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 3 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.

Mitochondrial function in hypoxic ischemic injury and influence of aging

“…It also is an alternative electron carrier that shuttles electrons between NADH and cytochrome c (cyt c ) so that electrons are rerouted with complex I and III inhibition thereby attenuating ROS overproduction. Its neuroprotective effects have also been attributed to mitophagy (Di et al , 2015), stimulation of HIF-1α (Ryou et al , 2015), and improved cerebral metabolic and hemodynamic function (Lin et al , 2012). MB significantly decreases infarct volume after middle cerebral artery occlusion in animal models (Di et al , 2015; Shen et al , 2013).…”

Experimental neuroprotection in ischemic stroke: a concise review

“…Furthermore, MB enhances cytochrome c oxidase activity while bypassing complexes 1-3. 37,75 It is also thought to augment HIF-1α activation and stabilization, 61 increase cerebral blood flow to mismatched areas, and promote autophagy via p53-AMPK-TSC2-mTOR, while downregulating apoptosis via the p53-Bax-Bcl2-caspase 3 pathway in perfusion-diffusion mismatched tissue. Shen et al 68 found that MB in a 60-minute rat MCA model of stroke had no effect on the initial MRI-demonstrated lesion volume; however, at 2 days after stroke, the final infarct volumes increased in the vehicle group but decreased in the MB group, yielding a 30% overall infarct difference.…”

Erythropoietin and mTOR: A “One-Two Punch” for Aging-Related Disorders Accompanied by Enhanced Life Expectancy

“…EPO oversees mTOR and down-stream signaling pathways that involve PRAS40 to increase neuronal survival during oxygen-glucose deprivation (95). EPO also increases mTOR activity during hypoxia-reoxygenation stress to protect hippocampus-derived neuronal cells (204). …”