目的 基于網(wǎng)絡(luò)藥理學(xué)探究17-羥-巖大戟內(nèi)酯B（HJB）對(duì)人肝癌HepG2細(xì)胞凋亡的作用機(jī)制，并進(jìn)行實(shí)驗(yàn)驗(yàn)證。方法 采用網(wǎng)絡(luò)藥理學(xué)對(duì)HJB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)HJB抗肝癌潛在的作用靶點(diǎn)及相關(guān)通路進(jìn)行預(yù)測(cè)。并用0（空白對(duì)照）、2.5、5、10、20、40、80 µmol/L的HJB作用于HepG2細(xì)胞24、48、72 h，采用細(xì)胞計(jì)數(shù)試劑盒-8（CCK-8）法檢測(cè)各組細(xì)胞的增殖活性。以0（空白對(duì)照）、5、10、20 µmol/L的HJB作用于HepG2細(xì)胞48 h后，膜聯(lián)蛋白V（Annexin V）–異硫氰酸熒光素（FITC）/碘化丙啶（PI）雙染法檢測(cè)各組細(xì)胞的凋亡，JC-1染色法觀察細(xì)胞的線粒體膜電位變化，DCFH-DA染色法觀察細(xì)胞的活性氧水平。Western blotting法檢測(cè)各組細(xì)胞線粒體凋亡及磷脂酰肌醇3激酶（PI3K）-蛋白激酶B（Akt）信號(hào)通路相關(guān)蛋白表達(dá)變化。結(jié)果 通過網(wǎng)絡(luò)藥理學(xué)預(yù)測(cè)，得出HJB與肝癌有291個(gè)共同靶點(diǎn)；京都基因與基因組百科全書（KEGG）富集分析結(jié)果顯示HJB治療肝癌結(jié)果主要在癌癥通路、癌癥中的蛋白多糖、血脂與動(dòng)脈粥樣硬化和PI3K-Akt信號(hào)通路等信號(hào)通路；HJB時(shí)間和劑量相關(guān)性地顯著抑制HepG2細(xì)胞增殖。與空白對(duì)照組相比，5、10、20 µmol/L HJB組細(xì)胞的活性氧水平、凋亡率、B淋巴細(xì)胞瘤-2（Bcl-2）相關(guān)X蛋白（Bax）、細(xì)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）蛋白水平呈濃度相關(guān)性升高（P＜0.05），線粒體膜電位、Bcl-2、磷酸化PI3K（p-PI3K）、磷酸化Akt（p-Akt）蛋白表達(dá)顯著降低（P＜0.05）。結(jié)論 HJB可抑制肝癌HepG2細(xì)胞增殖，促進(jìn)HepG2細(xì)胞凋亡，其機(jī)制可能與PI3K-Akt通路介導(dǎo)線粒體凋亡途徑有關(guān)。

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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黑龍江省省屬本科高?；究蒲袠I(yè)務(wù)費(fèi)科研項(xiàng)目（2023-KYYWF-0870）;黑龍江省衛(wèi)生健康委科研項(xiàng)目（20221313050620）;黑龍江省中醫(yī)藥管理局青年中醫(yī)藥科研課題（ZYH2024-297）