In our study, overexpression TLR4 or FASN reversed the inhibition effect of D-DNA on TLR4, NF-kB, NLRP3 and c-caspase3 expression in NEC IEC-6 cells, indicating that NF-κB-mediated NLRP3 activation is involved in the mitigation of NEC intestinal epithelial barrier damage by D-DNA. This evidence concerns the gene TLR4 and necrotizing enterocolitis.