抄録
Background: Hypoxia-inducible factor-1 (HIF-1) is the key transcription factor that regulates expression of many hypoxia-responsive genes. Under normoxic conditions, HIF-1α is degraded by HIF-1 prolyl hydroxylase enzymes (PHDs) which hydroxylate proline residues on HIF-1α causing ubiquitination and proteosomal degradation and consequently, constitutive levels of HIF-1α protein are very low. Hypoxia and drugs that inhibit PHD activity can result in HIF-1α accumulation and subsequently increase target gene expression. Previously, we have shown that pre- and post-injury treatment with hypoxia and PHD Inhibitors (cobalt chloride and desferrioxamine (DFX)) can protect against damage in adult and neonatal models of brain injury, and it is likely that this neuroprotective effect is mediated by HIF-1 and its target genes. DFX is already used clinically which makes it highly advantageous to study as a neuroprotective agent. Here, we examined whether HIF-1 was involved in DFX-induced neuroprotection in rat organotypic hippocampal slice cultures (OHSCs).
Methods: OHSCs were prepared from postnatal day 6 Sprague Dawley rats. Hippocampal tissue was dissected, 400μm slices collected and cultured for 6 days in vitro (div) prior to experimentation. On div 6, OHSCs were exposed to oxygen glucose deprivation (OGD) for 90 minutes followed by exposure to DFX (10-200μM) and propidium iodide (PI) fluorescence was used as an indicator of injury.
Results: Exposure to OGD resulted in increased PI fluorescence at 24h after OGD in OHSCs (15%), and this was significantly reduced by all DFX concentrations tested (4-8%, p<0.05 one-way ANOVA, Tukey's post-hoc test (n=15-20 slices obtained from 3-4 different animals)). DFX (30μM) was selected for all subsequent experiments as this concentration offered the most neuroprotection. Slices that were treated with DFX in the presence of HIF-1 inhibitor YC-1 (100μM) (3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole) immediately after OGD showed a reversal of the DFX-induced neuroprotection (from 5% to 14% PI fluorescence). DFX (30μM) also enhanced levels of HIF-1α, but no change in target gene expression was observed up to 24h after OGD.
Conclusions: Our results indicate an essential role for the HIF-1 pathway in the neuroprotective action of DFX, and suggest roles for HIF-1 as a viable neuroprotective target for acute brain injuries such as stroke.
