Background: Significant brain damage and loss of function are frequent outcomes of ischemic stroke. After ischemic damage, neural stem cells (NSCs) exhibit considerable potential to promote neuroprotection and facilitate neural repair. N-butylphthalide (NBP) efficiently increases the viability of NSCs under hypoxic conditions. Given that the underlying mechanisms remain unknown, this study aims to investigate the protective effects of NBP on NSCs both in vitro and in vivo.

Methods: This study assessed the neuroprotective effects of NBP using a middle cerebral artery occlusion/reperfusion (MCAO/R) model in mice. Neurobehavioral performance of MCAO/R mice was evaluated using neurobehavioral tests, and brain infarct volumes were analyzed. Western blot analysis was used to measure the expression of Nestin, Ki-67, glycogen synthase kinase-3β (GSK-3β), phosphorylation-GSK-3β (p-GSK-3β), and nuclear factor erythroid 2-related factor 2 (NRF2) in brain tissues. In vitro experiments were conducted using NSCs under Oxygen-Glucose Deprivation/Reoxygenation (OGD/R) conditions, followed by treatment with NBP and GSK-3β inhibitors to assess cell viability and the levels of the GSK-3β/NRF2 signaling pathway.

Results: NBP significantly improved the neurobehavioral scores and reduced infarct volumes in MCAO/R mice (p < 0.05). It also increased the expression levels of Nestin and Ki-67, indicating enhanced proliferation of NSCs (p < 0.05). Additionally, NBP upregulated the expression levels of GSK-3β and NRF2 in brain tissues (p < 0.05). In vitro, NBP promoted the viability of NSCs and proliferation under OGD/R conditions, with increased expression levels of Nestin and Ki-67 (p < 0.05). The effect of NBP was partially reversed by GSK-3β inhibitors, indicating that NBP's effects are partially mediated by the GSK-3β/NRF2 signaling pathway (p < 0.05).

Conclusion: By regulating the GSK-3β/NRF2 signaling pathway, this study shows that NBP stimulates proliferation of NSCs and enhances neurofunctional recovery after ischemic stroke. These findings indicate that NBP not only promotes cellular survival and repair but also has the potential to improve overall neurological outcomes.