TNF-alpha induces autophagy through ERK1/2 pathway to regulate apoptosis in neonatal necradzing enterocolitis model cells IEC-6

Necrotizing enterocolitis (NEC) is a potentially fatal illness in premature neonates. Tumor necrosis factor-alpha (TNF-alpha) and autophagy are associated with the pathogenesis of NEC. This study aimed to explore whether TNF-alpha might regulate apoptosis in neonatal NEC model cells IEC-6 via regulation of autophagy. NEC rat model was induced by hand feeding and exposure to asphyxia/cold-stress for histologic examination. The NEC in vitro model (IEC-6/NEC cells) was established by stimulating the intestinal epithelial cell line IEC-6 with lipopolysaccharide (LPS, 100 mu g/mL) for 3 h to investigate the effects of TNF-alpha on IEC-6 proliferation and apoptosis. In this study, NEC rats showed decreased proliferating cell nuclear antigen (PCNA) expression, increased TUNEL-positive cells, higher expression of TNF-alpha, p-ERK1/2, and autophagy-related proteins in rat small intestine compared with their controls. Additionally, the LPS-stimulated IEC-6/NEC cells showed a significantly decreased proliferation and increased apoptosis compared with the control cells. Furthermore, the LPS-stimulated IEC-6/NEC cells exhibited enhanced autophagy level, as evidenced by a dose-dependent increase in Beclin-1 protein expression, LC311/LC31 ratio and accumulation of MDC-positive autophagic vacuoles. Moreover, inhibition of autophagy by wortmannin or LY294002 significantly abolished the LPS-mediated decreased proliferation and increased apoptosis of IEC-6/NEC cells. Results also showed that inhibition of ERK1/2 pathway using UO126 significantly inhibited TNF-alpha-induced autophagy. Furthermore, the TNF-alpha-mediated inhibition of IEC-6 proliferation and promotion of IEC-6 apoptosis was abolished by UO126. Our findings demonstrated that TNF-alpha might induce autophagy through ERK1/2 pathway to regulate apoptosis in neonatal NEC cells IEC-6. Our study enhances our understanding of neonatal NEC pathogenesis.