Abstract: Immortalized mouse embryonic fibroblasts (MEFs) with wild-type (WT), Bak/Bax double knockout (Bak/Bax-DKO), Bak knockout (Bak-KO) and Bax knockout (Bax-KO) were used to investigate the role and possible mechanisms of pro-apoptotic protein Bak/Bax on the erastin-induced ferroptosis. The survival rates and reactive oxygen species (ROS) production were determined by CCK-8 and flow cytometry, the levels of GSH/GSSG were measured by testing kits. In addition, the expression levels of target genes and proteins were detected by real-time fluorescence quantitative PCR (RT-qPCR) and Western Blot. The results showed that knockout Bak and Bax inhibited erastin-induced ferroptosis significantly, and the expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2) protein, glutathione peroxidase 4 (GPX4) protein and mRNA were increased significantly in Bak/Bax-DKO cells. Further studies showed that the absence of Bax also inhibited erastin-induced ferroptosis and promoted the expression levels of GPX4. However, no significant changes on erastin-induced ferroptosis and GPX4 expression were found in Bak-KO cells. These results indicated that Bak and Bax promoted erastin-induced ferroptosis via Nrf2/GPX4 signaling pathway and Bax rather than Bak played a key role.

Key words: Bak/Bax, ferroptosis, reactive oxygen species, glutathione peroxidase 4, nuclear factor-erythroid 2-related factor 2