Sialylated human milk oligosaccharides prevent intestinal inflammation by inhibiting toll like receptor 4/NLRP3 inflammasome pathway in necrotizing enterocolitis rats

BACKGROUND: Necrotizing enterocolitis (NEC) remains a fatal gastrointestinal disorder in neonates and has very limited therapeutic options. Sialylated human milk oligosaccharides (SHMOs) improve pathological changes in experimental NEC models. The objectives of this study were to investigate the involvement of NLRP3 inflammasome in NEC pathology and to explore the effects of SHMOs on toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB)/NLRP3 inflammatory pathway in experimental NEC. METHODS: The intestinal-tissue segments were collected from NEC infants, NLRP3 and caspase-1 positive cell were examined by immunohistochemistry. Newborn rats were hand-fed with formula containing or non-containing SHMOs (1500 mg/L) and exposed to hypoxia/cold stress to induce experimental NEC. The NEC pathological scores were evaluated; ileum protein expression of membrane TLR4 (mTLR4), inhibitor κB-α (IκB-α), NF-κB p65 subunit and phospho-NF-κB p65, as well as NLRP3 and caspase-1 were analyzed; ileum concentrations of interleukin-1β, interleukin-6, tumor necrosis factor-α (TNF-α) were also measured. Human colon epithelial Caco-2 cells were pre-treated with or without SHMOs and stimulated with TLR4 activator, lipopolysaccharide. Cell viabilities, mitochondrial membrane potential and supernatant matrix metalloprotease 2 (MMP-2) activities were analyzed. RESULTS: Increased frequencies of NLRP3 and caspase-1 positive cells were found in the lamina propria of damaged intestinal area of NEC neonates. SHMOs supplementation reduced NEC incidence and pathological damage scores of rats challenged with hypoxia/cold stress. Accumulation of interleukin-1β, interleukin-6 and TNF-α in NEC group were attenuated in SHMOs + NEC group. Protein expression of mTLR4, NLRP3 and caspase-1 were elevated, cytoplasmic IκB-α were reduced, nuclear phospho-NF-κB p65 were increased in the ileum of NEC rats. SHMOs supplementation ameliorated the elevation of mTLR4, NLRP3 and caspase-1, restored IκB-α in the cytoplasmic fraction and reduced phospho-NF-κB p65 in the nuclear fraction in the ileum of NEC rats. SHMOs pre-treatment improved Caco-2 cell viability, mitigated loss of mitochondrial membrane potential and modulated MMP-2 activities in the presence of lipopolysaccharide in-vitro. CONCLUSIONS: This study provided clinical evidence of involvement of NLRP3 inflammasome in NEC pathology, and demonstrated the protective actions of SHMOs might be owing to the suppression of TLR4/NF-κB/NLRP3-mediated inflammation in NEC.