TY - JOUR
T1 - Methylene blue-induced neuronal protective mechanism against hypoxia-reoxygenation stress
AU - Ryou, M. G.
AU - Choudhury, G. R.
AU - Li, W.
AU - Winters, A.
AU - Yuan, F.
AU - Liu, R.
AU - Yang, S. H.
N1 - Funding Information:
This work was partly supported by National Institutes of Health grants R01NS054651 (SY), R01NS088596 (SY), Institute of Aging and Alzheimer’ s Disease Research grant RI6148 (MGR) American Heart Association grant SDG16960084 (RL), and National Natural Science Foundation of China Grant 81228009 (SY, FY).
Publisher Copyright:
© 2015 IBRO.
PY - 2015/8/1
Y1 - 2015/8/1
N2 - 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. Conclusion: 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.
AB - 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. Conclusion: 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.
KW - Hypoxia-inducible factor
KW - Ischemia and reperfusion injury
KW - Methylene blue
KW - Neuroprotection
KW - Oxygen and glucose deprivation
UR - http://www.scopus.com/inward/record.url?scp=84931275214&partnerID=8YFLogxK
U2 - 10.1016/j.neuroscience.2015.05.064
DO - 10.1016/j.neuroscience.2015.05.064
M3 - Article
C2 - 26047733
AN - SCOPUS:84931275214
SN - 0306-4522
VL - 301
SP - 193
EP - 203
JO - Neuroscience
JF - Neuroscience
ER -