目的 基于網絡藥理學、分子對接技術和實驗驗證探究羽扇豆醇對肝癌HepG2細胞的作用機制。方法 采用網絡藥理學對羽扇豆醇作用靶點進行篩選，構建靶點網絡及蛋白質–蛋白質相互作用（PPI）網絡，對羽扇豆醇抗肝癌潛在的作用靶點及相關通路進行預測。采用MTT法檢測羽扇豆醇對肝癌HepG2細胞的增殖抑制活性，采用酶標儀檢測細胞內Ca²⁺濃度，流式細胞術和激光共聚焦顯微鏡檢測細胞內細胞的凋亡情況、線粒體膜電位、活性氧水平的變化情況；通過Western blotting法檢測羽扇豆醇對鈣調蛋白（CaM）、蘭尼堿受體（RyR）、B淋巴細胞瘤-2（Bcl-2）、Bcl-2相關X蛋白（Bax）、細胞色素C（Cyt C）、磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B（Akt）、哺乳動物雷帕霉素靶蛋白（mTOR）蛋白表達水平的影響。結果 通過網絡藥理學預測，得出羽扇豆醇與肝癌有134個共同靶點；KEGG富集分析結果顯示羽扇豆醇治療肝癌潛在靶點主要富集在化學致癌–受體激活、鈣信號通路、PI3K/Akt信號通路等信號通路；MTT結果顯示，與對照組相比，羽扇豆醇組HepG2細胞的活性明顯降低，呈時間–濃度相關趨勢；羽扇豆醇能夠抑制肝癌細胞HepG2集落形成；采用50、75、100 μmol/L羽扇豆醇作用HepG2細胞48 h后，HepG2細胞內Ca²⁺濃度顯著升高，總凋亡率均顯著升高（P＜0.01、0.001），線粒體膜電位均顯著降低，活性氧水平均顯著升高；此外，HepG2細胞經過羽扇豆醇處理后，細胞內RyR、CaM、Cyt C、Bax的表達水平上升，p-PI3K、p/Akt、Bcl-2蛋白的表達水平下降。結論 羽扇豆醇可抑制肝癌HepG2細胞增殖，減少集落形成，其機制可能是鈣離子濃度升高，激活PI3K/Akt信號通路從而介導線粒體凋亡。

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

黑龍江省博士后科研啟動金項目（LBH-QY24003）