Inhibition of endothelial S1PR2 preserves blood-brain barrier integrity after traumatic brain injury through activating the PI3K-AKT signaling pathway.

Abstract

Traumatic brain injury (TBI) is a major cause of blood-brain barrier (BBB) disruption and neurological dysfunction, wherein endothelial dysfunction plays a critical pathogenic role. As a member of the G protein-coupled receptor family, sphingosine-1-phosphate receptor 2 (S1PR2) is known to regulate vascular homeostasis; however, its specific role in protecting the blood-brain barrier following TBI remains unclear. This study aims to elucidate the mechanism by which S1PR2 maintains blood-brain barrier integrity and to evaluate the therapeutic potential of S1PR2 inhibition after TBI. A mouse model of TBI was established using controlled cortical impact, while human umbilical vein endothelial cells (HUVECs) were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic ischemia-reperfusion injury in vitro. We employed shRNA technology to knock down S1PR2 expression and utilized single-cell RNA sequencing (dataset GSE269748) to characterize cell type-specific expression profiles. Endothelial function, blood-brain barrier permeability, inflammatory responses, and cell apoptosis were assessed using tube formation assays, transendothelial electrical resistance (TER) analysis, Western blotting, immunofluorescence, qPCR, ELISA, Evans blue staining, and brain water content measurements. Behavioral tests including open field test and novel object recognition test were used to evaluate the recovery of neurological function. At the same time, the PI3K-AKT pathway was interfered by S1PR2 knockdown mediated by AAV virus and pharmacological inhibitor (JTE-013/LY94002) or activator (Cyn). Single-cell analysis revealed that S1PR2 is specifically expressed in endothelial cells and is significantly upregulated following TBI. In vitro, S1PR2 knockdown counteracted the OGD/R-induced reduction in tube formation capacity and the elevation in transendothelial electrical resistance, and restored the expression of tight junction proteins Occludin and ZO-1. RNA-seq and KEGG enrichment analysis suggested that PI3K-AKT pathway was the key downstream target of S1PR2. In vivo experiments demonstrated that S1PR2 expression peaked at 72 h post-TBI and colocalized with CD31, while the ratios of p-PI3K/PI3K and p-AKT/AKT were markedly reduced. Intervention targeting S1PR2 significantly enhanced locomotor activity and novel object recognition, reduced brain lesion area, suppressed neuronal apoptosis and inflammatory cytokine levels, and restored BBB integrity in TBI mice. Mechanismally, activation of PI3K-AKT pathway could mimic the protective effect of S1PR2 knockdown, whereas inhibition of this pathway negated the improvements in BBB integrity and neurological function induced by S1PR2 knockdown. Endothelial S1PR2 is a critical regulator of vascular homeostasis after TBI. Inhibition of Endothelial S1PR2 preserves blood-brain barrier integrity, mitigates neuroinflammation and apoptosis, and promotes neurological recovery through activation of the PI3K-AKT signaling pathway, thereby offering a promising new strategy for targeted TBI therapy.