Regulation of FOXO3 on neuronal ferroptosis after intracerebral hemorrhage via modulating NOX4 transcription.

Abstract

Intracerebral hemorrhage (ICH) is known to trigger neuronal ferroptosis while forkhead box O3 (FOXO3) is implicated in ICH. This study aimed to determine the specific effect of FOXO3 on neuronal ferroptosis after ICH. The ICH mouse model was established through the injection of bacterial collagenase type IV and the cell model was established in Hemin-induced HT-22 cells. Subsequently, neurological functions, brain water content, and histopathological changes in mice were assessed. HT-22 cell activity was examined via cell counting kit-8 (CCK-8) method, and the levels of FOXO3, NADPH oxidase 4 (NOX4), and glutathione peroxidase 4 (GPX4) in brain tissues and HT-22 cells were measured. Fe2+ concentration and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) in the tissues and cells were examined. The binding relationship between FOXO3 and the NOX4 promoter region was determined via chromatin-immunoprecipitation (Ch-IP). Rescue experiments were designed to probe the role of NOX4 in the regulation of FOXO3 on neuronal ferroptosis. FOXO3 was highly-expressed in ICH models while silencing FOXO3 alleviated brain damage, edema, and inflammatory infiltration in ICH mice. Meanwhile, silencing FOXO3 enhanced cell activity, diminished ROS and MDA activities and Fe2+ concentration, and elevated GSH and GPX4 levels in the tissues or cells. FOXO3 could bind to the NOX4 promoter and upregulate NOX4 transcription. NOX4 overexpression partially neutralized the repressive role of silencing FOXO3 in neuronal ferroptosis. Silencing FOXO3 attenuated ICH-induced neuronal ferroptosis via down-regulating NOX4 transcription levels, thus ameliorating post-ICH brain damage.