目的 基于网络药理学探究17-羟-岩大戟内酯B（HJB）对人肝癌HepG2细胞凋亡的作用机制，并进行实验验证。方法 采用网络药理学对HJB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HJB抗肝癌潜在的作用靶点及相关通路进行预测。并用0（空白对照）、2.5、5、10、20、40、80 µmol/L的HJB作用于HepG2细胞24、48、72 h，采用细胞计数试剂盒-8（CCK-8）法检测各组细胞的增殖活性。以0（空白对照）、5、10、20 µmol/L的HJB作用于HepG2细胞48 h后，膜联蛋白V（Annexin V）–异硫氰酸荧光素（FITC）/碘化丙啶（PI）双染法检测各组细胞的凋亡，JC-1染色法观察细胞的线粒体膜电位变化，DCFH-DA染色法观察细胞的活性氧水平。Western blotting法检测各组细胞线粒体凋亡及磷脂酰肌醇3激酶（PI3K）-蛋白激酶B（Akt）信号通路相关蛋白表达变化。结果 通过网络药理学预测，得出HJB与肝癌有291个共同靶点；京都基因与基因组百科全书（KEGG）富集分析结果显示HJB治疗肝癌结果主要在癌症通路、癌症中的蛋白多糖、血脂与动脉粥样硬化和PI3K-Akt信号通路等信号通路；HJB时间和剂量相关性地显著抑制HepG2细胞增殖。与空白对照组相比，5、10、20 µmol/L HJB组细胞的活性氧水平、凋亡率、B淋巴细胞瘤-2（Bcl-2）相关X蛋白（Bax）、细胞色素C（Cyt C）、裂解胱天蛋白酶-9（cleaved Caspase-9）、裂解胱天蛋白酶-3（cleaved Caspase-3）蛋白水平呈浓度相关性升高（P＜0.05），线粒体膜电位、Bcl-2、磷酸化PI3K（p-PI3K）、磷酸化Akt（p-Akt）蛋白表达显著降低（P＜0.05）。结论 HJB可抑制肝癌HepG2细胞增殖，促进HepG2细胞凋亡，其机制可能与PI3K-Akt通路介导线粒体凋亡途径有关。

Objective To investigate the effects of 17-hydroxyjolkinolide B (HJB) on apoptosis of human hepatocellular carcinoma cells based on network pharmacology and to analyze the possible mechanisms. Methods Network pharmacology was used to screen the targets of HJB action, construct target networks and protein-protein interactions (PPI) networks, and predict the potential targets of HJB anti-hepatocellular carcinoma action and related pathways. HepG2 cells were treated with 0 (blank control), 2.5, 5, 10, 20, 40 and 80 µmol/L of HJB for 24, 48 and 72 h, and the proliferative activities of the cells in each group were detected by CCK-8 assay. After HepG2 cells were treated with 0 (blank control), 5, 10 and 20 µmol/L of HJB for 48 h, apoptosis of cells in each group was detected by Annexin V-FITC/PI double staining, mitochondrial membrane potential changes of the cells were observed by JC-1 staining, and reactive oxygen species levels of the cells were observed by DCFH-DA staining. Western blot assay was performed to detect mitochondrial apoptosis of cells and the proliferative activity of cells in each group. Western blotting assay was used to detect the mitochondrial apoptosis and protein expression changes related to PI3K-Akt signaling pathway in each group. Results The network pharmacological prediction yielded 291 common targets between HJB and hepatocellular carcinoma; KEGG enrichment analysis showed that the results of HJB treatment for hepatocellular carcinoma were mainly in the signaling pathways of cancer pathway, proteoglycans in cancer, lipids and atherosclerosis, and PI3K-Akt signaling pathway and the time-dose related significant inhibition of HepG2 cell proliferation by HJB. Compared with the blank control group, after the action of HJB, the cellular reactive oxygen species level, apoptosis rate, Bax, Cyt C, cleaved caspase-9, cleaved caspase-3 protein levels were increased in a concentration-dependent manner (P< 0.05), and the mitochondria’s membrane potential, Bcl-2, p-PI3K, and p-Akt protein expression were significantly reduced (P< 0.05). Conclusion The study showed that HJB inhibited the value-added of hepatocellular carcinoma HepG2 cells and promoted apoptosis of HepG2 cells, and the mechanism may be related to the PI3K-Akt pathway mediating the mitochondrial apoptotic pathway.

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黑龙江省省属本科高校基本科研业务费科研项目（2023-KYYWF-0870）;黑龙江省卫生健康委科研项目（20221313050620）;黑龙江省中医药管理局青年中医药科研课题（ZYH2024-297）