AATF alleviates cerebral ischemia-reperfusion injury by suppressing endoplasmic reticulum stress-induced ferroptosis through upregulating the PI3K/Akt pathway

Objective: The study aimed to investigate the role of apoptosis-antagonizing transcription factor (AATF) in regulating endoplasmic reticulum (ER) stress-ferroptosis interaction and its neuroprotective mechanism in cerebral ischemia-reperfusion injury (CIRI).

Methods: Two models were used: oxygen-glucose deprivation/reperfusion (OGD/R)-induced hippocampal neuron cells and transient middle cerebral artery occlusion/reperfusion (tMCAO/R)-induced rats. AATF overexpression and knockdown were respectively performed to assess its impact on neuronal injury. Ferroptosis biomarkers - Glutathione Peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), iron responsive element binding protein 2 (IREB2) and ER stress markers - phosphorylated eukaryotic initiation factor 2α (p-EIF2α)/EIF-2α, activating transcription factor 4 (ATF4) were analyzed by Western blot and enzyme-linked immunosorbent assay (ELISA). Pharmacological inhibitors or activators were utilized to explore the regulatory relationship between AATF, ER stress, and Ferroptosis.

Results: AATF was significantly upregulated during CIRI. AATF overexpression improved neuronal viability, reduced Reactive Oxygen Species (ROS), and alleviated ferrous ion (Fe²⁺) accumulation; conversely, endogenous knockdown of AATF reversed its neuroprotective effects. Pathologically, AATF reduced cerebral infarction volume, neurological deficits, and brain edema. Mechanistic analysis showed that AATF inhibited the ER stress-ferroptosis axis by regulating related proteins. Pharmacological phosphatidylinositol 3-kinase (PI3K) inhibition reversed the neuroprotective effects of AATF, indicating the essential role of the PI3K- protein kinase B (Akt) signaling pathway in AATF-mediated suppression of both Ferroptosis and ER stress.

Discussion: AATF mitigates CIRI injury by modulating the ER stress-ferroptosis axis, primarily through the activation of the PI3K-Akt signaling pathway. These findings position AATF as a promising therapeutic target for brain ischemia-reperfusion injury.

AATF; ER stress; Ferroptosis; PI3K-Akt signaling; cerebral infarction; hippocampal neurons; ischemia-reperfusion injury; neuroprotection.