H_(2)O_(2)抑制ERK通路诱导L-O2细胞铁死亡

Ferroptosis Induced by H_(2)O_(2)in L-O2 Cells Through Inhibition of the ERK Pathway

目的:探讨过氧化氢(H_(2)O_(2))诱导人肝实质细胞(L-O2细胞)铁死亡的作用及潜在机制。方法:采用不同浓度梯度H_(2)O_(2)刺激L-O2细胞,分别于处理12、24 h后使用CCK-8法检测细胞存活率,从而确定最有效的H_(2)O_(2)诱导条件。在实验中设立了以下处理组:空白对照组、H_(2)O_(2)组、Erastin组、H_(2)O_(2)+Fer-1组;确定H_(2)O_(2)可诱导L-O2细胞发生铁死亡,为了进一步明确具体的机制,细胞分为空白对照组、H_(2)O_(2)组、H_(2)O_(2)+不同浓度的ERK激动剂以及ERK抑制剂,使用试剂盒检测各处理组的细胞死亡情况、ROS水平和MMP水平,利用RT-qPCR检测铁死亡标记物PTGS2和ACSL4 mRNA表达水平。结果:800μmol/L H_(2)O_(2)刺激24 h可诱导L-O2细胞死亡;与空白对照组相比,H_(2)O_(2)组和Erastin组细胞受损明显,细胞内ROS水平升高、MMP水平显著降低,同时PTGS2和ACSL4 mRNA的表达水平升高;与H_(2)O_(2)组相比,H_(2)O_(2)+Fer-1组细胞受损减弱,细胞内ROS水平降低、MMP水平恢复,PTGS2和ACSL4 mRNA的表达水平降低。与H_(2)O_(2)组相比,H_(2)O_(2)+ERK激动剂组细胞受损减弱,细胞内ROS水平降低、MMP水平恢复、PTGS2和ACSL4 mRNA的表达水平降低;然而,加入ERK抑制剂组则出现了相反的结果。结论:800μmol/L的H_(2)O_(2)处理24 h能显著诱导L-O2细胞发生铁死亡,且此过程可能主要通过ERK通路的抑制作用来实现。

Objective To investigate the role and potential mechanism of hydrogen peroxide(H_(2)O_(2))inducing ferroptosis in human liver parenchymal cells(L-O2 cells).Methods Different concentration gradients of H_(2)O_(2)were used to stimulate L-O2 cells,and cell survival was detected using the CCK-8 assay after 12 and 24 hours of treatment to determine the opti⁃mal H_(2)O_(2)induction conditions.Treatment groups included a blank control group,H_(2)O_(2)group,Erastin group,and H_(2)O_(2)+Fer-1 group.It was determined that H_(2)O_(2)could induce ferroptosis in L-O2 cells,and to further clarify the specific mecha⁃nism,the cells were divided into the blank control group,the H_(2)O_(2)group,and the H_(2)O_(2)+ERK agonists of different con⁃centrations as well as ERK inhibitors.The kit was used to detect the cell death,ROS,and MMP in the treatment groups,and the mRNA expressions of ferroptosis markers PTGS2 and ACSL4 were detected using RT-qPCR.Results After stimu⁃lation with 800μmol/L H_(2)O_(2)for 24 hours,L-O2 cell death could be induced.Compared with the blank control group,the cells in the H_(2)O_(2)and Erastin groups were significantly damaged,with elevated intracellular ROS,significantly reduced MMP,and increased mRNA expressions of PTGS2 and ACSL4.Compared with the H_(2)O_(2)group,the cells in the H_(2)O_(2)+Fer-1 group had attenuated damage,reduced intracellular ROS,restored MMP,and down-regulated mRNA expressions of PTGS2 and ACSL4.Similarly,compared with the H_(2)O_(2)group,cell damage was weakened in the H_(2)O_(2)+ERK agonist group,with reduced ROS,restored MMP,and lowered mRNA expressions of PTGS2 and ACSL4.However,after the addi⁃tion of the ERK inhibitor,opposite results were observed.Conclusion Treatment with 800μmol/L H_(2)O_(2)for 24 hours sig⁃nificantly induced ferroptosis in L-O2 cells,and this process may be mainly through the inhibition of the ERK pathway.