目的 基于网络药理学、分子对接技术和实验验证探究羽扇豆醇对肝癌HepG2细胞的作用机制。方法 采用网络药理学对羽扇豆醇作用靶点进行筛选，构建靶点网络及蛋白质–蛋白质相互作用（PPI）网络，对羽扇豆醇抗肝癌潜在的作用靶点及相关通路进行预测。采用MTT法检测羽扇豆醇对肝癌HepG2细胞的增殖抑制活性，采用酶标仪检测细胞内Ca²⁺浓度，流式细胞术和激光共聚焦显微镜检测细胞内细胞的凋亡情况、线粒体膜电位、活性氧水平的变化情况；通过Western blotting法检测羽扇豆醇对钙调蛋白（CaM）、兰尼碱受体（RyR）、B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、细胞色素C（Cyt C）、磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B（Akt）、哺乳动物雷帕霉素靶蛋白（mTOR）蛋白表达水平的影响。结果 通过网络药理学预测，得出羽扇豆醇与肝癌有134个共同靶点；KEGG富集分析结果显示羽扇豆醇治疗肝癌潜在靶点主要富集在化学致癌–受体激活、钙信号通路、PI3K/Akt信号通路等信号通路；MTT结果显示，与对照组相比，羽扇豆醇组HepG2细胞的活性明显降低，呈时间–浓度相关趋势；羽扇豆醇能够抑制肝癌细胞HepG2集落形成；采用50、75、100 μmol/L羽扇豆醇作用HepG2细胞48 h后，HepG2细胞内Ca²⁺浓度显著升高，总凋亡率均显著升高（P＜0.01、0.001），线粒体膜电位均显著降低，活性氧水平均显著升高；此外，HepG2细胞经过羽扇豆醇处理后，细胞内RyR、CaM、Cyt C、Bax的表达水平上升，p-PI3K、p/Akt、Bcl-2蛋白的表达水平下降。结论 羽扇豆醇可抑制肝癌HepG2细胞增殖，减少集落形成，其机制可能是钙离子浓度升高，激活PI3K/Akt信号通路从而介导线粒体凋亡。

Objective To investigate the targets and mechanisms of lupeol on hepatocellular carcinoma HepG2 cells based on network pharmacology, molecular docking technology and experimental verification. Methods Network pharmacology was employed to screen the potential targets of lupeol, construct target networks and protein-protein interaction (PPI) networks, and predict the potential targets and related pathways of lupeol in anti-liver cancer activity. The inhibitory effect of lupeol on the proliferation of HepG2 liver cancer cells was assessed using the MTT assay. Intracellular Ca²⁺ concentration was measured using a microplate reader. Flow cytometry and confocal laser microscopy were used to detect changes in cell apoptosis, mitochondrial membrane potential, and reactive oxygen species (ROS) levels. Western blotting was performed to examine the effects of lupeol on the expression levels of calmodulin (CaM), ryanodine receptor (RyR), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), cytochrome C (Cyt C), phosphoinositide 3-kinase (PI3K), serine-threonine kinase (Akt), and mammalian target of rapamycin (mTOR). Results Network pharmacology analysis predicted 134 common targets between lupeol and liver cancer. KEGG enrichment analysis revealed that the potential targets of lupeol in liver cancer treatment were mainly enriched in pathways such as chemical carcinogenesis-receptor activation, calcium signaling pathway, and PI3K/Akt signaling pathway. MTT results demonstrated that compared to the control group, the viability of HepG2 cells in the lupeol group significantly decreased in a time- and concentration-dependent manner. Lupeol inhibited the colony formation of HepG2 cells. After treating HepG2 cells with 50, 75, and 100 μmol/L lupeol for 48 hours, intracellular Ca²⁺ concentration, total apoptosis rate, and ROS levels significantly increased, while mitochondrial membrane potential significantly decreased. Additionally, after lupeol treatment, the expression levels of RyR, CaM, Cyt C, and Bax in HepG2 cells were upregulated, while the expression levels of p-PI3K, p-Akt, and Bcl-2 were downregulated. Conclusion Lupeol inhibits the proliferation and colony formation of HepG2 liver cancer cells. Its mechanism may involve increased intracellular Ca²⁺ concentration, activation of the PI3K/Akt signaling pathway, and induction of mitochondrial apoptosis.

R965

黑龙江省博士后科研启动金项目（LBH-QY24003）