Preconditioning induces ischemic tolerance by attenuating ferroptosis: involvement of the m6A-dependent FTO-IGF2BP1/3-ACSL4 regulatory Axis.

Cerebral ischemic preconditioning (CIP) confers robust protection against subsequent ischemic stroke by enhancing brain ischemic tolerance. Although ferroptosis is increasingly recognized as a critical pathogenic mechanism in stroke, the role of N6-methyladenosine (m6A) RNA modification in its regulation remains poorly understood. This study investigated the involvement of m6A signaling in the anti-ferroptotic mechanism of CIP using complementary in vivo mouse middle cerebral artery occlusion (MCAO) and in vitro oxygen-glucose deprivation (OGD) models. We found that CIP significantly attenuated ischemia-induced neuronal ferroptosis in vivo, whereas OGD preconditioning (OGDP) recapitulated these anti-ferroptotic effects in HT22 cells. Acyl-CoA synthetase long-chain family member 4 (ACSL4), a key executor of ferroptosis, was markedly upregulated following ischemic injury, whereas CIP effectively suppressed this increase. Similarly, OGDP attenuated OGD-induced ACSL4 upregulation in HT22 cells. Importantly, ACSL4 overexpression significantly attenuated the protective effects of OGDP against OGD-induced ferroptosis. Mechanistic analyses in HT22 cells revealed that OGD downregulated the m6A demethylase fat mass and obesity-associated protein (FTO) and upregulated m6A readers insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs), including IGF2BP1 and IGF2BP3, thereby stabilizing ACSL4 mRNA and promoting ferroptosis. OGDP alleviated these effects by restoring FTO expression, reducing IGF2BP1 and IGF2BP3 levels, and enhancing m6A-dependent ACSL4 mRNA decay. Together, these findings demonstrate that CIP induces ischemic tolerance and attenuates ACSL4-associated ferroptosis. Mechanistic studies in neuronal cells further identify the m6A-dependent FTO-IGF2BP1/3-ACSL4 axis as a potential regulatory mechanism contributing to OGDP-induced ferroptosis resistance.