Comparative Analysis of Ferroptosis- and Mitochondria-Related Genes in Ischemic Stroke via Bioinformatics Analysis...

Ischemic stroke (IS) is a major cause of death and long-term disability worldwide. Multiple complex biological processes contribute to IS-related neuronal death, among which oxidative stress plays a central role in disease progression. Increasing evidence suggests that oxidative stress-induced neuronal injury is closely associated with ferroptosis and mitochondrial dysfunction, both of which contribute to excessive reactive oxygen species accumulation, lipid peroxidation, and impaired cellular energy metabolism during cerebral ischemia. However, their relative contributions and associated molecular signatures in IS have not been systematically compared. Therefore, this study aimed to identify ferroptosis- and mitochondria-associated genes involved in oxidative stress and neuronal injury in IS and to explore their potential as therapeutic targets for ischemic brain injury.

Two public microarray datasets (GSE22255 and GSE58294) were integrated and analyzed using weighted gene co-expression network analysis (WGCNA) to identify IS-associated gene modules. Ferroptosis-related genes from FerrDb and mitochondria-associated genes from MitoCarta3.0 were intersected with key modules to screen candidate genes. Protein-protein interaction analysis and CytoHubba were applied to identify hub genes. Logistic regression models were constructed to compare the diagnostic performance of ferroptosis- and mitochondria-related gene signatures. Functional enrichment analyses were conducted using Gene Ontology and KEGG. Key genes were further validated in a rat middle cerebral artery occlusion/reperfusion (MCAO/R) model treated with the ferroptosis inhibitor Ferrostatin-1 (Fer-1).

Nine ferroptosis-related hub genes and nine mitochondria-related hub genes were identified. The ferroptosis-based diagnostic model showed significantly higher discriminatory power than the mitochondrial model (AUC = 0.949 vs. 0.829). Among these genes, GSK3B, IDH1, and PRDX1 exhibited the most prominent differential expression and were selected as core genes. In vivo experiments demonstrated that Fer-1 markedly reduced infarct volume, improved neurological function, attenuated oxidative stress, and restored the ACSL4/GPX4/TFR1 signaling axis. Fer-1 also reversed MCAO-induced dysregulation of GSK3B phosphorylation, IDH1, and PRDX1 expression, indicating effective suppression of ferroptosis.

Ferroptosis-related gene signatures outperform mitochondria-associated genes in the diagnosis of ischemic stroke. GSK3B, IDH1, and PRDX1 represent key molecular regulators linking oxidative stress to ferroptotic neuronal injury and may serve as promising biomarkers and therapeutic targets for ischemic stroke.