ACT001 suppresses ox-LDL-mediated macrophage ferroptosis via activating the NRF2 pathway: An in vitro mechanistic study relevant to atherosclerosis

Abstract

Oxidized low-density lipoprotein (ox-LDL) is a key inducer of ferroptosis, which is associated with the progression of atherosclerosis. Macrophages play a notable role in atherosclerosis development, and ferroptosis in these cells contributes to the pathological process of atherosclerosis. However, the potential intervention strategy for ox-LDL-induced RAW264.7 cell ferroptosis remains unclear. ACT001 has emerged as a novel anti-inflammation agent; therefore, in vitro experiments were conducted to elucidate the specific effects and mechanism of ACT001 on ox-LDL-induced ferroptosis in RAW264.7 cells. RAW264.7 cells were treated with ox-LDL to establish an in vitro ferroptosis model and then co-treated with ACT001. Consequently, the expression of ferroptosis-related markers were evaluated via western blotting, PCR/detection of lipid peroxidation markers (malondialdehyde, glutathione; SOD: Superoxide dismutase; 4-HNE: 4-Hydroxynonenal; lipid reactive oxygen species (ROS) content)/transmission electron microscopy/iron content assay/mitochondrial membrane potential assay and ferrous ion staining. The potential molecular mechanism of ACT001 was further explored by detecting the expression and nuclear translocation of nuclear factor E2-related factor 2 (NRF2) via western blotting, confocal immunofluorescence/CHIP-PCR. Ox-LDL significantly mediated ferroptosis in RAW264.7 cells, as evidenced by increased lipid peroxidation, decreased glutathione content, elevated ROS production and downregulated expression of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (xCT); however, co-treatment with ACT001 effectively reversed these changes. Mechanistically, ACT001 treatment significantly increased the nuclear translocation of NRF2 and enhanced the enrichment of NRF2 level in the promoter regions of GPX4 and xCT via binding with NRF2, thereby upregulating the expression of GPX4 and xCT. In conclusion, ACT001 effectively ameliorated ox-LDL-triggered ferroptosis in RAW264.7 macrophages through facilitating NRF2 nuclear translocation and upregulating GPX4/xCT expression. The present in vitro findings revealed a molecular mechanism linking ACT001 to the inhibition of macrophage ferroptosis, offering preliminary cell-level evidence of the ACT001 therapeutic potential against macrophage-driven pathological events in atherosclerosis.