Abstract WP262: Neuroglobin Mediates Neuroprotection of Hypoxic Postconditioning Against Transient Global Cerebral Ischemia in Rats via Preserving the Activity of Na + /K + ATPases

Abstract

Introduction: Hypoxic postconditioning (HPC) is an innovative neuroprotective strategy with pro-survival effects on hippocampal neurons against transient global cerebral ischemia (tGCI), but the mechanisms have not yet been completely elucidated. Neuroglobin (Ngb) is an endogenous neuroprotectant with hypoxia-inducible property whose role in experimental stroke has increasingly been attractive. In this study, we explored the role and underlying mechanism of Ngb in HPC-mediated neuroprotection after tGCI. Methods: HPC was carried out with air containing 8% O 2 and 92% N 2 24 h after 10 min tGCI induced by a four-vessel occlusion method in adult male Wistar rats. The level of Ngb and Na + /K + ATPases β1 subunit (Atp1b1) were measured by immunohistochemistry, western blot and immunofluorescence after tGCI and HPC with or without the administration of Ngb antisense oligodeoxynucleotide (AS-ODNs) and lentivirus. Then reactive oxygen species (ROS) was detected via DCFH-DA and immunoprecipitation was performed to measure the glutathionylation of Atp1b1. Finally, the activity of Na + /K + ATPases (NKA) was detected by a Na + K + -ATPase assay kit. Results: HPC increased Ngb expression in CA1 subregion after tGCI. Inhibition of Ngb expression with Ngb AS-ODNs abolished HPC-mediated neuroprotection, whereas overexpression of Ngb ameliorated neuronal damage in hippocampal CA1 subregion after tGCI, confirming that Ngb upregulation is involved in the neuroprotection of HPC against tGCI. Similar to the tendency of Ngb expression, the membranous level of Atp1b1, a Ngb-interacting protein, reduced after tGCI while increased after HPC. Notably, knockdown of Ngb reversed the increase of membranous Atp1b1 mediated by HPC, whereas Ngb overexpression induced an opposite result. Furthermore, we found that Ngb upregulation after HPC maintained membranous Atp1b1 through the direct Ngb-Atp1b1 interaction and reduced the glutathionylation of membranous Atp1b1 via suppression of ROS, finally enhancing the activity of NKA. Conclusions: Our results suggested that Ngb preserved the activity of NKA after tGCI via its regulation on both the level and the glutathionylation of membranous Atp1b1, which contributed to HPC-mediated neuroprotection against tGCI.