Inhibition of DNA demethylation attenuates experimental necrotizing enterocolitis via suppression of TLR4-mediated inflammation

Background: Necrotizing enterocolitis (NEC) is a serious inflammatory bowel disease that primarily affects preterm infants. NEC is characterized by damage to the intestinal epithelium, inflammation, and a high mortality rate. Alterations in gene regulation, particularly DNA methylation, have been linked to the development of NEC in preterm infants. However, the significance of DNA demethylation and its potential as a therapeutic target require further investigation. Methods: We investigated DNA methylation and demethylation changes in experimental NEC using neonatal mouse models in vivo and mouse and human enteroids in vitro. We measured global DNA methylation (5-mC), hydroxymethylation (5-hmC), and the expression of the ten-eleven translocation (TET) enzymes. We also evaluated the effects of the DNA demethylation inhibitor Bobcat339 on intestinal injury, inflammation, and the Toll-like receptor 4 (TLR4) signaling pathway. Results: Neonatal mice with NEC exhibited reduced levels of 5-hmC and increased levels of 5-mC, indicating global hypermethylation of the intestinal epithelium. Treatment with the DNA demethylation inhibitor Bobcat339 significantly reduced the severity of NEC and decreased proinflammatory mediators, including interleukin 1beta ( Il1b ), tumor necrosis factor alpha ( Tnfa ), and lipocalin-2 ( Lcn2 ), following LPS-induced endotoxemia. Conclusions: These findings advance our understanding of the role of abnormal DNA demethylation in NEC development. Moreover, our results demonstrate that a DNA demethylation inhibitor attenuates experimental NEC by decreasing TLR4-driven immune responses and intestinal inflammation. Targeting DNA demethylation by modulating TLR4-associated inflammatory pathways may represent a promising therapeutic strategy for NEC.