Methylene blue-induced neuronal protective mechanism against hypoxia-reoxygenation stress.
Methylene blue-induced neuronal protective mechanism against hypoxia-reoxygenation stress.
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DOI：
10.1016/j.neuroscience.2015.05.064
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发表时间：
2015-08-20
期刊：
影响因子：
3.3
通讯作者：
Yang SH
中科院分区：
文献类型：
--
作者：
Ryou MG;Choudhury GR;Li W;Winters A;Yuan F;Liu R;Yang SH
脑缺血再灌注（I/R）损伤发生在多种病理状态下，但目前临床上尚无有效的治疗方法。亚甲蓝（MB）是一种具有世纪历史的药物，在缺血性卒中模型中具有新发现的保护作用。本研究通过体外氧糖剥夺（OGD）-复氧模型，研究了缺氧诱导因子-1 α（HIF-1α）的稳定和激活对MB神经保护作用的机制。HT 22细胞暴露于OGD（0.1%O2，6 h）和复氧（21%O2，24 h）。用钙黄绿素AM测定法测定细胞活力。荧光寿命成像显微镜（FLTIM）监测细胞内O2浓度的动态变化。使用2-[N-（7-硝基苯并-2-氧杂-1，3-二唑-4-基）氨基]-2-脱氧-D-葡萄糖（2-NBDG）试验定量葡萄糖摄取。用分光光度法测定ATP浓度和糖酵解酶活性。通过免疫印迹法测量蛋白质含量变化：HIF-1α、脯氨酰羟化酶2（PHD 2）、促红细胞生成素（EPO）、Akt、mTOR和PIP 5 K。通过用2-甲氧基乙烯-2（2-MeOE 2）阻断HIF-1α活化和瞬时阻断组成型活性HIF-1α，证实了HIF-1α活化在MB诱导的神经保护机制中的贡献。与OGD对照相比，MB使细胞活力增加约50%。与相应对照组相比，MB增加细胞内O2浓度和葡萄糖摄取以及己糖激酶和G-6-PDH的活性和ATP浓度。MB激活EPO信号通路，相应增加HIF-1α。MB处理后，Akt的磷酸化显著增加，随后激活mTOR途径。重要的是，我们观察到，与对照组相比，MB增加了HIF-1α的核转位（约3倍），这通过细胞核：细胞质HIF-1α蛋白含量的比率来显示。结论：MB通过增强能量代谢、增加HIF-1α蛋白含量及激活EPO信号通路，保护海马神经元细胞免受OGD-复氧损伤。
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. 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. 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. 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.