Ferroptosis Inducer Induced Ferroptosis In Jurkat Cells And Mechanism

Objective Ferroptosis inducer Erastin can induce ferroptosis in cells.The study was to explore whether Erastin can induce ferroptosis in in human ALL cell line Jurkat cells and whether autophagy inhibitor 3-MA is involved as a synergistic effect,to explore the signaling pathways that may be involved in ferroptosis,and to provide a basis for further elucidation of the mechanism of ferroptosis.Methods Jurkat cells were collected when they were in the logarithmic growth stage and treated with Erastin of 1.25,2.5,5,10,15,20 and 40 mol/L,respectively.OD value was detected by CCK-8 kit after 24 hours.Finally,Erastin concentration(10 mol/L)with cell inhibition rate of 50% was selected for the follow-up study.Treated with Erastin Jurkat cells at the same time points to join Fer-1(ferroptosis inhibitor),Z-VAD-fmk(apoptosis inhibition),Necrostatin-1(necrosis inhibitor),3-MA(autophagy inhibitor),use of CCK-8 method to detect value-added,GSH kits and MDA was detected in the kits Jurkat intracellular GSH content and paper peroxide level after 24 hours,so as to determine whether the cell death of iron.GPX4 and SLC7A11 were tested at the gene level to further determine iron death in Jurkat cells.The expression of ferroptosis pathway P62-Keap1-NRF2 at the gene level was detected by Real-time PCR,and protein expression of ferroptosis pathway P62-Keap1-NRF2 after Erastin treatment was detected by Western-Blotting.Results(1)the ferroptosis inducer Erastin had a toxic effect on Jurkat cells,and the inhibition rate increased with the increase of Erastin exposure concentration.(2)Jurkat cells were treated with five drug combinations of Erastin,Erastin+ fer-1,Erastin+Z-VAD-fmk,Erastin+Necrostatin-1,Erastin+3-MA,respectively.After 24 hours,proliferation toxicity of cells was detected by CCK-8 method,glutathione content and paper peroxidation level in Jurkat cells were detected by GSH kit and MDA kit respectively.The experimental results indicated that the cell inhibition rate of the Erastin+fer-1 group was lower,the intracellular GSH content was higher,the MDA level was lower,and the cell inhibition rate of the Erastin+3-VA group was higher,the intracellular GSH content was lower,and the MDA level was higher,compared with Erastin group.(3)Jurkat cells were treated with 10umol/L Erastin,1umol/L Fer-1,and5mmol/L 3-MA,respectively,and the effects of Erastin on GPX4 and SLC7A11 levelsin cells were detected by real-time PCR 24 hours later.The results showed that the relative expressions of GPX4 and SLC7A11 m RNA in Erastin group were lower and higher compared with Erastin+3-MA group.(4)Real-time PCR results showed that the m RNA contents of P62,Keap1 and NRF2 in the Erastin group were higher than that in the Erastin+Fer-1 group and lower compared with the Erastin+3-MA group.Western-blotting results indicated that the expressions of P62 and NRF2 in Erastin+3-MA group were higher compared with the control group,and the expression of Keap1 was lower compared with the control group.In the Erastin group,the expressions of P62 and NRF2 were higher than those of the Erastin+Fer-1 group,and the expressions of Keap1 were lower than those of the Erastin+Fer-1 group.Conclusion Ferroptosis inducer Erastin can induce ferroptosis in Jurkat cells,and autophagy inhibitor 3-MA is likely to be a synergistic factor for ferroptosis.In Jurkat cells,the pathway P62-Keap1-NRF2 is activated when ferroptosis was induced by Erastin.