A New Discovery of Hirudin in Regulating Ferroptosis to Alleviate Ischemic Stroke

Background: Ischemic stroke is a major global cause of neurological disability and mortality, triggered by cerebral blood flow occlusion that leads to irreversible brain damage. Ferroptosis, an iron-dependent, lipid peroxidation-driven cell death pathway, contributes significantly to this injury. Hirudin has demonstrated neuroprotective potential in cerebrovascular diseases. This study experimentally validated the molecular mechanism by which hirudin mitigates ischemic stroke through the regulation of ferroptosis, focusing on key signaling pathways and vascular repair. Methods: In vivo, middle cerebral artery occlusion (MCAO) rats were treated with hirudin. Brain injury was evaluated using 2,3,5-Triphenyltetrazolium Chloride (TTC)/terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Western blotting was performed to detect ferroptosis (glutathione peroxidase 4 (GPX4) and Solute Carrier Family 7 Member 11 (SLC7A11)) and plasminogen activator, urokinase (PLAU)/nuclear factor kappa-B (NF-κB) proteins. Ferroptosis-related biomarkers (Fe2+, malondialdehyde (MDA), glutathione/glutathione disulfide (GSH/GSSG)) were measured in brain tissues using commercial assay kits. In vitro, oxygen-glucose deprivation/reoxygenation (OGD/R)-induced mouse brain endothelial cells (bEnd.3) were treated with hirudin. Cell counting kit-8 (CCK-8) was used to determine optimal hirudin concentration. Lactate dehydrogenase (LDH) release/scratch/tube formation assays were performed to evaluate cell function, while Western blotting and enzyme-linked immunosorbent assay (ELISA) were used to detect related proteins and factors. PLAU-silenced cells were used to verify hirudin's underlying mechanism. Results: Hirudin reduced MCAO rats' cerebral infarct size and neuronal apoptosis, upregulated GPX4, SLC7A11 and GSH/GSSG, decreased Fe2+, MDA, PLAU, p-65/p65, and inhibited inflammatory factor level (p < 0.01). In OGD/R-induced bEnd.3 cells, 1 IU/mL hirudin reduced LDH, promoted cell viability/migration and tube formation, upregulated angiogenesis factors and GPX4/SLC7A11, and downregulated inflammatory factor, PLAU and p-65/p65 levels (p < 0.01). Notably, the protective effects achieved by PLAU silencing were comparable to those of hirudin treatment, and their combination did not yield significant additive effects, suggesting that hirudin acts primarily via regulating PLAU. Conclusion: Hirudin alleviates cerebral ischemic injury and improves ischemic stroke, accompanied by suppression of the PLAU/NF-κB pathway, attenuation of ferroptosis, and enhancement of angiogenesis. These findings clarify hirudin's neuroprotective mechanism and support its potential as a therapeutic agent for ischemic stroke.