目的 利用斑馬魚(yú)模型結(jié)合網(wǎng)絡(luò)藥理學(xué)，研究甘草酸拮抗烏頭堿心臟毒性及作用機(jī)制，探討附子-甘草配伍減毒的現(xiàn)代科學(xué)內(nèi)涵。方法 選用受精后48 h（48 hpf）的健康野生型AB系斑馬魚(yú)，脫膜后隨機(jī)移入6孔板中，每孔30枚。對(duì)照組加入新鮮養(yǎng)魚(yú)水；模型組和各藥物處理組均給予烏頭堿溶液，終濃度均為10 μmol/L；各藥物組同時(shí)分別給予低、中、高濃度（10、50、100 μmol/L）的甘草酸、甘草素、甘草苷和低、中、高濃度（1、5、10 μmol/L）的異甘草素，各組斑馬魚(yú)處理后放入恒溫光照培養(yǎng)箱中培養(yǎng)至72 hpf，在顯微鏡下記錄斑馬魚(yú)20 s心跳并拍照，計(jì)算心率，并利用Image-Pro Plus5.0軟件計(jì)算心包面積，篩選甘草中拮抗烏頭堿心臟毒性的成分。通過(guò)心臟結(jié)構(gòu)與功能分析、活性氧（ROS）和凋亡細(xì)胞檢測(cè)，進(jìn)一步驗(yàn)證甘草酸拮抗烏頭堿心臟毒性作用。基于PharmMapper、Swiss Target Prediction、STITCH數(shù)據(jù)庫(kù)收集烏頭堿毒性作用靶點(diǎn)，利用CTD、GeneCards、DisGeNET數(shù)據(jù)庫(kù)，以“心臟毒性”“心臟損傷”“心衰”為關(guān)鍵詞檢索靶點(diǎn)，將上述兩組靶點(diǎn)導(dǎo)入Draw venn在線繪圖取交集，得到烏頭堿心臟毒性作用靶點(diǎn)集；從PharmMapper數(shù)據(jù)庫(kù)中收集甘草酸活性靶點(diǎn)集；將2個(gè)靶點(diǎn)集共有靶點(diǎn)導(dǎo)入String數(shù)據(jù)庫(kù)進(jìn)行蛋白互作分析；使用Cytoscape 3.6.0軟件對(duì)結(jié)果進(jìn)行拓?fù)浞治觯怨?jié)點(diǎn)度值中位數(shù)為標(biāo)準(zhǔn)篩選重要靶點(diǎn)；得到的靶點(diǎn)導(dǎo)入DAVID數(shù)據(jù)庫(kù)，進(jìn)行GO和KEGG分析。結(jié)果 與對(duì)照組比較，模型組幼魚(yú)心包水腫顯著（P<0.01），心率顯著升高（P<0.01）；與模型組比較，甘草酸與異甘草素各濃度組、甘草苷高濃度組心包面積顯著下降（P<0.01），甘草酸效果更加明顯且呈濃度相關(guān)性；甘草酸低和高濃度、甘草素高濃度和異甘草素低濃度拮抗烏頭堿所致心率加快效果顯著（P<0.05、0.01）。甘草酸可濃度相關(guān)性地拮抗烏頭堿導(dǎo)致的斑馬魚(yú)心室短軸縮短率降低，且高濃度組具有顯著性差異（P<0.01）；甘草酸顯著抑制烏頭堿誘導(dǎo)的斑馬魚(yú)體內(nèi)活性氧生成（P<0.01）；明顯減弱烏頭堿導(dǎo)致的細(xì)胞凋亡。甘草酸拮抗烏頭堿心臟毒性的作用機(jī)制可能與MAPK、GRB2、CDC42、EGFR、GSK3B、SRC等關(guān)鍵靶點(diǎn)，以及PI3K-Akt、Ras、FoxO等信號(hào)通路相關(guān)。結(jié)論 甘草酸可以顯著拮抗烏頭堿心臟毒性，可能通過(guò)調(diào)控PI3K-Akt、Ras信號(hào)通路等發(fā)揮作用。

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 10 μmol/L. Each drug group was given glycyrrhizic acid, glycyrrhizin, and liquiritin at low, medium and high concentrations (10, 50 and 100 μmol/L) and isoglycyrrhizin at low, medium and high concentrations (1, 5 and 10 μmol/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.

山東省優(yōu)秀青年人才基金項(xiàng)目（ZR2020YQ60）；齊魯工業(yè)大學(xué)（山東省科學(xué)院）科教產(chǎn)融合創(chuàng)新試點(diǎn)工程項(xiàng)目（2020KJCZD08），濟(jì)南市“高校20條”資助項(xiàng)目（2020GXRC053）