目的 探讨葫芦素B通过影响铁死亡途径提高结直肠癌细胞对5-氟尿嘧啶（5-fluorouracil，5-FU）的敏感性并研究其潜在机制。方法 构建结直肠癌耐药细胞株HCT116/5-FU，用低、高剂量（5、15μg/mL）的葫芦素B分别处理HCT116和HCT116/5-FU细胞不同时间（12、24、48、72 h），细胞计数试剂盒-8（cell counting kit-8，CCK-8）实验检测细胞活力。利用生物信息学数据库探究外核苷酸焦磷酸酶/磷酸二酯酶2（ectonucleotide pyrophosphatase/phosphodiesterase 2，ENPP2）在结直肠癌中的表达情况及与预后的关系。免疫荧光检测葫芦素B对HCT116/5-FU细胞中ENPP2及铁死亡相关标志物Fe²⁺含量、脂质过氧化水平等表达的影响。在HCT116/5-FU细胞中过表达ENPP2，检测细胞活力及铁死亡相关分子的表达。结果 成功构建了HCT-116/5-FU耐药细胞株，并发现葫芦素B对该细胞株的生长具有较好的抑制作用，表现出明显的时间-剂量相关性，15 μg/mL葫芦素B作用48 h抑制率显著升高（P＜0.05）。生物信息学分析显示，ENPP2基因在结直肠癌组织中高表达，且与患者预后不良密切相关。葫芦素B处理可升高HCT-116/5-FU细胞内的Fe²⁺含量及脂质过氧化水平（P＜0.05），并影响铁死亡标志分子谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）、环氧化酶2（cyclooxygenase 2，COX2）的表达（P＜0.01）。ENPP2过表达可降低葫芦素B对Fe²⁺含量、脂质过氧化和铁死亡的上调作用（P＜0.05、0.01）。结论 葫芦素B通过抑制ENPP2的表达影响铁死亡提高结直肠癌细胞耐药的敏感性。

Objective To investigate whether cucurbitacin B (CuB) enhances the sensitivity of colorectal cancer cells to 5-fluorouracil (5-FU) by influencing the ferroptosis pathway and explore its potential mechanisms. Methods A colorectal cancer drug-resistant cell line HCT116/5-FU was established. HCT116 and HCT116/5-FU cells were treated with different concentrations of CuB (5, 15 μg/mL) for various durations (12, 24, 48, 72 h) and cell viability was assessed using the cell counting kit-8 (CCK-8) assay. Public bioinformatics databases were utilized to investigate the expression of ectonucleotide pyrophosphatase/phosphodiesterase (ENPP2) in colorectal cancer and its correlation with prognosis. Immunofluorescence techniques were employed to detect the effects of CuB on the ENPP2 expression and ferroptosis-related markers Fe²⁺ concentration and lipid peroxidation levels in HCT116/5-FU cells. ENPP2 was overexpressed in HCT116/5-FU cells, and then cell viability and the expression of ferroptosis-related molecules were evaluated. Results The HCT116/5-FU drug-resistant cell line was successfully established, and CuB was found to significantly inhibit the growth of this cell line with a clear time-dose dependency (P < 0.05). Notably, the inhibition rate of CuB significantly increased at a concentration of 15 μg/mL for 48h (P < 0.05). Bioinformatics analysis revealed that ENPP2 gene was highly expressed in colorectal cancer tissues and closely associated with poor patient prognosis. The Fe²⁺ concentration and lipid peroxidation levels in HCT116/5-FU cells were increased by CuB treatment (P < 0.05), with the expression of ferroptosis-related markers glutathione peroxidase 4 (GPX4) and cyclooxygenase 2 (COX2) was influenced (P < 0.01). Furthermore, overexpression of ENPP2 significantly reduced the up-regulation effects of CuB on intracellular Fe²⁺ concentration, lipid peroxidation levels and ferroptosis (P < 0.05, 0.01). Conclusion CuB enhances the sensitivity of colorectal cancer cells to drug resistance by influencing ferroptosis through the inhibition of ENPP2 expression.

R285.5

河南省科技攻关项目（242102310142）；河南省中医药科学研究专项课题（2023ZY2084）；河南省中医药拔尖人才培养项目（2022ZYBJ11）