Background and aim The importance of Toll-like receptor-4 (TLR4) signalling in necrotizing enterocolitis (NEC) is well-documented, but the potential mechanisms that regulate enterocyte apoptosis remain unclear. We investigated the role of apoptosis factors-Caspases in NEC and its pathway (endogenous and/or exogenous).
Methods TLR4-deficient C57BL/10ScNJ mice and Lentivirus-mediated stable TLR4-silent cell line (IEC-6) were used. NEC was induced by formula gavage, cold, hypoxia, combined with LPS in vivo or LPS stimulation in vitro. NEC severity was evaluated by histology. Enterocyte apoptosis was evaluated by TUNEL or Annexin analysis. The expression of TLR4, Caspases8, Caspase9, and Caspase3 were detected by qRT-PCR and western blot. Inflammatory factors including TNF-α, IL-6, IL-10 and IL-2 were examined by Luminex.
Results In TLR4-deficient mice, the severity of NEC was reduced, the expression of caspase8 was decreased, caspase9 was increased, and Caspase3 did not change significantly. In TLR4-silent IEC-6 cell line, expression of caspase3 was decreased and apoptosis rate were significantly reduced. Cytokine level of TNF-α and IL-2 were decreased.