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Abstract
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.
Conclusion TLR4 induced apoptosis plays a critical role in the pathogenesis in NEC. Defect of TLR4 inhibits enterocytes from apoptosis in NEC, predominantly through Caspase8-mediated apoptosis pathway.
Sequences of PCR primers
TLR4-deficient mice were protected from the development of NEC. Abdominal anatomy of mice in the different groups. Wild type C57BL/10j mice (A) or TLR4-deficient C57BL/10ScNJ mice (C) were served as breast-fed controls. NEC was induced in newborn Wild type C57BL/10j mice (B) or TLR4-deficient C57BL/10ScNJ mice (D).
FCM detection of apoptosis after stimulation of LPS (50 μg/ml) at different times in vitro. (a) IEC-WT control group; (b) 12 h after stimulation with LPS in IEC-WT group; (c) 24 h after stimulation with LPS in IEC-WT group; (d) IEC-6 transfected with vector only (Non Target, NT) control group; (e) 12 h after stimulation with LPS in IEC-NT group; (f) 24 h after stimulation with LPS in IEC-NT group; (g) TLR4-silent-IEC-6 control group; (h) 12 h after stimulation with LPS in TLR4-silent-IEC-6 group; (i) 24 h after stimulation with LPS in TLR4-silent-IEC-6 group; The Q4 quadrant (annexin V+/7-AAD-) indicate the percentage of apoptosis. (j) Quantification of apoptosis percentage determined by a blinded observer. *P < 0.05 by ANOVA compared with IEC-6-WT and IEC-6-NT group at the same time point, #P < 0.05 by ANOVA compared with control within IEC-6-WT group, **P < 0.05 by ANOVA compared with control within IEC-6-NT group, †P < 0.05 by ANOVA compared with control within TLR4-silent-IEC-6 group. Representative data from four separate experiments with three times per group.