Osteopontin attenuates microglia-mediated neuroinflammation and blood–brain barrier damage after pontine infarction

Abstract Pontine infarction, a subtype of posterior circulation stroke, often leads to severe neurological deficits because of its strategic brainstem location. However, its pathological mechanisms remain incompletely understood. In this study, bioinformatics analysis of single-cell RNA sequencing revealed extensive neutrophil infiltration, pronounced activation of microglia/macrophages, and significant loss of pericytes after pontine infarction, which was further validated by immunofluorescence. Network topology analysis identified microglia as crucial hub cells in the pontine infarction signaling network, exhibiting enhanced interactions with macrophages, endothelial cells, astrocytes, and neutrophils. Notably, secreted phosphoprotein 1, the gene encoding osteopontin, was markedly upregulated in microglia from PI mice compared with normal controls, as confirmed by western blotting and immunofluorescence. Serum analysis from patients with pontine infarction showed significantly elevated osteopontin levels, which positively correlated with infarct volume. In vitro knockdown of secreted phosphoprotein 1 in BV2 microglia aggravated the secretion of proinflammatory cytokines following oxygen glucose deprivation/reoxygenation stimulation. Conversely, administration of recombinant osteopontin in vivo attenuated blood–brain barrier disruption, reduced inflammatory cell infiltration, and improved neurological and behavioral outcomes in pontine infarction mice. Mechanistically, multi-omics analyses revealed that osteopontin exerts neuroprotective effects by modulating purine metabolism, enhancing adenosine signaling, and dampening neuroinflammation. This study investigated the molecular drivers of pontine infarction and revealed osteopontin is a key regulator of microglial activation and blood–brain barrier integrity. By integrating single-cell transcriptomics, multi-omics analysis, animal models, and patient samples, we provide comprehensive evidence for an immunometabolic role of osteopontin. These findings not only establish the dual role of osteopontin as a mechanistic link between neuroinflammation and vascular dysfunction but also position it as a potential therapeutic target for pontine infarction.