目的 通过网络药理学、分子对接技术和实验验证探究17-羟-岩大戟内酯B-4（HJB-4）对膀胱癌T24细胞的作用机制。方法 运用网络药理学对HJB-4的作用靶点进行筛选，构建蛋白质相互作用（PPI）网络，进行基因本体（GO）功能和京都基因与基因组百科全书（KEGG）通路富集分析，并进行分子对接验证。采用CCK-8检测HJB-4对膀胱癌T24细胞的增殖抑制活性，采用流式细胞术和激光共聚焦显微镜检测细胞凋亡情况，采用JC-1流式细胞术检测细胞线粒体膜电位变化情况；Western blotting检测B细胞淋巴瘤2（Bcl-2）、Bcl-2相关X蛋白（Bax）、Bcl-2相互作用细胞死亡介质（Bim）、细胞色素C（Cyt-C、裂解化半胱氨酸蛋白酶3（cleaved-Caspase-3）cleaved Caspase-9、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水平。结果 通过网络药理学预测，共获取315个共同靶点；GO分析和KEGG通路富集分析结果显示HJB-4可能通过丝裂原活化蛋白激酶（MAPK）信号通路、PI3K/Akt通路及凋亡途径等发挥作用。分子对接显示HJB-4与核心靶点的结合能均小于−7.0 kcal/mol。体外实验结果显示，HJB-4以增加细胞增殖抑制率、细胞凋亡率（P＜0.05、0.01）。HJB-4（1.0、2.0、4.0 μmol/L）组JC-1单体的比值增加（P＜0.01）。Western blotting实验结果发现，HJB-4能下调抗凋亡蛋白Bcl-2、p-PI3K、p-Akt表达，上调促凋亡蛋白Bax、Cyt-C、cleaved Caspase-9和cleaved Caspase-3的表达水平（P＜0.05、0.01）。结论 HJB-4通过多靶点抑制膀胱癌T24细胞增殖，通过抑制PI3K/Akt信号通路，诱导线粒体功能障碍，激活内源性凋亡通路，从而发挥抗膀胱癌作用。

Objective To investigate the targets and mechanisms of HJB-4 on bladder cancer T24 cells based on network pharmacology, molecular docking technology and experimental verification. Methods Network pharmacology was employed to screen the potential targets of HJB-4, construct a PPI network, conduct GO function and KEGG pathway enrichment analysis, and perform molecular docking verification. Cell viability was detected by CCK-8 assay. Flow cytometry and confocal laser microscopy were used to detect changes in cell apoptosis, mitochondrial membrane potential levels. Western blotting was performed to examine the effects of HJB-4 on the expression levels of Bcl-2, Bax, Bim, Cyt-C, cleaved Caspase-9, cleaved Caspase-3, and PI3K/Akt pathway. Results Network pharmacology analysis predicted 315 common targets between HJB-4 and bladder cancer. GO and KEGG enrichment analysis indicated that HJB-4 may exert its effects through pathways such as the MAPK signaling pathway, PI3K/Akt pathway, and apoptosis pathway. Molecular docking demonstrated that the binding energy of between HJB-4 and core targets was all below −7.0 kcal/mol. The results of the in vitro experiments showed that HJB-4 increased the inhibition rate of cell proliferation and the apoptosis rate (P < 0.05, 0.01). The ratio of JC-1 monomers in the HJB-4 (1.0, 2.0, 4.0 μmol/L) groups increased (P < 0.01). The results of the Western blotting experiment revealed that HJB-4 could down-regulate the expression of anti-apoptotic proteins Bcl-2, p-PI3K, and p-Akt, and up-regulate the expression levels of pro-apoptotic proteins Bax, Cyt-C, cleaved Caspase-9, and cleaved Caspase-3 (P < 0.05, 0.01). Conclusion HJB-4 inhibits the proliferation of bladder cancer T24 cells through multiple targets, induces mitochondrial dysfunction, activates the intrinsic apoptosis pathway, and suppresses cell migration, thereby exerting anti-bladder cancer effects.

R287.3

国家自然科学基金资助项目（82174207）；齐齐哈尔医学科学院项目（ QMSI2023B-05）；黑龙江省省属本科高校基本科研业务费科研项目（2023-KYYWF-0857）