目的 基于網(wǎng)絡(luò)藥理學(xué)結(jié)合分子對接技術(shù)探討雷公藤多苷片（TPT）生物堿類成分抗肝癌作用機(jī)制以及細(xì)胞實驗驗證TPT生物堿類成分誘導(dǎo)肝癌細(xì)胞毒性的作用。方法 利用Swiss Target Prediction和Targetnet數(shù)據(jù)庫預(yù)測TPT中9個生物堿類成分［雷公藤晉堿、雷公藤次堿、雷公藤春堿、雷公藤定堿、雷公藤寧堿A、18-O-(3-糠酰)雷公藤春堿、衛(wèi)矛堿、peritassine A、雷公藤新堿］的作用靶點。借助Genecards、OMIM和TTD數(shù)據(jù)庫檢索肝癌的相關(guān)靶點，將兩者輸入Venny在線工具交集得到生物堿類成分治療肝癌的作用靶點，通過Cytoscape軟件構(gòu)建“雷公藤多苷片-活性成分-靶點-肝癌”相互作用網(wǎng)絡(luò)。運用String數(shù)據(jù)庫對交集靶點進(jìn)行蛋白質(zhì)-蛋白質(zhì)相互作用（PPI）網(wǎng)絡(luò)分析，并使用Metascape數(shù)據(jù)庫進(jìn)行京都基因與基因組百科全書（KEGG）通路和基因本體（GO）富集分析。借助AutoDockTools和PyMol軟件對核心靶點及9種生物堿類成分進(jìn)行分子對接驗證。通過體外人肝癌HepG2細(xì)胞CCK-8實驗驗證TPT種9種生物堿的細(xì)胞毒作用。結(jié)果 經(jīng)篩選得到TPT生物堿成分潛在作用靶點119個，肝癌疾病靶點1 168個，交集靶點23個。經(jīng)PPI分析篩選出10個關(guān)鍵靶點，包括EGFR、CASP3、HSP90AA1等，KEGG富集分析主要涉及通路有Pathways in cancer、Prostate cancer、PI3K-Akt signalingpathway等。GO富集發(fā)現(xiàn)TPT生物堿類成分促肝癌細(xì)胞凋亡與改變細(xì)胞形態(tài)和調(diào)節(jié)蛋白激酶活性有關(guān)。分子對接顯示多種成分與多個關(guān)鍵靶點有較好的結(jié)合能力。細(xì)胞驗證實驗發(fā)現(xiàn)5種生物堿［雷公藤晉堿、雷公藤次堿、雷公藤寧堿A、18-O-(3-糠酰)雷公藤春堿、peritassine A］對HepG2細(xì)胞均具有細(xì)胞毒性。結(jié)論 TPT中9種生物堿類成分可能通過作用于EGFR、CASP3、HSP90AA1等關(guān)鍵靶點和調(diào)節(jié)APAF1/CASP9/CASP3信號通路，從而降低肝癌細(xì)胞增殖、激活體內(nèi)內(nèi)在凋亡途徑，最終促進(jìn)肝癌細(xì)胞凋亡，具有潛在的抗肝癌活性。

Objective To investigate the mechanism of anti-liver cancer effects of alkaloidal components of Tripterygium Polyglycoside Tablet (TPT) based on network pharmacology combined with molecular docking technique and to validate the cytotoxicity-inducing effect of TPT alkaloidal components in liver cancer by cellular experiment. Methods The Swiss Target Prediction and Targetnet databases were used to predict the targets of nine alkaloidal components (wilforgine, wilforine, wilfortrine, wilfordine, wilfornine A, triptonine B, euonymine, peritassine A, euonine) in TPT. The targets of liver cancer were retrieved from the Genecards, OMIM and TTD databases, and the targets of alkaloidal components for the treatment of liver cancer was were obtained by inputting the two into the Venny online tool for intersection. The interaction network of "TPT-active ingredient-target-liver cancer" was constructed by Cytoscape software. Protein-protein interaction (PPI) network analysis of the intersecting targets was performed using the String database, Protein-protein interaction (PPI) network analysis of the intersecting targets was performed using the String database, and the enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathways was performed using the Metascape database. Molecular docking validation of the core targets and nine alkaloidal components was performed by using AutoDockTools and PyMol software. Verification of the cytotoxicity of nine alkaloids of TPT by CCK-8 assay in human liver cancer HepG2 cells in vitro. Results The screening yielded 119 potential targets of TPT alkaloidal components, 1 168 liver cancer disease targets, and 23 intersecting targets. The PPI analysis identified ten key targets, including EGFR, CASP3, HSP90AA1, etc. The KEGG enrichment analysis mainly involved Pathways in cancer, Prostate cancer, PI3K-Akt signaling pathway, etc. The GO enrichment revealed that the TPT alkaloidal components promote apoptosis in liver cancer cells was associated with alteration of cell morphology and regulation of protein kinase activity. The molecular docking showed that multiple components have good binding ability to multiple key targets. Cellular validation experiments showed that five alkaloids (wilforgine, wilforine, wilfornine A, triptonine B, peritassine A) have cytotoxicity to HepG2 cells. Conclusion The nine alkaloidal components of TPT may act on key targets such as EGFR, CASP3, HSP90AA1 and modulate the APAF1/CASP9/CASP3 signaling pathway, thereby reducing cancer cell proliferation, activating the intrinsic apoptotic pathway in vivo and ultimately promoting apoptosis in liver cancer cells, having potential anti-liver cancer activity.

國家自然科學(xué)基金資助項目（82174209）