目的 探讨雷公藤红素调控肝癌细胞死亡的分子机制。方法 以人源肝癌细胞HepG2、鼠源肝癌细胞H22为实验对象，采用CCK-8法筛选出合适的药物浓度；运用EdU实验、流式细胞术、细胞划痕实验分别检测雷公藤红素对肝癌细胞增殖、凋亡和迁移能力的影响；通过比色法检测细胞内Cu²⁺浓度、荧光探针法检测线粒体膜电位（JC-1）的变化评估铜死亡的发生情况；采用免疫印迹法检测细胞中铜代谢调节蛋白铁氧还蛋白1（FDX1）和二氢硫辛酰胺转乙酰基酶（DLAT）的表达水平。结果 经不同浓度雷公藤红素处理24 h后，2种肝癌细胞（HepG2、H22）和2种正常肝细胞（LO2、AML12）的活力均显著下降（P＜0.05、0.01、0.001）。在有效浓度范围内，雷公藤红素（0.5、2.5、8.0 μmol/L）可显著抑制肝癌细胞增殖、促进肝癌细胞凋亡、降低肝癌细胞迁移能力（P＜0.05、0.01、0.001），且均呈一定的浓度相关性。雷公藤红素能够打破肝癌细胞内的铜代谢平衡，上调胞内Cu²⁺水平并引发Cu²⁺蓄积（P＜0.05、0.01、0.001），且呈一定的浓度相关性。为维持铜代谢平衡而代偿性上调表达的FDX1、DLAT并未能扭转铜代谢失衡，反而促使胞内毒性Cu⁺累积，导致线粒体膜电位降低、线粒体功能损伤，最终导致FDX1、DLAT蛋白表达损伤性下调，引起细胞死亡。加入铜离子螯合剂TTM后，雷公藤红素诱导的胞内Cu²⁺蓄积可被逆转，FDX1、DLAT异常表达被阻断，肝癌细胞活力回升（P＜0.05、0.01、0.001）。结论 在体外肝癌细胞模型中，雷公藤红素通过破坏肝癌细胞内铜代谢稳态，调控下游FDX1/DLAT表达，进而诱导肝癌细胞发生铜死亡。

Objective To explore the molecular mechanism by which celastrol induces cuproptosis in hepatocellular carcinoma cells. Methods Human hepatocellular carcinoma cell line HepG2 and mice hepatocellular carcinoma cell line H22 were used as experimental models. The CCK-8 assay was employed to screen for appropriate drug concentrations for subsequent experiments. EdU proliferation assay, flow cytometry, and wound healing assay were used to evaluate the effects of celastrol on HCC cell proliferation, apoptosis, and migration, respectively. Cuproptosis was assessed by measuring intracellular Cu²⁺ concentration via colorimetric assay and detecting changes in mitochondrial membrane potential by JC-1 fluorescence probe assay. The expression levels of copper metabolism regulatory proteins FDX1 and DLAT were determined by Western blotting. Results After being treated with different concentrations of celastrol for 24 h, the viability of both liver cancer cells (HepG2, H22) and normal liver cells (LO2, AML12) significantly decreased (P < 0.05, 0.01, 0.001). Within the effective concentration range, celastrol could significantly inhibit the proliferation of liver cancer cells, promote their apoptosis, and reduce their migration ability (P < 0.05, 0.01, 0.001), and all of these showed a certain concentration correlation. Celastrol （0.5, 2.5, 8.0 μmol/L）could disrupt the copper metabolism balance within liver cancer cells, upregulate the intracellular Cu²⁺ level and trigger Cu²⁺ accumulation (P < 0.05, 0.01, 0.001), and also showed a certain concentration correlation. To maintain the copper metabolism balance, the compensatory upregulation of FDX1 and DLAT expression failed to reverse the copper imbalance, instead promoting the intracellular accumulation of toxic Cu⁺, resulting in a decrease in mitochondrial membrane potential, mitochondrial dysfunction, and ultimately causing the damaging downregulation of FDX1 and DLAT protein expression, leading to cell death. After adding the copper ion chelator TTM, the intracellular Cu²⁺ accumulation induced by celastrol could be reversed, the abnormal expression of FDX1 and DLAT was blocked, and the viability of liver cancer cells recovered (P < 0.05, 0.01, 0.001). Conclusion In an in vitro HCC cell model, celastrol disrupts intracellular copper homeostasis in HCC cells, modulates the downstream expression of FDX1/DLAT, and further induces cuproptosis.

R286.5

青海省2022年“高端创新人才千人计划”项目（k9923195）