TPEN attenuates neuronal ferroptosis in ischemic brain injury via GPR39-mediated PI3K/AKT signaling activation

Brain injury involves neuronal ferroptosis, and the zinc chelator TPEN exhibits neuroprotective potential. However, its mechanisms remain unclear. GPR39, a G protein-coupled receptor, participates in cell survival pathways. This study aimed to investigate whether TPEN protects against ischemic neuronal injury by modulating GPR39 and its downstream PI3K/AKT signaling pathway.

PC12 cells were subjected to oxygen-glucose deprivation (OGD) to mimic brain injury, and a rat model of middle cerebral artery occlusion (MCAO) was used to evaluate infarct volume (TTC staining) and oxidative stress markers. The effects of TPEN on cell viability, ferroptosis-related markers, and key protein expression were evaluated using CCK-8 assay, biochemical kits, Western blot, TUNEL staining, and transmission electron microscopy. GPR39 expression and PI3K/AKT/mTOR phosphorylation were analyzed using Western blot and phospho-kinase array. PI3K inhibitor LY294002 was utilized to investigate the signaling mechanisms involved.

TPEN treatment improved cell viability in OGD-injured PC12 cells and reduced infarct volume in MCAO rats. It attenuated ferroptosis by decreasing intracellular Fe^{2 +}, MDA, and lipid ROS levels, while restoring GSH content and modulating the expression of NRF2, GPX4, and ACSL4. TPEN also upregulated GPR39 expression and activated the PI3K/AKT/mTOR pathway. GPR39 knockdown abolished TPEN-induced PI3K/AKT activation and cytoprotection, whereas GPR39 overexpression mimicked these effects. Moreover, the anti-ferroptotic benefits of TPEN were reversed by the PI3K inhibitor LY294002.

TPEN attenuates neuronal ferroptosis an brain injury by activating the GPR39/PI3K/AKT signaling axis. This identifies GPR39 as a novel therapeutic target for brain injury treatment.