Protection against stroke-induced blood-brain barrier disruption by Guanxinning injection and its active-component combination via TLR4/NF-κB/MMP9-mediated neuroinflammation.

BACKGROUNDThe blood-brain barrier (BBB) is essential for central nervous system (CNS) homeostasis, yet neuroinflammatory mechanisms driving BBB disruption remain poorly understood.

PURPOSETo explore the oxygen-glucose deprivation/reoxygenation (OGD/R)-induced BBB dysfunction and evaluate the therapeutic effects of Guanxinning injection (GXNI), a Danshen-Chuanxiong herbal combination, targeting neuroinflammatory pathways.

METHODSA 3D-BBB organoid composed of human brain microvascular endothelial cells, human astrocytes, and primary human brain microvascular pericytes was constructed, and conditions for OGD/R that simulate ischemic stroke were established. Structure and function of the in vitro BBB were evaluated by morphology, paracellular permeability, and tight junction proteins ZO-1, claudin-5, and occludin expression. In vivo, infarct volume and BBB leakage were measured in a mid-cerebral artery occlusion-induced cerebral ischemia-reperfusion injury model. RNA-seq and network pharmacology analysis were used to identify key genes and pathways for ischemic BBB disruption. HPLC-MS was performed to identify and quantify active components. Molecular docking, SPR, and molecular dynamics were performed to predict and confirm the interaction of active compounds and target proteins.

RESULTSA Danshen-Chuanxiong double herbal medicine, GXNI, mitigated these effects, restoring transport capacity, reducing oxidative stress (ROS), and enhancing basement membrane components (laminin, collagen IV). In vivo, GXNI alleviated cerebral ischemia-reperfusion injury (CIRI), decreasing BBB leakage, infarct volume, and neurological deficits. The pivotal role of TLR4/NF-κB/MMP9 neuroinflammatory axis for GXNI BBB protection was identified through transcriptomic analysis and validated via immunofluorescence in BBB spheroids. Molecular docking revealed Danshen-derived salvianolic acid B (SAB) as a high-affinity MMP9 binder, confirmed by quantitative binding assays. The SAB and Chuanxiong-derived senkyunolide I (SI) combination achieved more prominent upregulation of tight junction proteins and suppression of MMP9.

CONCLUSIONOur findings further confirm neuroinflammation as a central driver of ischemic BBB damage and demonstrate that GXNI preserves BBB integrity by targeting TLR4/NF-κB/MMP9 signaling in 3D models and CIRI mice, with SAB-SI synergistically contributing to enhanced therapeutic efficacy.