Figure 8.

Roxadustat lowers the vulnerability of brain-resident cells towards ischemic stress through glucose metabolism reprogramming. (A) Neurons and astrocytes were treated with roxadustat in the absence or presence of 5 mM DCA for 24 h prior to exposure to OGD conditions (glucose-free aCSF; 0.2% O₂) for 4 h. DMSO pre-treated cells exposed to aCSF (+Glc) under normoxic conditions for 4 h are used as control (Ctrl). Cell viability was determined using a dead-cell protease activity assay. Values are expressed as fold change of OGD-subjected DMSO pre-treated cells (n = 7-12 per group; One-way ANOVA with Holm-Sidak's multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.001). (B-D) Neurons and astrocytes were treated with roxadustat for 6 h or 24 h. DMSO treated cells were used as Ctrl. (B) Real-time RT-PCR and Western blotting were applied to determine transcript and protein levels of PFKFB3. Values are normalized to Rps12 (mRNA) or β-tubulin (protein) and expressed as fold change of Ctrl (n = 4-9 per group; One-way ANOVA with Holm-Sidak's multiple comparisons test; ** p < 0.01, *** p < 0.001). (C) Cellular NADP⁺ and NADPH levels were measured by LC-MS/MS technique and the NADPH/NADP⁺ ratio was calculated (n = 4-7 per group; unpaired two-tailed Student's t test; ** p < 0.01). (D) Levels of total (GSx_tot), reduced (GSH), and oxidized (GSSG) glutathione were quantified by UPLC and the ratios of GSH to GSSG and GSSG to GSx_tot, respectively, were calculated (n = 6 per group; unpaired two-tailed Student's t test; ** p < 0.01, *** p < 0.001). (E) Neurons were treated with roxadustat and/or 10 nM AZ-PFKFB3-67 (AZ67) for 24 h and 4 h, respectively, prior to exposure to OGD conditions (glucose-free aCSF; 0.2% O₂) for 4 h. DMSO pre-treated cells exposed to OGD conditions for 4 h are used as Ctrl. Cell viability was determined using a dead-cell protease activity assay. Values are expressed as fold change of Ctrl (n = 6 per group; One-way ANOVA with Holm-Sidak's multiple comparisons test; ** p < 0.01, *** p < 0.001). (F) OHSCs were treated with 10 µM syrosingopine (Syro) and/or roxadustat for 24 h before exposure to OGD (glucose-free aCSF; 0.2% O₂) for 30 min followed by 24 h of reoxygenation. DMSO pre-treated OHSCs exposed to OGD/R are used as Ctrl. Cellular uptake of propidium iodide (PI) was used to estimate neuronal cell death within the hippocampal CA sub region. (n = 5-6 per group; One-way ANOVA with Holm-Sidak's multiple comparisons test; ** p < 0.01). Representative microphotographs: PI (red), DAPI (blue). Scale bar, 500 µm.