title: Methylene blue-induced neuronal protective mechanism against hypoxia-reoxygenation stress.
abstract: Brain ischemia and reperfusion (I/R) injury occurs in various pathological conditions, but there is no effective treatment currently available in clinical practice. Methylene blue (MB) is a century-old drug with a newly discovered protective function in the ischemic stroke model. In the current investigation we studied the MB-induced neuroprotective mechanism focusing on stabilization and activation of hypoxia-inducible factor-1α (HIF-1α) in an in vitro oxygen and glucose deprivation (OGD)-reoxygenation model.
  
  
  METHODS
  HT22 cells were exposed to OGD (0.1% O2, 6h) and reoxygenation (21% O2, 24h). Cell viability was determined with the calcein AM assay. The dynamic change of intracellular O2 concentration was monitored by fluorescence lifetime imaging microscopy (FLTIM). Glucose uptake was quantified using the 2-[N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino]-2-Deoxy-d-Glucose (2-NBDG) assay. ATP concentration and glycolytic enzyme activity were examined by spectrophotometry. Protein content changes were measured by immunoblot: HIF-1α, prolyl hydroxylase 2 (PHD2), erythropoietin (EPO), Akt, mTOR, and PIP5K. The contribution of HIF-1α activation in the MB-induced neuroprotective mechanism was confirmed by blocking HIF-1α activation with 2-methoxyestradiol-2 (2-MeOE2) and by transiently transfecting constitutively active HIF-1α.
  
  
  RESULTS
  MB increases cell viability by about 50% vs. OGD control. Compared to the corresponding control, MB increases intracellular O2 concentration and glucose uptake as well as the activities of hexokinase and G-6-PDH, and ATP concentration. MB activates the EPO signaling pathway with a corresponding increase in HIF-1α. Phosphorylation of Akt was significantly increased with MB treatment followed by activation of the mTOR pathway. Importantly, we observed, MB increased nuclear translocation of HIF-1α vs. control (about three folds), which was shown by a ratio of nuclear:cytoplasmic HIF-1α protein content.
  
  
  CONCLUSIONS
  We conclude that MB protects the hippocampus-derived neuronal cells against OGD-reoxygenation injury by enhancing energy metabolism and increasing HIF-1α protein content accompanied by an activation of the EPO signaling pathway.
authors.affiliation: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA; Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, TX, USA. Electronic address: myoung-gwi.ryou@unthsc.edu.
authors.forename: M-G
authors.initials: MG
authors.lastname: Ryou
authors.affiliation: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA.
authors.forename: G R
authors.initials: GR
authors.lastname: Choudhury
authors.affiliation: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA.
authors.forename: W
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA.
authors.forename: A
authors.initials: A
authors.lastname: Winters
authors.affiliation: Department of Neurosurgery, Beijing Tiantan Hospital, Beijing Neurosurgical Institute, Capital Medical University, Beijing 100050, China.
authors.forename: F
authors.initials: F
authors.lastname: Yuan
authors.affiliation: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA.
authors.forename: R
authors.initials: R
authors.lastname: Liu
authors.affiliation: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX, USA; Cardiovascular Research Institute, University of North Texas Health Science Center, Fort Worth, TX, USA; Department of Neurosurgery, Beijing Tiantan Hospital, Beijing Neurosurgical Institute, Capital Medical University, Beijing 100050, China. Electronic address: shaohua.yang@unthsc.edu.
authors.forename: S-H
authors.initials: SH
authors.lastname: Yang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015687:Cell Hypoxia / Q000187:drug effects; D002460:Cell Line; D005947:Glucose / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism* / Q000517:prevention & control; D008751:Methylene Blue / Q000008:administration & dosage*; D008810:Mice, Inbred C57BL; D018696:Neuroprotective Agents / Q000008:administration & dosage*; D010100:Oxygen / Q000378:metabolism; D015427:Reperfusion Injury / Q000378:metabolism* / Q000517:prevention & control; D013312:Stress, Physiological / Q000187:drug effects*
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D018696:Neuroprotective Agents; D000255:Adenosine Triphosphate; D005947:Glucose; D010100:Oxygen; D008751:Methylene Blue
keywords: hypoxia-inducible factor; ischemia and reperfusion injury; methylene blue; neuroprotection; oxygen and glucose deprivation