目的 探讨西黄丸调控铁死亡途径抑制肝癌的作用机制。方法 选用HepG2细胞采用CCK-8法研究西黄丸低、中、高剂量（0.65、1.30、2.60 g·kg^-1）制备的10%含药血清干预给药24、48 h对细胞增殖的抑制作用，以获得最佳给药浓度及时间，并采用透射电镜观察西黄丸各剂量含药血清对细胞线粒体形态的影响。HepG2细胞分为对照组、西黄丸组、质粒空载组（NC）、溶质载体家族7成员11（SLC7A11）过表达质粒转染组（OE-SLC7A11）、西黄丸＋OE-SLC7A11组。采用实时荧光定量PCR（qRT-PCR）和Western blotting法检测SLC7A11转染效率；采用Edu法检测细胞增殖率；采用二氢乙锭（DHE）荧光探针染色法及试剂盒法检测细胞活性氧（ROS）含量及活力水平；比色法检测丙二醛（MDA）水平和谷胱甘肽（GSH）水平；分别采用普鲁士蓝染色法观察铁沉积及比色法测定细胞内亚铁离子（Fe²⁺）水平；采用Western blotting法及qRT-PCR法检测各组细胞SLC7A11、GPX4蛋白及mRNA水平。结果 CCK-8实验研究结果表明，与对照组相比，西黄丸含药血清干预给药24、48 h可显著抑制细胞增殖（P＜0.05、0.01），其中高剂量组作用效果最显著，干预给药24 h抑制增殖效果最佳；透射电镜观察结果显示，西黄丸含药血清干预可引起HepG2线粒体肿胀、线粒体嵴模糊甚至消失等细胞铁死亡的表现，高剂量组线粒体损伤最显著。SLC7A11质粒转染引起的铁死亡抑制细胞实验中，与对照组比较，OE-SLC7A11组SLC7A11蛋白及mRNA水平均显著提高（P＜0.05、0.01），说明转染成功。与对照组比较，西黄丸组Edu阳性细胞率、GSH水平下降，MDA、ROS、Fe²⁺水平显著上升（P＜0.01），SLC7A11、GPX4蛋白及mRNA表达水平显著下降（P＜0.05、0.01）；与对照组比较，OE-SLC7A11组Edu阳性细胞率、GSH水平显著升高（P＜0.05、0.01），MDA、ROS水平显著下降（P＜0.01），Fe²⁺水平无明显差异，SLC7A11、GPX4蛋白及mRNA表达水平显著升高（P＜0.01）。与OE-SLC7A11组比较，西黄丸＋OE-SLC7A11组MDA、ROS、Fe²⁺水平显著上升（P＜0.05、0.01），Edu阳性细胞率、GSH水平下降（P＜0.01）； SLC7A11、GPX4蛋白及mRNA表达水平显著上升（P＜0.01）。结论 西黄丸可通过下调SLC7A11/GPX4轴，诱导肝癌细胞铁死亡，从而发挥治疗肝癌作用。

Objective To investigate the mechanism by which Xihuang Pill inhibits hepatocellular carcinoma through modulation of the ferroptosis pathway. Methods HepG2 cells were treated with low-, medium-, and high-dose (0.65, 1.30, and 2.60 g·kg^-1) Xihuang Pill-medicated serum. The CCK-8 assay was employed to evaluate the inhibition of cell proliferation at 24 h and 48 h, thereby identifying the optimal concentration and treatment duration. Subsequently, transmission electron microscopy was utilized to observe changes in mitochondrial morphology induced by each dose of the medicated serum. A ferroptosis-inhibited model was established by transfecting cells with an SLC7A11 overexpression plasmid. HepG2 cells were divided into the following groups: control group, Xihuang Pill high-dose-medicated serum group, empty plasmid group, SLC7A11 overexpression plasmid transfection group (OESLC7A11), and SLC7A11 overexpression plasmid transfection combined with Xihuang Pill-medicated serum intervention group (OE-SLC7A11+Xihuang Pill). Western blotting and qRT-PCR were used to detect transfection efficiency; the EdU assay was adopted to determine cell proliferation rate. The levels of reactive oxygen species (ROS) were measured using the dihydroethidium(DHE) fluorescent probe staining method; Malondialdehyde (MDA) and glutathione (GSH) levels were measured using colorimetric assays; Iron deposition was observed using Perls Prussian blue staining, and intracellular Fe²⁺ levels were determined by colorimetric assay; Western blotting and qRT-PCR were used to detect the protein and mRNA levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) in each group. Results The results of the CCK-8 assay demonstrated that, compared with the control group, Xihuang Pill-medicated serum significantly inhibited HepG2 cell proliferation at 24 and 48 h (P<0.05, 0.01). The high-dose group exhibited the most pronounced inhibitory effect, with optimal inhibition observed at 24 h. Transmission electron microscopy (TEM) revealed that Xihuang Pill-medicated serum treatment induced mitochondrial swelling, cristae blurring, and even cristae loss in HepG2 cells, which are characteristic morphological features of ferroptosis. These mitochondrial alterations were most prominent in the high-dose group. In the ferroptosis inhibition experiment induced by SLC7A11 plasmid transfection, the following results were observed. Compared with the NC group, both SLC7A11 protein and mRNA levels were significantly elevated in the OESLC7A11 group (P<0.05, 0.01), indicating successful transfection. Compared with the control group, the Xihuang Pill group exhibited a significant decrease in GSH levels and Edu-positive cell rate, along with marked increases in MDA, ROS, and Fe²⁺ levels (P<0.01). The protein and mRNA expression levels of SLC7A11 and GPX4 were also significantly downregulated in the Xihuang Pill group (P<0.05, 0.01). Compared with the NC group, the OE-SLC7A11 group showed significantly elevated GSH levels and Edupositive cell rate (P<0.01), reduced MDA and ROS levels (P<0.01) and significantly upregulated protein and mRNA expression of SLC7A11 and GPX4 (P<0.05, 0.01), and there was no significant difference in intracellular Fe²⁺ levels of the OE-SLC7A11 group. Compared with the OE-SLC7A11 group, the OE-SLC7A11 + Xihuang Pill group displayed significantly increased MDA, ROS, and Fe²⁺ levels (P<0.05, 0.01), decreased GSH levels and Edu-positive cell rate (P<0.01), and significantly downregulated protein and mRNA expression of SLC7A11 and GPX4 (P<0.05, 0.01). Conclusion Xihuang Pill exerts its therapeutic effect on hepatocellular carcinoma by down-regulating the SLC7A11/GPX4 axis to induce ferroptosis in hepatocellular carcinoma cells.

R965

山西省自然科学基金面上项目（202203021221205）；山西中医药大学中药药剂学科建设项目（2026XK36）