Background: N-Butylphthalide (NBP) can effectively improve cerebral vascular microcirculation and cellular energy metabolism in patients with ischemic stroke. This study aims to explore the underlying mechanism by which NBP exerts protective effects in ischemic stroke.

Methods: After exposure of primary neuronal cells to different concentrations of NBP (0, 0.1, 1, 10 μM), the optimal concentration for promoting cell survival was determined using a cell counting kit 8 (CCK8) assay. The primary neuronal cell damage model was established with oxygen-glucose deprivation/re-oxygenation (OGD/R), after which treatment with NBP alone or in combination with glycogen synthase kinase 3 beta (GSK-3β) inhibitors was performed. Cell viability and levels of oxidative stress (lactate dehydrogenase (LDH) and reactive oxygen species (ROS)) were detected. The changes of 4-Hydroxynonenal (4-HNE) and malondialdehyde (MDA) were examined by enzyme-linked immunosorbent assay (ELISA). Fe²⁺ levels, proteins associated with ferroptosis, and the GSK-3β/nuclear factor erythroid 2-related factor 2 (NRF2) signaling were tested.

Results: Both 1 μM and 10 μM NBP effectively alleviated OGD/R-induced cell damage. NBP increased cell viability, inhibited oxidative stress, and down-regulated LDH, ROS, 4-HNE and MDA levels. The cellular Fe²⁺ content was also significantly decreased after NBP treatment. NBP up-regulated ferroptosis-related proteins (glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11)) and activated the GSK-3β/NRF2 signaling. GSK-3β/NRF2 inhibitors reversed the effects of NBP on OGD/R-induced cells.

Conclusion: NBP effectively ameliorates neuronal ferroptosis in ischemic stroke by activating the GSK-3β/NRF2 signaling pathway, which provides a promising therapeutic strategy and a novel mechanistic basis for clinical management of stroke.