METTL3-mediated m6A modification targets the regulation of PCBP1 expression, thereby modulating GPX4 levels and intracellular Fe2⁺ homeostasis to influence ferroptosis and contribute to juvenile sepsis-associated brain injury

Juvenile sepsis-associated brain injury (SABI) lacks effective targeted therapies, and ferroptosis has emerged as a critical pathogenic mechanism. N⁶-methyladenosine (m⁶A) modification is increasingly recognized as a key epitranscriptomic regulator in inflammatory and neurological disorders, yet its role in ferroptosis regulation during SABI remains unexplored. Here, we identify a novel METTL3–PCBP1–ferroptosis axis that drives hippocampal neuronal injury in SABI. Using a cecal ligation and puncture (CLP)-induced rat SABI model and an in vitro co-culture system of lipopolysaccharide (LPS)-stimulated microglia with primary hippocampal neurons, we demonstrate that SABI significantly upregulates methyltransferase-like 3 (METTL3) expression and global m⁶A modification levels in the hippocampus. MeRIP-seq reveals that poly(rC)-binding protein 1 ( Pcbp1 ) mRNA—encoding an iron chaperone that maintains cellular iron homeostasis—exhibits the most pronounced increase in m⁶A modification among cell death-related genes, with site-specific methylation at position 1151 (GGACA motif). This modification recruits the m⁶A reader YTH domain-containing family protein 2 (YTHDF2), promoting Pcbp1 mRNA decay and subsequent PCBP1 protein downregulation. Loss of PCBP1 reduces glutathione peroxidase 4 (GPX4) expression—likely through impaired mRNA binding—and disrupts intracellular Fe 2 ⁺ homeostasis, ultimately triggering ferroptosis characterized by lipid peroxidation and mitochondrial damage. Notably, METTL3 knockdown restores PCBP1 expression, normalizes Fe 2 ⁺ levels, upregulates GPX4, and attenuates ferroptosis and hippocampal injury; however, concurrent PCBP1 knockdown reverses these protective effects. Furthermore, PCBP1 overexpression in SABI rats rescues ferroptosis and neurological deficits. Collectively, our findings establish that METTL3-mediated m⁶A modification of Pcbp1 mRNA exacerbates ferroptosis and SABI pathogenesis, identifying the METTL3/PCBP1 axis as a promising therapeutic target for juvenile SABI.