ETV4 Improves Cerebral Ischemia-Reperfusion Injury by Restraining YBX1-GPX4-Ferroptosis Cascades.

BACKGROUND

Cerebral ischemia-reperfusion injury (CIRI) is a critical pathological process following ischemic stroke, with ferroptosis being increasingly recognized as a key contributor to neuronal damage. However, the regulatory mechanisms, particularly the role of specific transcription factors like E26 transformation-specific variant 4 (ETV4), remain poorly understood. This study aimed to investigate the function and underlying mechanism of ETV4 in neuronal ferroptosis during CIRI.

METHODS

In vitro, SH-SY5Y cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were used to model CIRI. Cell viability and ferroptosis markers (Fe2 +, malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), reactive oxygen species (ROS)) were assessed. Molecular expressions were measured by Real-time Quantitative PCR (RT-qPCR) and/or western blot. In vivo, a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established, and brain injury was evaluated via 2% solution of 2,3,5-triphenyl tetrazolium chloride (TTC), hematoxylin-eosin (HE), and TdT-mediated dUTP nick-end labeling (TUNEL) staining. Chromatin immunoprecipitation (ChIP), dual-luciferase reporter, RNA pull-down, RNA immunoprecipitation (RIP), and actinomycin D assays were employed to validate the molecular interactions within the ETV4/Y-box binding protein 1 (YBX1)/glutathione peroxidase-4 (GPX4) axis.

RESULTS

ETV4 was significantly down regulated in both the MCAO/R model and OGD/R-treated cells. Overexpression of ETV4 markedly attenuated OGD/R-induced oxidative stress, ferroptosis in vitro, and ameliorated brain injury in vivo. Mechanistically, ETV4 transcriptionally activated YBX1 by directly binding to its promoter. YBX1, in turn, stabilized GPX4 mRNA, which was modified by NSUN2-mediated methylation. Crucially, the protective effects of ETV4 in vitro were abolished upon YBX1 or GPX4 knockdown.

CONCLUSION

Our findings demonstrate that ETV4 transcriptionally up regulates YBX1 to stabilize GPX4 mRNA in an NSUN2-m5Cmethylation dependent manner, thus suppressing neuronal ferroptosis. This reveals a novel ETV4/YBX1/GPX4 axis as a potential therapeutic target for CIRI.