title: Minocycline ameliorates hypoxia-induced blood-brain barrier damage by inhibition of HIF-1α through SIRT-3/PHD-2 degradation pathway.
abstract: 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 1h 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.
authors.affiliation: Department of Pathophysiology and High Altitude Pathology, College of High Altitude Military Medicine, Third Military Medical University, 30 Gaotanyan, Shapingba, Chongqing 400038, China; Key Laboratory of High Altitude Medicine (Third Military Medical University), Ministry of Education, China; Key Laboratory of High Altitude Medicine, PLA, China.
authors.forename: F
authors.initials: F
authors.lastname: Yang
authors.affiliation: Department of Pharmacology, Xinqiao Hospital & The Second Affiliated Hospital, The Third Military Medical University, Chongqing, China.
authors.forename: L
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Department of Pathophysiology and High Altitude Pathology, College of High Altitude Military Medicine, Third Military Medical University, 30 Gaotanyan, Shapingba, Chongqing 400038, China; Key Laboratory of High Altitude Medicine (Third Military Medical University), Ministry of Education, China; Key Laboratory of High Altitude Medicine, PLA, China.
authors.forename: D
authors.initials: D
authors.lastname: Wang
authors.affiliation: Department of Neurosurgery and Key Laboratory of Neurotrauma, Southwest Hospital, Third Military Medical University, Chongqing 400038, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Pathophysiology and High Altitude Pathology, College of High Altitude Military Medicine, Third Military Medical University, 30 Gaotanyan, Shapingba, Chongqing 400038, China; Key Laboratory of High Altitude Medicine (Third Military Medical University), Ministry of Education, China; Key Laboratory of High Altitude Medicine, PLA, China. Electronic address: huangqingyuan@tmmu.edu.cn.
authors.forename: Q-Y
authors.initials: QY
authors.lastname: Huang
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D015687:Cell Hypoxia / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D055785:Gene Knockdown Techniques; D002534:Hypoxia, Brain / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors* / Q000378:metabolism; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases / Q000378:metabolism; D008297:Male; D008911:Minocycline / Q000494:pharmacology*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D056566:Sirtuin 3 / Q000235:genetics / Q000378:metabolism; D037761:Sirtuins / Q000235:genetics / Q000378:metabolism
chemical_list: C497442:HIF1A protein, human; C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D018696:Neuroprotective Agents; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C000607791:SIRT3 protein, rat; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; C580731:Egln1 protein, rat; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; C466093:SIRT3 protein, human; D056566:Sirtuin 3; D037761:Sirtuins; D008911:Minocycline
keywords: HIF-1α; SIRT-3; blood–brain barrier; hypoxia; minocycline; neuro-inflammation