Silencing of SOX4 prevents endothelial injury from ox-LDL stimulation by inhibiting NF-κB pathway and ferroptosis

Objective: Over the past decades, Ferroptosis has been reported to be associated with atherosclerotic endothelial injury, while the mechanism remains incompletely understood. The purpose of the study was to investigate the role of SOX4 in ferroptosis-related endothelial injury.

Methods: Human umbilical vein endothelial cells (HUVECs) were treated with 100 µg/ml oxidized low-density lipoprotein (ox-LDL) for 24 h to induce endothelial injury. Silencing of SOX4 was achieved by transfection of siRNA targeting SOX4. To induce activation of NF-κB pathway and Ferroptosis, HUVECs were treated with NF-κB pathway activator PMA and Ferroptosis inducer erastin for 48 h. The viability of HUVECs following oxidized low-density lipoprotein (ox-LDL) stimulation was examined by CCK8 assays, production of Reactive Oxygen Species (ROS) and nitric oxide (NO) by using DCFH-DA and DAF-FM DA fluorescence probes, mitochondrial membrane potential (MMP) changes by JC-1 assays, opening of mitochondrial permeability transition pore (MPTP) by calcein AM staining, mitochondrial ROS (mito-ROS) by MitoSOX Red dye staining, intracellular Fe(2 +) level by Cellular Red Fluorescent Ferrous Ion Assay Kit with RhoNox-6, lipid ROS level by C11-BODIPY 581/591 staining, and lipid droplet by Oil Red O staining. The quantitative Real-Time PCR and Western blotting were employed to determine expressions of SOX4, eNOS, ferroptosis-related markers, and changes of NF-κB pathway RESULTS: The mRNA and protein expressions of SOX4 increased as the ox-LDL concentration increased. Silencing of SOX4 was found to decrease intracellular ROS production, increase intracellular NO production, improve the destruction of MMP, prevent the opening of MPTP, and reduce mito-ROS production in ox-LDL-treated HUVECs. Importantly, silencing of SOX4 significantly reduced the Fe(2 +) level, lipid peroxidation, expressions of pro-ferroptosis markers TFR1 and ACSL4, and increased expressions of anti-ferroptosis markers GPX4 and SLC7A11 in ox-LDL-treated HUVECs. The mechanistic studies demonstrated treatment with PMA could counteract the effects of silencing of SOX4 on ox-LDL-induced oxidative stress, mitochondrial damage, and Ferroptosis in HUVECs. Similarly, treatment with erastin could counteract the effects of silencing of SOX4 on ox-LDL-induced oxidative stress and mitochondrial damage in HUVECs.

Conclusion: These findings suggest SOX4 may be a candidate target to inhibit Ferroptosis and thus improve atherosclerotic endothelial injury.

Atherosclerosis; Endothelial injury; Ferroptosis; NF-κB pathway; Oxidized low-density lipoprotein; SOX4.