Pulmonary neutrophil-derived circulating MMP9 mediates secondary brain injury after lung ischemia-reperfusion by disrupting the blood-brain barrier

Lung ischemia‑reperfusion injury (LIRI) frequently causes persistent cognitive decline. Although emerging studies have implicated neuroimmune interactions and pulmonary microbiota in lung‑brain crosstalk, the specific circulating mediators by which the acutely injured lung acts as a reservoir of systemic inflammation to disrupt the blood‑brain barrier (BBB) remain completely undefined. Using a rat left hilar occlusion model of LIRI, we performed unbiased dual-organ proteomic profiling, single-cell RNA sequencing, and a novel lung-restricted Flag-tagged adeno-associated virus 6 (AAV6) tracing strategy. Lung‑specific genetic knockdown and peripheral pharmacological inhibition of matrix metalloproteinase-9 (MMP9) were also employed. BBB integrity was assessed by Evans blue/IgG extravasation and transmission electron microscopy; neuroinflammation and synaptic integrity were evaluated by flow cytometry, immunofluorescence, Golgi‑Cox staining, and western blot; cognitive functions were measured via Y‑maze/novel object recognition tests. In vitro experiments on brain microvascular endothelial cells (BMECs) were also conducted to verify the direct effect of MMP9 on tight junctions. Proteomics identified MMP9 as the prominent upregulated effector in the injured lung, temporally correlated with degradation of BBB tight junctions (Occludin, ZO‑1). In vitro, recombinant MMP9 directly disrupted tight junction integrity in BMECs, an effect reversed by MMP-9-IN-1. Physical tracing with Flag‑tagged AAV6 showed that lung‑derived MMP9 entered the circulation and co‑localized with CD31⁺ cerebral microvessels, confirming its vascular accumulation. Both lung‑specific MMP9 knockdown and systemic pharmacological inhibition robustly attenuated BBB permeability, suppressed peripheral macrophage infiltration and microglial activation, preserved perineuronal net integrity and dendritic spine density, and significantly ameliorated spatial working memory, spatial reference memory, and recognition memory deficits induced by LIRI. Furthermore, single-cell transcriptomics combined with flow cytometric analysis identified recruited neutrophils as the predominant cellular source of the pulmonary MMP9 surge, a process transcriptionally driven by the AP-1 complex. Pulmonary neutrophil‑derived MMP9 acts as a critical systemic conduit in the lung‑brain axis, physically bridging acute pulmonary insult to remote BBB disruption and subsequent cognitive impairment. Circulating MMP9 therefore represents a promising, peripherally accessible therapeutic target for mitigating neurological complications following LIRI.