Systems biology analysis uncovers a ROS-associated gene signature and immunomodulatory role of CLEC4E in ischemic stroke.

Reactive oxygen species (ROS) are critically implicated in ischemic stroke (IS), yet the transcriptional networks and predictive biomarkers underlying ROS dysregulation remain incompletely understood. We integrated two independent microarray cohorts (GSE58294 and GSE16561) to comprehensively analyze ROS-related pathways in IS. Single-sample gene set enrichment analysis (ssGSEA) was used to quantify pathway activity, and weighted gene co-expression network analysis (WGCNA) identified modules associated with ROS dysregulation. Functional enrichment and protein-protein interaction (PPI) network analyses characterized the biological functions of module genes. Elastic Net regression modeling, receiver operating characteristic (ROC) analysis, calibration, and decision curve analysis (DCA) were employed to construct and validate a predictive risk score model. SHapley Additive exPlanations (SHAP) analysis was further applied to interpret gene contributions. Immune cell infiltration was assessed using multiple algorithms, and CLEC4E, the top-ranked gene, was functionally investigated through single-gene GSEA. The OGD/R-treated SH-SY5Y cells and mouse ischemia-reperfusion (I/R) models were established for in vivo and in vitro validation. ROS-related pathways were consistently upregulated in IS across both cohorts. WGCNA revealed a robust ROS-associated module (brown module), enriched in immune activation and inflammatory signaling processes. Elastic Net regression identified seven key genes (CLEC4E, SLC8A1, HIST1H4H, BMX, MCEMP1, KREMEN1, ZFP36L2) with strong predictive ability (AUC = 0.81-0.86 across datasets). SHAP analysis highlighted CLEC4E as the most influential contributor, positively associated with IS risk. Immune deconvolution indicated that CLEC4E expression was negatively correlated with B- and T-cell infiltration, while functional analysis linked it to MAPK signaling, RNA degradation, and neutrophil activation pathways. Finally, CLEC4E was significantly elevated in IS. Knockdown of CLEC4E could alleviate the effect of OGD/R on SH-SY5Y cells. Our study demonstrates pervasive activation of ROS-related transcriptional programs in IS and identifies a novel seven-gene signature predictive of disease risk. Among these, CLEC4E emerges as a key mediator connecting ROS dysregulation to immune infiltration and inflammatory signaling, providing new insights into IS pathophysiology and potential therapeutic targets.