Stroke is a major neurological disorder characterized by an increase in the Glu (glutamate) concentration resulting in excitotoxicity and eventually cellular damage and death in the brain. HIF-1 (hypoxia-inducible factor-1), a transcription factor, plays an important protective role in promoting cellular adaptation to hypoxic conditions. It is known that HIF-1α, the regulatable subunit of HIF-1, is expressed by astrocytes under severe ischaemia. However, the effect of HIF-1 on astrocytes following Glu toxicity during ischaemia has not been well studied. We investigated the role of HIF-1 in protecting ischaemic astrocytes against Glu toxicity. Immunostaining with GFAP (glial fibrillary acidic protein) confirmed the morphological modification of astrocytes in the presence of 1 mM Glu under normoxia. Interestingly, when the astrocytes were exposed to severe hypoxia (0.1% O2), the altered cell morphology was ameliorated with up-regulation of HIF-1α. To ascertain HIF-1's protective role, effects of two HIF-1α inhibitors, YC-1 [3-(50-hydroxymethyl-20-furyl)-1-benzylindazole] and 2Me2 (2-methoxyoestradiol), were tested. Both the inhibitors decreased the recovery in astrocyte morphology and increased cell death. Given that ischaemia increases ROS (reactive oxygen species), we examined the role of GSH (reduced glutathione) in the mechanism for this protection. GSH was increased under hypoxia, and this correlated with an increase in HIF-1α stabilization in the astrocytes. Furthermore, inhibition of GSH with BSO (l-butathione sulfoximine) decreased HIF-1α expression, suggesting its role in the stabilization of HIF-1α. Overall, our results indicate that the expression of HIF-1α under hypoxia has a protective effect on astrocytes in maintaining cell morphology and viability in response to Glu toxicity.
brain ischaemia; cell viability; glutamate; glutathione; hypoxia-inducible factor 1; stroke; BSO, l-butathione sulfoximine; CNS, central nervous system; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; ENT-1, equilibrative nucleoside transporter 1; EPO, erythropoietin; FBS, fetal bovine serum; GFAP, glial fibrillary acidic protein; Glu, glutamate; GSH, reduced glutathione; HBSS, Hanks balanced salt solution; HIF-1, hypoxia-inducible factor-1; HO-1, haem oxygenase 1; HSP, heat-shock protein; LDH, lactate dehydrogenase; MCB, monochlorobimane; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor; YC-1, 3-(50-hydroxymethyl-20-furyl)-1-benzylindazole; 2Me2, 2-methoxyoestradiol