肝细胞癌目前是世界上死亡率第三高的癌症。肝细胞癌患者呈上升趋势且年轻化，但对癌症药理作用的研究多为单成分，天然植物产品可以具有相加或协同作用，可以更好地放大对癌症的干预效果。评估6-姜烯酚和姜黄素对肝癌细胞系HepG2的协同治疗作用。本研究采用网络药理学方法预测和验证6-姜烯酚和姜黄素治疗肝细胞癌（HCC）的分子靶点和通路。姜黄素的组合并研究其作用机制。结果还通过细胞测定得到验证。 HepG2 细胞用 6-姜烯酚和姜黄素以及两者的组合处理。使用Compusyn软件根据Chou-Talalay方程计算HepG2细胞中6-姜烯酚和姜黄素的结合指数。接下来通过MTT实验、细胞凋亡实验和细胞周期实验研究其协同抗癌作用。采用蛋白印迹法分析Ras介导的PI3K/AKT和MAPK信号通路的联合抗肝细胞癌作用。基于网络药理学的筛选确定了6-姜黄素和姜黄素在肝细胞癌中的72个核心靶点，并预测主要信号通路是Ras信号通路。 6-姜烯酚和姜黄素的抗癌作用在基于细胞的测定中得到验证，并确定了 6-姜烯酚 5 µmoL/L 和姜黄素 30 µmoL/L 的最佳协同浓度。 6-姜烯酚和姜黄素协同阻断细胞周期G2/M期并促进细胞凋亡。免疫印迹分析首次证实了两者在下调 Ras 介导的 PI3K/AKT 和 MAPK 信号通路中的联合作用。此外，6-姜烯酚和姜黄素共同作用，下调Cyclin-B、CDK-1、Bcl-2，上调BAX。6-姜烯酚和姜黄素协同作用，改变肝细胞癌细胞形态，阻断细胞周期于G2 /M期，抑制增殖和分裂，有效促进晚期凋亡。这两种成分的联合作用为进一步开发新型抗肝癌产品提供了理论基础。版权所有©2024 Jin，Jiao，Lian，Chitrakar，Sang 和 Wang。

Hepatocellular carcinoma currently has the third highest mortality rate in the world. Patients with hepatocellular carcinoma are on the rise and at a younger age, but research into the pharmacological effects of cancer is mostly single-component, and natural plant products can have additive or synergistic effects that can better amplify the effects of intervention in cancer.To evaluate the synergistic therapeutic effects of 6-shogaol and curcumin against hepatocellular carcinoma line HepG2 cells.In this study, a network pharmacology approach was used to predict and validate the mol ecular targets and pathways of the hepatocellular carcinoma (HCC) of 6-shogaol and curcumin in combination and to investigate their mechanism of action. The results were also validated by cellular assays. HepG2 cells were treated with 6-shogaol and curcumin as well as the combination of the two. The combination index of 6-shogaol and curcumin in HepG2 cells was calculated using Compusyn software according to the Chou-Talalay equation. The synergistic anti-cancer effect was next investigated by MTT assay, apoptosis assay and cell cycle assay. The combined anti-hepatocellular carcinoma effect of the Ras-mediated PI3K/AKT and MAPK signalling pathways was analysed using protein blotting assays.A network pharmacology-based screening identified 72 core targets of 6-curcumin and curcumin in hepatocellular carcinoma, and predicted that the main signalling pathway is the Ras signalling pathway. The anti-cancer effects of 6-shogaol and curcumin were validated in cell-based assays and the optimal synergistic concentrations of 5 μmoL/L for 6-shogaol and 30 μmoL/L for curcumin were determined. 6-shogaol and curcumin synergistically blocked the cell cycle in the G2/M phase and promoted apoptosis. Immunoblot analysis confirmed for the first time the combined action of both in down-regulating the Ras-mediated PI3K/AKT and MAPK signaling pathways. In addition, 6-shogaol and curcumin acting together downregulated Cyclin-B, CDK-1, Bcl-2, and upregulated BAX.6-shogaol and curcumin act synergistically to alter the morphology of hepatocellular carcinoma cells, block the cell cycle in the G2/M phase, inhibit proliferation and division, and effectively promote late apoptosis. The combined action of these two components provides a theoretical basis for the further development of novel anti-liver cancer products.Copyright © 2024 Jin, Jiao, Lian, Chitrakar, Sang and Wang.