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AIM The aim of this study was to elucidate the molecular mechanisms underlying disruption of the blood-brain-barrier in diabetes and to determine whether activation of AMP-activated protein kinase prevents diabetes-induced blood-brain-barrier dysfunction.METHODS Human brain microvascular endothelial cells (HBMEC) were cultured in endothelial cell media mixed with astrocyteconditioned medium (1∶ 1).Confluent cells were maintained in 1% fetal calf serum and exposed to normal glucose (NG, 5 mmol· L-1)or high glucose (HG, D-glucose 25 mmol·L-1) for up to 1 week, control groups were exposed to L-glucose (LG, 20 mmol·L-1) in normal medium containing D-glucose (5 mmol·L-1).Intracellular O2-was measured using the dihydroethidium (DHE) fluorescence assay.Trans-endothelial electrical resistance (TEER) was determined by the Millicell-ERS.And the inter related proteins were examined by Western blot.RESULTS HBMEC exposure to HG for 2 h to 1 week significantly increased the permeability of blood-brainbarrier in parallel with lowered expression levels of zonula occludens-1, occludin, and claudin-5, three proteins that are essential to maintain endothelial cell tight junctions.In addition, HG significantly increased generation of superoxide anions.Adenoviral overex pression of superoxide dismutase or catalase significantly attenuated the HG-induced reduction of endothelial cell tight junction proteins.Furthermore, administration of apocynin reversed the effects of HG on endothelial cell tight junction proteins.Finally, activation of AMP-activated protein kinase with 5-amino-4-imidazole carboxamide riboside (AICAR) or adenoviral overexpression of constitutively active AMP-activated protein kinase mutants (Ad-AMPK-CA) abolished both induction of NAD(P) H oxidase-derived superoxid anions and tight junction protein degradation induced by HG.CONCLUSION We conclude that HG increases blood-brain-barrier dysfunction in diabetes through induction of superoxide anions and that the activation of AMP-activated protein kinase protects the integrity of the blood-brain-barrier by suppressing the induction of NAD(P) H oxidase-derived superoxide anions.