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<p>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 (<i>Pcbp1</i>) 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&#xa0;<i>Pcbp1</i>&#xa0;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<sup>2</sup>⁺ homeostasis, ultimately triggering ferroptosis characterized by lipid peroxidation and mitochondrial damage. Notably, METTL3 knockdown restores PCBP1 expression, normalizes Fe<sup>2</sup>⁺ 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&#xa0;<i>Pcbp1</i>&#xa0;mRNA exacerbates ferroptosis and SABI pathogenesis, identifying the METTL3/PCBP1 axis as a promising therapeutic target for juvenile SABI.</p>

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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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年8月3日

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  • Fajuan Tang,
  • Hu Gao,
  • Bin Chen,
  • Junjie Ning,
  • Min Yang,
  • Xihong Li,
  • Lina Qiao

摘要

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 Fe2⁺ homeostasis, ultimately triggering ferroptosis characterized by lipid peroxidation and mitochondrial damage. Notably, METTL3 knockdown restores PCBP1 expression, normalizes Fe2⁺ 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.