Gardenoside restores blood–brain barrier integrity following ischemic stroke via AMPK-dependent ZO-1 preservation

Background Blood-brain barrier (BBB) disruption is a critical pathological feature of ischemic stroke, yet effective therapies targeting BBB repair remain limited. Gardenoside, an iridoid glycoside from Gardenia jasminoides Ellis, has shown neuroprotective properties, but its role in post-stroke BBB restoration is unclear. This study investigated whether Gardenoside preserves BBB integrity after ischemic stroke via AMPK-dependent regulation of the tight junction protein ZO-1. Methods Male and female C57BL/6 mice (8–10 weeks) underwent transient middle cerebral artery occlusion (MCAO; 60 min ischemia, 24 h reperfusion). Gardenoside (20 mg/kg/day, i.p.) was administered for 21 days pre-MCAO and continued post-reperfusion. Neurological deficits, BBB permeability (sodium fluorescein, Evans blue), infarct volume (TTC), endothelial activation markers (ICAM-1, VCAM-1), ZO-1 expression (qPCR, western blot, immunofluorescence), and AMPK phosphorylation were assessed. In parallel, human brain microvascular endothelial cells (HBMVECs) were subjected to oxygen-glucose deprivation/reperfusion (OGD/R; 6 h OGD, 24 h reoxygenation) with Gardenoside (5–10 μM) ± the AMPK inhibitor compound C. Outcomes included cell viability (CCK-8), LDH release, permeability (FITC-dextran, TEER), and ZO-1/AMPK signaling. Results In MCAO mice, Gardenoside significantly reduced neurological deficit scores (by ~50%), BBB permeability (28–32% reduction), and infarct volume (45% reduction) compared to vehicle-treated controls. It suppressed ICAM-1 and VCAM-1 expression (mRNA: 43–52%; protein: 30–34%) and restored ZO-1 levels to near-sham values. Gardenoside increased AMPK phosphorylation (p-AMPK/AMPK ratio: 2.1-fold). Co-administration of compound C (10 mg/kg) abolished these protective effects, reversing infarct volume, BBB leakage, neurological scores, and ZO-1 restoration. In vitro , Gardenoside (5–10 μM) dose-dependently improved HBMVEC viability (from 42% to 68–88% of control), reduced LDH release (27–47%), decreased permeability (25–40%), and restored TEER (40–66%) and ZO-1 expression (50–98%) following OGD/R. These effects were associated with enhanced AMPK phosphorylation and were completely reversed by compound C (10 μM). Comparable efficacy was observed in female mice. Conclusion Gardenoside preserves BBB integrity and improves neurological outcomes after ischemic stroke through AMPK-dependent restoration of ZO-1. These findings identify AMPK-mediated tight junction preservation as a previously unrecognized mechanism of Gardenoside, supporting its potential as a vascular-targeted therapy for ischemic stroke.