目的 基于網(wǎng)絡(luò)藥理學(xué)、分子對(duì)接技術(shù)和實(shí)驗(yàn)驗(yàn)證探究羽扇豆醇對(duì)肝癌HepG2細(xì)胞的作用機(jī)制。方法 采用網(wǎng)絡(luò)藥理學(xué)對(duì)羽扇豆醇作用靶點(diǎn)進(jìn)行篩選，構(gòu)建靶點(diǎn)網(wǎng)絡(luò)及蛋白質(zhì)–蛋白質(zhì)相互作用（PPI）網(wǎng)絡(luò)，對(duì)羽扇豆醇抗肝癌潛在的作用靶點(diǎn)及相關(guān)通路進(jìn)行預(yù)測(cè)。采用MTT法檢測(cè)羽扇豆醇對(duì)肝癌HepG2細(xì)胞的增殖抑制活性，采用酶標(biāo)儀檢測(cè)細(xì)胞內(nèi)Ca²⁺濃度，流式細(xì)胞術(shù)和激光共聚焦顯微鏡檢測(cè)細(xì)胞內(nèi)細(xì)胞的凋亡情況、線粒體膜電位、活性氧水平的變化情況；通過(guò)Western blotting法檢測(cè)羽扇豆醇對(duì)鈣調(diào)蛋白（CaM）、蘭尼堿受體（RyR）、B淋巴細(xì)胞瘤-2（Bcl-2）、Bcl-2相關(guān)X蛋白（Bax）、細(xì)胞色素C（Cyt C）、磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B（Akt）、哺乳動(dòng)物雷帕霉素靶蛋白（mTOR）蛋白表達(dá)水平的影響。結(jié)果 通過(guò)網(wǎng)絡(luò)藥理學(xué)預(yù)測(cè)，得出羽扇豆醇與肝癌有134個(gè)共同靶點(diǎn)；KEGG富集分析結(jié)果顯示羽扇豆醇治療肝癌潛在靶點(diǎn)主要富集在化學(xué)致癌–受體激活、鈣信號(hào)通路、PI3K/Akt信號(hào)通路等信號(hào)通路；MTT結(jié)果顯示，與對(duì)照組相比，羽扇豆醇組HepG2細(xì)胞的活性明顯降低，呈時(shí)間–濃度相關(guān)趨勢(shì)；羽扇豆醇能夠抑制肝癌細(xì)胞HepG2集落形成；采用50、75、100 μmol/L羽扇豆醇作用HepG2細(xì)胞48 h后，HepG2細(xì)胞內(nèi)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(xì)胞經(jīng)過(guò)羽扇豆醇處理后，細(xì)胞內(nèi)RyR、CaM、Cyt C、Bax的表達(dá)水平上升，p-PI3K、p/Akt、Bcl-2蛋白的表達(dá)水平下降。結(jié)論 羽扇豆醇可抑制肝癌HepG2細(xì)胞增殖，減少集落形成，其機(jī)制可能是鈣離子濃度升高，激活PI3K/Akt信號(hào)通路從而介導(dǎo)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(dòng)金項(xiàng)目（LBH-QY24003）