Ischemic Postconditioning Attenuates Cerebral Ischemic Injury by Suppressing the Ferroptosis-associated Gene NOX4

INTRODUCTION: This study investigated the neuroprotective mechanisms of ischemic postconditioning (IPostC) in ischemic stroke, focusing on ferroptosis and the regulatory role of the ferroptosis-related gene NADPH oxidase 4 (NOX4). METHODS: Male C57BL/6 mice underwent 45-minute middle cerebral artery occlusion (MCAO), followed by IPostC (three 15s/30s ischemia/reperfusion cycles after initial 2-minute reperfusion). RNA sequencing, combined with the least absolute shrinkage and selection operator (LASSO) and random forest machine learning, quantitative real-time PCR (qRT-PCR), infarct size measurement, and neurological tests, was used to identify ferroptosis-related genes and validate their roles in IPostC-induced neuroprotection. RESULTS: RNA sequencing revealed that 42 ferroptosis-associated differentially expressed genes underlie the neuroprotective effects of IPostC. Among them, NOX4 emerged as a central pathogenic regulator through LASSO and random forest machine learning analyses. IPostC reduced cerebral infarct size and improved foot-fault rate compared to MCAO mice. Notably, the ferroptosis inducer Erastin abolished the protective effects of IPostC. qRT-PCR validation revealed that IPostC downregulated NOX4 mRNA expression compared to MCAO controls, while Erastin upregulated NOX4 expression. In addition, pharmacological inhibition of NOX4 with GLX351322 reduced its mRNA expression, decreased infarct size, and improved neurological function, further confirming its critical role in mediating ferroptosis-driven brain injury after ischemic stroke. DISCUSSION: The inhibition of ferroptosis-associated gene NOX4 by IPostC may be a novel mechanism for treating ischemic stroke. CONCLUSION: Our study indicates that IPostC attenuates cerebral ischemic injury by suppressing ferroptosis-associated gene NOX4.