目的 基于网络药理学和分子对接技术并结合体内外实验研究炮制对苍耳子肝毒性的影响。方法 通过中药系统药理学数据库和分析平台(Traditional Chinese Medicine Systems Pharmacology，TCMSP)、毒性与基因比较数据库(The Comparative Toxicogenomics Database，CTD)筛选苍耳子的毒性成分，利用Swiss Target Prediction数据库获取毒性成分作用靶点;检索Gene cards数据库和DisGeNET数据库中肝相关疾病靶点，通过绘制韦恩图获取苍耳子毒性成分靶点和肝疾病相关靶点的交集;通过Cytoscape软件构建"药物-毒性成分-靶点"网络，利用STRING数据库对苍耳子肝毒性作用靶点进行蛋白质相互作用(protein-protein interaction，PPI)分析并筛选关键靶点;利用Metascape数据库对上述关键靶点进行基因本体论(Gene Ontology，GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)通路富集分析。使用AutoDocktools和PyMOL软件对苍耳子的肝毒性成分和核心靶点进行分子对接验证。体外实验采用MTS法比较苍耳子不同炮制品对正常肝细胞株(L-02)毒性，并测定培养液中乳酸脱氢酶(lactate dehydrogenase，LDH)释放，利用流式细胞仪检测细胞的凋亡情况。体内实验检测灌服苍耳子不同炮制品小鼠血清中谷丙转氨酶(Alanine aminotransferase，ALT)和谷草转氨酶(Aspartate aminotransferase，AST)活性。结果 网络药理学分析苍耳子肝毒性成分主要包括羧基苍术苷(carboxyatractyloside)、谷甾醇(sitosterol)、豆甾醇(stigmasterol)等，其核心靶点主要为PPARG、PPARA、NR1H3等。GO功能富集到脂质定位和储存的正、负调节和脂质代谢过程调节等生物过程;转录调节复合物、神经细胞体和受体复合物等细胞组分，转录因子结合、核受体结合和蛋白质结构域特异性结合等分子功能。KEGG通路富集到非酒精性脂肪肝、脂质和动脉粥样硬化、酒精肝、胰岛素抵抗和PPAR等信号通路。分子对接结果显示，肝毒性化合物与核心靶点能较好地结合。体外实验结果显示，苍耳子生品明显促进L-02细胞LDH释放，显示具有明显的细胞毒性，并能诱导细胞凋亡(P＜0。05)，炮制品能减少LDH释放，降低细胞毒性，诱导细胞凋亡作用减弱，其中酒炙品效果更明显。体内实验结果显示，苍耳子生品显著升高正常小鼠血清中AST和ALT的活性(P＜0。05)，炮制品无显著影响(P＞0。05)。结论 炒制和酒炙炮制方法可能通过减弱PPARA、PPARG和NR1H3等肝毒性靶点效应改善脂质代谢信号通路异常，从而降低苍耳子肝毒性，且酒炙法减毒效果更好。
Effect of Processing on Hepatotoxicity of Cang'erzi(Xanthii Fructus):Network Pharmacology and Experiment Validation
Objective Based on network pharmacology and molecular docking technology,combined with in vivo and in vitro experiments,the effects of processing on the hepatotoxicity of Cang'erzi(Xanthii Fructus)were studied.Methods Screening the toxic components of Cang'erzi(Xanthii Fructus)through Traditional Chinese Medicine Systems Pharmacology(TCMSP)and The Comparative Toxicogenomics Database(CTD)databases.The action targets of the toxic components were obtained using the Swiss Target Prediction database.Liver related disease targets were retrieved in the Gene cards database and DisGeNET database,and the intersection of Cang'erzi(Xanthii Fructus)toxic component action targets and liver disease related targets were obtained by drawing Venn diagrams.The"drug-toxic component-target"network was constructed by Cytoscape software,and STRING da-tabase was used to analyze protein-protein interaction(PPI)and screen key targets for hepatotoxicity of Cang'erzi(Xanthii Fructus).Metascape database was employed to conduct Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis on key targets.AutoDocktools and PyMOL software were used to perform molecular doc-king validation on the hepatotoxic components and key targets of Cang'erzi(Xanthii Fructus).The toxicity of different processed products of Cang'erzi(Xanthii Fructus)on normal liver cell lines(L-02)was tested by MTS method,and the release of lactate dehydrogenase(LDH)in the culture medium was measured.At the same time,cell apoptosis was detected by flow cytometry in vitro.The activities of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum of mice were detected in vivo.Results Network pharmacology analysis showed that the hepatotoxic components of Cang'erzi(Xanthii Fructus)mainly in-cluded carboxyatractyloside,sitosterol,stigmasterol,etc.Its key targets were PPARG,PPARA,NR1H3,etc.GO functions were enriched in biological processes(positive and negative regulation of lipid localization and storage,as well as regulation of lipid me-tabolism processes),cell components(transcriptional regulatory complexes,neural cell bodies,and receptor complexes)and mo-lecular functions(transcription factor binding,nuclear receptor binding,and protein domain specific binding).KEGG pathway was enriched to nonalcoholic fatty liver,lipid and atherosclerosis,alcoholic liver disease,insulin resistance and PPAR signaling pathways.The molecular docking results showed that the hepatotoxic compounds of Cang'erzi(Xanthii Fructus)could bind well to the key targets.The in vitro experimental results showed that the raw product of Cang'erzi(Xanthii Fructus)significantly pro-moted the release of LDH in L-02 cells,showing significant cytotoxicity and inducing cell apoptosis(P＜0.05).Processed products could reduce LDH release,attenuate cell toxicity and weaken the induction of cell apoptosis,and the wine-processing product had a more significant effect.The in vivo experimental results showed that the raw product of Cang'erzi(Xanthii Fruc-tus)significantly increased the activities of ALT and AST in the serum of normal mice(P＜0.05),while the processed product had no significant effect on it(P＞0.05).Conclusion The methods of stir-frying and wine processing may improve the abnormal lipid metabolism signaling pathway by reducing the effects of Cang'erzi(Xanthii Fructus)on liver toxicity targets such as PPARA,PPARG and NR1H3,thereby reducing liver toxicity,and wine processing has a better detoxification effect.
万方数据
维普
