Hirudin promotes cerebral angiogenesis and exerts neuroprotective effects in MCAO/R rats by activating the Wnt/β-catenin pathway

Objective: Hirudin has shown potential in promoting angiogenesis and providing neuroprotection in ischemic stroke; however, its therapeutic role in promoting cerebrovascular angiogenesis remains unclear. In this study, we aimed to investigate whether hirudin exerts neuroprotective effects by promoting angiogenesis through the regulation of the Wnt/(3-catenin signaling pathway. Methods: An in vitro model of glucose and oxygen deprivation/reperfusion (OGD/R) was established using rat brain microvascular endothelial cells (BMECs). The effects of hirudin on OGD/R cell viability were assessed using the cell counting kit-8 (CCK-8) assay. The angiogenic potential of hirudin was evaluated using Transwell and tube formation assays. In vivo, a middle cerebral artery occlusion/reperfusion (MCAO/R) model was created in rats. The neuroprotective effects of hirudin were assessed using the modified neurological severity score (mNSS), Hematoxylin and eosin (H&E) staining, 2,3,5-Triphenyltetrazolium chloride (TTC) staining, and immunofluorescence staining. Dickkopf-1 (DKK1), a specific inhibitor of this pathway, was introduced in order to investigate the role of the Wnt/(3-catenin pathway. The effects of hirudin on the Wnt/(3-catenin pathway were examined through immunohistochemistry, western blotting, and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results: Hirudin significantly improved BMEC survival and enhanced both cell migration and tube formation in the OGD/R model. In the MCAO/R model, hirudin reduced the mNSS score, alleviated pathological damage, decreased infarction volume, and increased the expression of key angiogenic factors, including CD34, vascular endothelial growth factor (VEGF), and angiopoietin-2 (Ang-2). In addition, hirudin activated the Wnt/(3-catenin pathway, leading to elevated levels of Wnt3a and (3- catenin. Conclusion: Hirudin has substantial neuroprotective effects associated with the promotion of angiogenesis in the ischemic penumbra. This mechanism is mediated by the regulation of the Wnt/(3-catenin pathway.