Butylphthalide enhances neurorestoration following ischemic stroke by restructuring microvasculature through vimentin modulation

INTRODUCTION: Ischemic stroke severely threatens human health. Rapid restoration of cerebral blood flow (CBF) in ischemic microvessels is significant as it enhances neurovascular function, prevents neuronal death, and minimises cerebrovascular injury. Although butylphthalide (NBP) is commonly used to treat ischemic stroke, its exact molecular target remains unclear. OBJECTIVE: Our research aims to explore NBP's molecular target and mechanism in ischemic stroke treatment, providing more evidence for its clinical application. METHODS: We confirmed NBP's protective effect on the neurovascular network by performing MCAO/R surgery on rats and using immunofluorescence and optical coherence tomography angiography for monitoring. We then employed photoaffinity labeling click chemistry for activity-based protein profiling (PAL-CC-ABPP) to identify the NBP's target protein. Molecular docking and dynamics simulations were conducted to investigate NBP-protein interactions. Additionally, we examined the effect of VIM knockdown in vascular endothelium and neurons on cerebral microvessels. The Notch pathway was explored to understand the mechanism of NBP-induced post-stroke neovascularization and neurorestoration. RESULTS: Our research demonstrated that NBP can enhance CBF, increase microvessel density in the ischemic brains of rats, repair microvascular injuries and foster neurological recovery. Through PAL-CC-ABPP, vimentin (VIM) was identified as the target protein of NBP. And NBP could provide protective effects by targeting the amino acid residue Arg-304 in VIM's active site. Furthermore, our findings indicate that VIM knockdown within the vascular endothelium interferes with properly forming cerebral microvessels and neurons. In contrast, VIM knockdown in neurons primarily impacts electrical signals and brain development, rather than angiogenesis. This suggests that VIM in blood vessels is crucial in maintaining the neurovascular network. Additionally, NBP enhances post-stroke neovascularization and neurorestoration by targeting VIM, which affects the Notch pathway. CONCLUSION: Our study reveals NBP can effectively treat ischemic stroke by targeting VIM to revitalise microvasculature, facilitating neurorestoration.