circAFF1 enhances intracerebral hemorrhage induced neuronal ferroptosis by targeting miR-140–5p to regulate GSK-3β mediated Wnt/β-catenin signal pathway

Ferroptosis is a newly emerged form of cell apoptosis and one of the characters of intracerebral hemorrhage (ICH). Currently there are limited therapeutic approaches for ICH. This study aims to explore the possible regulatory mechanism of ferroptosis in ICH. Hemoglobin (Hb) was used to treat neurons to mimic ICH cell model. The cell viability was assessed by CCK-8 assay. The contents of iron ion, reactive oxygen species (ROS), malondialdehyde (MDA) and glutathione (GSH) were also measured. The expressions of ferroptosis related proteins were determined by qRT-PCR and Western blot. The interaction among circAFF1, GSK-3β and miR-140–5p was verified. In vivo ICH models were established and assessed using mNSS. The morphology and wet/dry ratio of brain were also observed and calculated. circAFF1 was highly expressed in ICH cell model. Knockdown of circAFF1 attenuated Hb-induced neuronal ferroptosis, as evidenced by inhibiting cell viability, ROS, MDA and iron ion, and promoting GDH levels, which can be counteracted by miR-140–5p knockdown. circAFF1 can target miR-140–5p, and GSK-3β was a target gene of miR-140–5p. The effect of miR-140–5p on neuronal ferroptosis can be reversed by GSK-3β overexpression. In vivo experiments identified knockdown of circAFF1 suppress ICH injury and inhibits neuronal ferroptosis through regulating miR-140–5p/GSK-3β axis. circAFF1 knockdown can suppress neuronal ferroptosis in vivo to attenuate ICH injury, which was associated with its targeting with miR-140–5p to up-regulate GSK-3β and to suppress Wnt/β-catenin signal pathway.