CKLF1 Serves as a Pivotal Mediator of Blood-Brain Barrier Disruption and Neuroinflammation via PI3K/AKT/mTOR Signaling in Ischemic Stroke.

Ischemic stroke (IS) is accompanied by blood-brain barrier (BBB) disruption and neuroinflammatory activation, but the upstream regulatory mechanisms remain incompletely defined. Here, integrated single-cell RNA sequencing, regulatory network analysis, and machine-learning screening identified Chemokine-like factor 1 (CKLF1) as a candidate endothelial regulator associated with IS. Functional validation showed that CKLF1 upregulation impaired endothelial barrier integrity, reduced tight-junction protein expression, enhanced apoptosis-associated activity, and activated PI3K/AKT/mTOR signaling under ischemic stress. CKLF1 also promoted chemokine amplification, microglial pro-inflammatory activation, and peripheral neutrophil recruitment. In a tMCAO/R mouse model, pharmacological inhibition of CKLF1 improved neurological outcomes, reduced infarct volume, preserved BBB integrity, and attenuated inflammatory activation, particularly when combined with CCL2 blockade. Conversely, CKLF1⁺ extracellular vesicle (EV)-related treatment aggravated BBB permeability and neuroinflammation. These findings support CKLF1 as a mediator of post-stroke endothelial injury and immune-inflammatory crosstalk and suggest that CKLF1-targeted intervention may represent a potential strategy for limiting BBB disruption after IS.