Metalloproteinase-9 as a Pyroptosis-Hypoxia Synergy Effector Drives Immune Remodeling in Ischemic Stroke: A Multi-Omics Validated Diagnostic Biomarker and Therapeutic Target.

Purpose: As a predominant contributor to disability and premature mortality worldwide, ischemic stroke (IS) urgently requires breakthroughs in early diagnostic biomarkers. The synergistic regulatory roles of pyroptosis and hypoxia, two critical pathological mechanisms in IS, require systematic exploration. Patients and Methods: We integrated two IS peripheral blood transcriptomic datasets (GSE66724 and GSE58294) to identify differentially expressed genes (DEGs) linked to pyroptosis and hypoxia. Co-expression networks were constructed using weighted gene co-expression network analysis. Diagnostic biomarkers were identified through the Least Absolute Shrinkage and Selection Operator, Support Vector Machine, and Random Forest algorithms, with validation performed in an independent cohort (GSE16561) and real-time quantitative PCR (RT-qPCR). Single-cell sequencing (GSE174574) was used to map cellular expression patterns of diagnostic genes. Candidate drugs were identified through the Connectivity Map (CMAP) database, with molecular docking validating their target protein interactions. Results: We identified 32 pyroptosis-related and 50 hypoxia-related DEGs, with enrichment analyses indicating their involvement in inflammatory responses, NF-κB signaling, and tumor necrosis factor pathways. Cross-algorithm validation identified matrix metalloproteinase-9 (MMP9) as a diagnostic biomarker. RT-qPCR revealed significantly elevated MMP9 levels in the peripheral blood of IS patients (p < 0.01). Immune microenvironment profiling showed positive correlations between MMP9 expression and macrophage/neutrophil infiltration. Single-cell sequencing confirmed predominant MMP9 expression in granulocytes. Drug prediction via CMAP and molecular docking identified Benperidol and Fluspirilene as high-affinity ligands for MMP9. Conclusion: This study employed multi-omics analysis followed by experimental validation to provide robust and systematic evidence supporting the diagnostic value and therapeutic potential of MMP9 in IS.