基于谷胱甘肽/谷胱甘肽过氧化物酶4轴探讨雷公藤甲素引起肝细胞铁死亡的作用机制

目的 观察雷公藤甲素对铁代谢和脂质过氧化的影响，探讨雷公藤甲素引起肝细胞铁死亡的可能机制。方法 利用雷公藤甲素处理人正常肝细胞HL7702和C57BL/6J小鼠，或利用铁死亡抑制剂（ferrostatin-1，Fer-1）和雷公藤甲素共同处理HL7702细胞，CCK-8法检测细胞存活率；普鲁士蓝染色观察铁沉积；试剂盒检测铁离子、谷胱甘肽（glutathione，GSH）、丙二醛（malondialdehyde，MDA）含量以及超氧化物歧化酶（superoxide dismutase，SOD）、丙氨酸氨基转移酶（alanine aminotransferase，ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase，AST）活性；qRT-PCR或Western blotting检测转铁蛋白受体1（transferrin receptor 1，TFR1）、谷胱甘肽过氧化物酶4（glutathione peroxidase 4，GPX4）、前列腺素内过氧化物合成酶2（prostaglandin-endoperoxide synthase 2，PTGS2）、长链脂酰辅酶A合成酶4（acyl-CoA synthetase long-chain family member 4，ACSL4）的表达。结果 雷公藤甲素显著降低细胞存活率（P＜0.05），引起铁沉积，升高血清中ALT、AST活性（P＜0.05），增加HL7702细胞和小鼠肝组织中铁离子、MDA含量（P＜0.05），降低GSH含量、SOD活性和GPX4表达（P＜0.05），上调TFR1、PTGS2及ACSL4表达（P＜0.05）。Fer-1显著上调细胞存活率和GPX4表达量（P＜0.05），显著下调铁离子含量、TFR1、PTGS2及ACSL4表达（P＜0.05）。结论 雷公藤甲素可能通过GSH/GPX4轴引起铁代谢异常和脂质过氧化，诱导肝细胞铁死亡。

Mechanism of triptolide-induced ferroptosis in liver cells based on glutathione/glutathione peroxidase 4 axis

Objective To investigate the effect of triptolide on iron metabolism and lipid peroxidation and evaluate the underlying mechanism of triptolide-induced ferroptosis in hepatic cells. Methods HL7702 cells and C57BL/6J mice were treated with triptolide, or HL7702 cells were co-treated with ferroptosis inhibitor ferrostatin-1 (Fer-1) and triptolide, and then cell survival rate was detected by CCK-8 method; Iron deposition was detected by Prussian blue staining; Iron, glutathione (GSH), malondialdehyde (MDA) contents and superoxide dismutase (SOD), alanine aminotransferase (ALT), aspartate aminotransferase (AST) activities were detected by kit; Transferrin receptor 1 (TFR1), glutathione peroxidase 4 (GPX4), prostaglandin-endoperoxide synthase 2 (PTGS2), acyl-CoA synthetase long-chain family member 4 (ACSL4) expressions were detected by qRT-PCR or Western blotting. Results Triptolide significantly decreased cell viability (P < 0.05), increased ALT and AST activities in serum (P < 0.05), and resulted in iron deposition, increased iron, MDA contents (P < 0.05), decreased GSH content, SOD activity and GPX4 expression (P < 0.05), and up-regulated TFR1, PTGS2 and ACSL4 expressions in HL7702 cells and liver tissues (P < 0.05). Fer-1 significantly reversed the decreased cell viability induced by triptolide, and increased GPX4 expression (P < 0.05), as well as down-regulated iron content, TFR1, PTGS2 and ACSL4 expressions (P < 0.05). Conclusion Triptolide may induce iron overload and lipid peroxidation via GSH/GPX4 axis which ultimately leading to ferroptosis in hepatic cells.