Target protein numbers and docking results.

Objective
This study aimed to elucidate the effects of Hederagenin (HG) on hepatocellular carcinoma (HCC) and explore its potential molecular mechanisms.
Materials and methods
Virtual screening was employed to identify potential targets within core pathways of liver cancer and to analyze the possible mechanisms of HG. CCK-8 assays were used to assess the viability of HCC cells, while Hoechst 33342/PI staining was utilized to evaluate apoptosis. The migration and invasion abilities of HCC cells were examined using Transwell and scratch assays, and single-cell cloning ability was assessed via colony formation assays. Subsequent qRT-PCR was conducted to determine the mRNA expression levels of FOXO1 and FOXO6 following HG treatment. Western blot (WB) analysis was employed to measure the protein expression levels of IGF1R, FOXO1, FOXO6, MMP2, MMP9, and VEGFA, as well as the phosphorylation status of FOXO1 Ser249.
Results
Virtual screening indicated that HG might exert antitumor effects through the FOXO signaling pathway. Experimental results demonstrated that HG induces apoptosis in a dose-dependent manner and inhibits the proliferation, migration, invasion, and single-cell cloning ability of HCC cells. After HG treatment, FOXO1 expression was upregulated, while the expression levels of IGF1R, phosphorylated FOXO1 Ser249, MMP2, MMP9, and VEGFA were downregulated.
Conclusion
In summary, our study is the first to demonstrate that HG regulates the phosphorylation of FOXO1, affecting the proliferation, migration, and invasion of HCC cells. The findings suggest that HG can inhibit the migration of HCC cells in vitro. The data indicate that HG-mediated targeting of the FOXO1/FOXO6 pathway holds promise as a novel therapeutic approach.

研究目的
本研究旨在阐明常春藤皂苷元（Hederagenin, HG）对肝细胞癌（hepatocellular carcinoma, HCC）的作用，并探讨其潜在分子机制。
材料与方法
本研究采用虚拟筛选技术，鉴定肝癌核心通路中的潜在靶点，并分析HG的潜在作用机制。采用CCK-8实验检测肝癌细胞的增殖活性；通过Hoechst 33342/PI双染色法评估细胞凋亡水平。利用Transwell实验与划痕实验检测肝癌细胞的迁移与侵袭能力，采用集落形成实验评估单细胞克隆能力。后续通过实时荧光定量PCR（qRT-PCR）检测HG处理后叉头框O转录因子1（FOXO1）与叉头框O转录因子6（FOXO6）的mRNA表达水平。采用蛋白质印迹法（Western blot, WB）检测胰岛素样生长因子1受体（IGF1R）、FOXO1、FOXO6、基质金属蛋白酶2（MMP2）、基质金属蛋白酶9（MMP9）及血管内皮生长因子A（VEGFA）的蛋白表达水平，同时检测FOXO1 Ser249位点的磷酸化状态。
实验结果
虚拟筛选结果显示，HG可能通过叉头框O转录因子（FOXO）信号通路发挥抗肿瘤作用。实验结果表明，HG可呈剂量依赖性诱导肝癌细胞凋亡，并抑制肝癌细胞的增殖、迁移、侵袭及单细胞克隆能力。经HG处理后，FOXO1的表达水平上调，而IGF1R、磷酸化FOXO1 Ser249、MMP2、MMP9及VEGFA的表达水平均下调。
结论
综上，本研究首次证实HG可通过调控FOXO1的磷酸化状态，影响肝癌细胞的增殖、迁移与侵袭能力。研究结果表明，HG可在体外抑制肝癌细胞的迁移。数据显示，HG介导的FOXO1/FOXO6通路靶向调控有望成为一种全新的治疗策略。