Minocycline ameliorates hypoxia-induced blood-brain barrier damage by inhibition of HIF-1α through SIRT-3/PHD-2 degradation pathway

Background: Minocycline, a second-generation tetracycline alleviates neuro-inflammation and protects the blood-brain barrier (BBB) in ischemia stroke. However, the effect of minocycline in hypoxia-induced BBB damage is unclear. Here, we have investigated the effect of minocycline under hypoxia and explored its possible underlying mechanisms. Methods: The effect of minocycline was examined in vitro in Human Brain Microvascular Endothelial Cells (HBMECs) using Trans Epithelial Electric Resistance (TEER). Protein and mRNA expression of Hypoxia-Inducible Factors-1α (HIF-1α), matrix metalloproteinases (MMP-2 and MMP-9) and tight junction proteins (TJs) were detected by using Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The translocation and transcription of HIF-1α were detected by using immunocytochemistry and luciferase reporter assay. In vivo, to adult male Sprague Dawley (SD) rats under hypobaric hypoxia were administered minocycline for 1 h and BBB permeability was tested by using Evans Blue and Transmission Electron Microscopy (TEM). Also, reduction of NAD-dependent deacetylase sirtuin-3 (SIRT-3)/proline hydroxylase-2 (PHD-2) signaling pathway was evaluated. Results: Minocycline increased TEER in HBMECs after hypoxia (P < 0.05), and reduced the extravasation of Evans Blue (P < 0.05) and colloidal gold nanoparticles in rats. Minocycline administration significantly reduced HIF-1α expression, protein and mRNA expression of MMP-2, MMP-9 and Vascular Endothelial Growth Factor (VEGF) (P < 0.05), and increased TJs (ZO-1, claudin-5 and occluding) (P < 0.05) in HBMECs after hypoxia. Furthermore, minocycline reversed the hypoxia-induced reduction of PHD-2 (P < 0.05) and SIRT-3 (P < 0.05). Effects of minocycline were abolished by siRNA-mediated knockdown of SIRT-3 in the brain. Conclusions: Minocycline inhibits HIF-1α-mediated cellular responses and protects BBB integrity through SIRT-3/PHD-2 pathway, proving to be a potential drug for the prevention and treatment of hypoxic brain injuries.