m6A-Dependent Upregulation of PFKFB3 Drives Macrophage-Mediated Inflammation in Necrotizing Enterocolitis

<b><i>Aims:</i></b> This study aimed to elucidate the role of N6-methyladenosine (m6A) methylation in necrotizing enterocolitis (NEC) pathogenesis, focusing on its regulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) expression, and to evaluate PFKFB3 as a therapeutic target for NEC. <b><i>Results:</i></b> We observed a significant reduction in N6-methyladenosine (m6A) methylation within the 3'-untranslated region (3'-UTR) of PFKFB3 mRNA in human NEC tissues. This epigenetic change stabilized PFKFB3 mRNA, increased protein levels, and accelerated glycolytic flux. In both in vivo (lipopolysaccharide-hypoxia-cold stress) and in vitro (THP-1-differentiated macrophage) NEC models, PFKFB3-driven glycolysis was found to promote M1 macrophage polarization through reactive oxygen species (ROS) accumulation, thereby intensifying intestinal inflammation. Importantly, pharmacological inhibition of PFKFB3 using 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one significantly reduced ROS production, limited macrophage infiltration, and mitigated mucosal injury. <b><i>Innovation and Conclusion:</i></b> This study identifies a critical metabolic-epigenetic axis in NEC pathogenesis, wherein reduced m6A methylation of PFKFB3 mRNA drives intestinal inflammation. Our results demonstrate that pharmacological inhibition of PFKFB3 effectively reduces inflammation and tissue injury in NEC models, positioning PFKFB3 as a novel therapeutic target. This work provides the first evidence of an m6A-mediated mechanism in NEC and highlights the potential of targeting PFKFB3 for clinical intervention. <i>Antioxid. Redox Signal.</i> 43, 765-781.