二氢青蒿素通过诱导铁死亡抑制肝癌细胞生长

二氢青蒿素（dihydroartemisinin,DHA）是青蒿素的一种衍生物，在多种肿瘤中表现出明显的抗肿瘤活性，但其具体机制不详。本文探讨了DHA对肝癌细胞的毒性作用机制。利用CCK-8试剂检测DHA对肝癌细胞株活力的影响，通过荧光探针染色及流式细胞术分析细胞内ROS及脂质过氧化物水平的变化；通过谷胱甘肽测定试剂盒检测细胞内还原型谷胱甘肽含量的变化，并通过免疫印迹分析DHA作用下细胞内铁死亡通路蛋白质中GPX4的变化。结果发现，DHA能显著抑制SMMC-7721及Huh-7细胞活力，其半数抑制浓度分别为23.74 μmol/L及26.92 μmol/L。 在35 μmol/L DHA 处理下，SMMC-7721及Huh-7细胞内ROS分别升高2.6倍和2.1倍，脂质过氧化物升高2.3倍和1.7倍。DHA可诱导细胞内GSH含量下降，并能下调铁死亡相关蛋白质GPX4蛋白水平。通过利用小分子抑制剂进行功能恢复实验发现，ROS抑制剂、铁螯合剂及铁死亡抑制剂都可不同程度恢复DHA引起的细胞活力下降。进一步检测发现，铁死亡抑制剂可抑制DHA诱导的脂质过氧化，并恢复GSH含量及GPX4蛋白水平。结果表明，在肝癌细胞中，DHA可通过诱导细胞发生铁死亡抑制肝癌细胞生长。

DHA Inhibits Proliferation of Human Hepatocellular Carcinoma Cells by Inducing Ferroptosis

Dihydroartemisinin (DHA), a derivative of artemisinin, has been identified as critical and promising agent for targeting human cancers, including hepatocellular carcinoma (HCC). However, the anticancer role of DHA in HCC and its underlying mechanism are still illusive. The aim of this study was to explore the toxic mechanism of DHA to hepatocellular carcinoma cells. CCK-8 reagent was used to detect the effect of DHA on human hepatocellular carcinoma cell viability. The changes of intracellular ROS and lipid peroxide levels were analyzed by fluorescence probe staining and flow cytometry. The glutathione assay kit was used to determine cellular reduced glutathione (GSH) content and the protein level of GPX4, a marker protein in ferroptosis, was tested by Western blot. The results showed that DHA can significantly inhibit SMMC-7721 and Huh-7 cell viabilities, with the half-inhibitory concentrations of 23.74 μmol/L and 26.92 μmol/L, respectively. Under the treatment of 35 μmol/L DHA, the ROS in SMMC-7721 and Huh-7 cells increased by 2.6-fold and 2.1-fold, respectively, and lipid peroxides increased by 2.3-fold and 1.7-fold. DHA induced a decrease of GSH amount and GPX4 protein level. Further rescue experiments exhibited that different small molecule inhibitors, like ROS inhibitors, iron chelating agents or ferroptosis inhibitors, could restore the decreased cell viability caused by DHA to different degrees. More tests demonstrated that ferroptosis inhibitor diminished the lipid peroxidation induced by DHA and reversed the GSH content and GPX4 protein levels. These results suggest that DHA inhibits hepatocellular carcinoma cell growth by inducing ferroptosis.