒温光照培养箱中培养至72 hpf,在显微镜下记录斑马鱼20 s心跳并拍照,计算心率,并利用Image-Pro Plus 5.0软件计算心包面积,筛选甘草中拮抗乌头碱心脏毒性的成分。通过心脏结构与功能分析、活性氧(ROS)和凋亡细胞检测,进一步验证甘草酸拮抗乌头碱心脏毒性作用。基于PharmMapper、Swiss Target Prediction、STITCH数据库收集乌头碱毒性作用靶点,利用CTD、GeneCards、DisGeNET数据库,以“心脏毒性”“心脏损伤”“心衰”为关键词检索靶点,将上述两组靶点导入Draw venn在线绘图取交集,得到乌头碱心脏毒性作用靶点集;从PharmMapper数据库中收集甘草酸活性靶点集;将2个靶点集共有靶点导入String数据库进行蛋白互作分析;使用Cytoscape 3.6.0软件对结果进行拓扑分析,以节点度值中位数为标准筛选重要靶点;得到的靶点导入DAVID数据库,进行GO和KEGG分析。结果与对照组比较,模型组幼鱼心包水肿显著(P<0.01),心率显著升高(P<0.01);与模型组比较,甘草酸与异甘草素各浓度组、甘草苷高浓度组心包面积显著下降(P<0.01),甘草酸效果更加明显且呈浓度相关性;甘草酸低和高浓度、甘草素高浓度和异甘草素低浓度拮抗乌头碱所致心率加快效果显著(P<0.05、0.01)。甘草酸可浓度相关性地拮抗乌头碱导致的斑马鱼心室短轴缩短率降低,且高浓度组具有显著性差异(P<0.01);甘草酸显著抑制乌头碱诱导的斑马鱼体内活性氧生成(P<0.01);明显减弱乌头碱导致的细胞凋亡。甘草酸拮抗乌头碱心脏毒性的作用机制可能与MAPK、GRB2、CDC42、EGFR、GSK3B、SRC等关键靶点,以及PI3K-Akt、Ras、FoxO等信号通路相关。结论甘草酸可以显著拮抗乌头碱心脏毒性,可能通过调控PI3K-Akt、Ras信号通路等发挥作用。

Objective The zebrafish model and network pharmacology were used to study the effect of glycyrrhizic acid ameliorated the cardiotoxicity of aconitine and its mechanism,and to explore the modern scientific connotation of the compatibility to reduce the poison of Radix Aconiti Laterlis Preparata and Radix Glycyrrhizae.Methods Healthy wild-type AB zebrafish 48 h post fertilization(hpf)were randomly transplanted into 6-well plates with 30 fish per well after demiltration.The control group was added with fresh fish water.Model group and each drug treatment group were given aconitine solution,the final concentration was 10μmol/L.Each drug group was given glycyrrhizic acid,glycyrrhizin,and liquiritin at low,medium and high concentrations(10,50 and 100μmol/L)and isoglycyrrhizin at low,medium and high concentrations(1,5 and 10μmol/L),respectively.Zebrfish in each group were treated and cultured in an incubator with constant temperature and light until 72 hpf.The zebrafish heart rate was recorded and photographed for 20 s under a microscope,and the heart rate was calculated.Image-Pro Plus 5.0 software was used to calculate the pericardial area,and the components antagonizing aconitine cardiotoxicity in licorice were screened.The antagonistic effect of glycyrrhizic acid against aconitine cardiotoxicity was further verified by analyzing cardiac structure and function,detecting reactive oxygen species(ROS)and apoptosis cells.Toxicity targets of aconitine were collected based on Pharmapper,Swiss Target Prediction and Stitch database,and"cardiotoxicity","heart injury"and"heart failure"were used as keywords for Target retrieval by using CTD,GeneCards and DisgeNet databases.The above two groups of targets were imported into Draw Venn online mapping to obtain the intersection of the cardiac toxicity target set of aconitine.Glycyrrhizin active target sets were collected from PharmMapper database;The common targets of two target sets were imported into the STRING database for protein interaction analysis.Cytoscape 3.6.0 software was used to analyze the topology of the results,and the median of node degree was used as the standard to screen important targets.The obtained targets were imported into David database for GO and KEGG analysis.Results Compared with the control group,the edema of pericardium in model group was significantly higher(P<0.01),and the heart rate was significantly higher(P<0.01).Compared with model group,the pericardial area of glycyrrhizin and isoglycyrrhizin concentration groups and glycyrrhizin high concentration group were significantly decreased(P<0.01),and the effect of glycyrrhizin was more obvious and concentration dependent.The effect of low and high concentrations of glycyrrhizic acid,high and low concentrations of glycyrrhizin and isoglycyrrhizin on the increased heart rate induced by aconitine was significant(P<0.05,0.01).Glycyrrhizin could concentrationdependent antagonize the decreased rate of ventricle shortening caused by aconitine,and the difference was significant in high concentration group(P<0.01).Glycyrrhizin significantly inhibited the production of reactive oxygen species(ROS)in zebrafish induced by aconitine(P<0.01).The apoptosis induced by aconitine was significantly reduced.The mechanism of glycyrrhizin antagonizing aconitine cardiotoxicity may be related to MAPK,GRB2,CDC42,EGFR,GSK3b,SRC and other key targets,as well as the PI3K-Akt,Ras,FoxO and other signaling pathways.Conclusion Glycyrrhizic acid significantly alleviated the cardiotoxicity of aconitine via regulating PI3K-Akt and Ras signaling pathways.