| 摘要: |
| 目的 基于网络药理学和体外实验探讨衢枳壳黄酮（PTFC）治疗肝癌的活性成分、潜在靶点和作用机制。方法 通过网络药理学方法筛选出PTFC抗肝癌的活性成分与基因靶点，利用Cytoscape软件绘制蛋白质-蛋白质相互作用（PPI）网络；对靶点基因进行基因本体论（GO）功能富集和京都基因与基因组百科全书（KEGG）信号通路富集分析；构建活性成分-靶点-通路可视化网络并筛选出关键活性成分和核心靶点，利用AutoDockTools软件进行分子对接；最后分别通过CCK-8、Western blot实验验证关键活性成分对HepG2细胞存活及EGFR-PI3K-Akt通路蛋白表达的影响。结果 共筛选出8个PTFC活性成分，15672个肝癌靶点，二者交集靶点256个。GO分析显示潜在治疗靶点参与的主要生物学过程包括烯烃化合物、花生四烯酸和类固醇的代谢过程等；KEGG分析显示主要信号通路包括PI3K-Akt信号通路、EGFR酪氨酸激酶抑制剂耐药信号通路和化学致癌-活性氧相关信号通路等。核心靶点关联程度由高到低依次为PIK3R1、PIK3CB、PIK3CA、AKT1、PIK3CD、SRC、EGFR、IGF1R、PTK2、MTOR。分子对接结果表明柚皮素、橙皮素与PIK3R1具有较好的亲和力。体外实验证实，柚皮素和橙皮素不仅能以剂量依赖性抑制HepG2细胞增殖，还能下调EGFR、PI3K、Akt蛋白表达（均P < 0.05）。结论 PTFC的潜在抗肿瘤作用可能与负向调控EGFR-PI3K-Akt信号通路异常活化密切相关，研究可为PTFC治疗肝癌提供理论依据。 |
| 关键词: 衢枳壳黄酮 肝癌 网络药理学 分子对接 体外实验 |
| DOI： |
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| 基金项目:浙江省中医药科技计划项目(2024ZR075) |
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| Mechanism of flavonoids of Citrus Paradisi cv. Changshan-huyou in the treatment of liver cancer |
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GAN Jinglu, YAO Wendong, XIE Xianze, JIANG Jiali
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The First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Chinese Medicine)
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| Abstract: |
| Objective To explore the active ingredients, potential targets, and mechanism of action of total flavonoids from Citrus Paradisi cv. Changshan-huyou (Quzhiqiao in Chinese, PTFC) in the treatment of liver cancer based on network pharmacology and in vitro experiments. Methods Network pharmacology was used to screen the active ingredients and gene targets of PTFC against liver cancer. Cytoscape software was employed to construct the protein-protein interaction (PPI) network. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis were performed on the target genes. A visual network of active ingredient-target-pathway was constructed to screen key active ingredients and hub targets, and molecular docking was conducted using AutoDockTools software. Finally, CCK-8 and Western blot assays were used to verify the effects of key active ingredients on HepG2 cell survival and the protein expression of EGFR-PI3K-Akt pathway. Results A total of 8 active ingredients of PTFC, 15672 liver cancer targets, and 256 potential therapeutic targets of PTFC against were identified. GO analysis showed that the potential therapeutic targets were involved in biological processes such as the metabolic processes of olefinic compounds, arachidonic acid, and steroids. KEGG analysis revealed that major signaling pathways including the PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, and chemical carcinogenesis-reactive oxygen species. Top 10 genes were identified as hub targets according to their degree values, which including PIK3R1, PIK3CB, PIK3CA, AKT1, PIK3CD, SRC, EGFR, IGF1R, PTK2, and MTOR. Molecular docking indicated that naringenin and hesperetin had good affinity with PIK3R1. In vitro experiments confirmed that naringenin and hesperetin not only inhibited HepG2 cell proliferation in a dose-dependent manner but also down-regulated the protein expression of EGFR, PI3K, and Akt (all P < 0.05). Conclusion The potential anti-tumor effect of PTFC may be closely related to the negative modulation of aberrant activation of the EGFR-PI3K-Akt signaling pathway, laying a theoretical foundation for PTFC intervention against liver cancer. |
| Key words: Flavonoids of Citrus Paradisi cv. Changshan-huyou Hepatocellular carcinoma Network pharmacology Molecular docking In vitro experiment |