Human milk oligosaccharide disialyllacto-n-tetraose protects human intestinal epithelium integrity and permeability against mast cell chymase-induced disruption by stabilizing ZO-1/FAK/P38 pathway of intestinal epithelial cell.

CONTEXT: Inflammatory bowel disease (IBD) is a chronic gut disease with intestinal-epithelium disruption. Mast cell (MC) has been discussed in IBD studies, but its subset MCTC (chymase/tryptase) and MC-chymase have not been well-explored extensively. Human-milk-oligosaccharide-Disialyllacto-N-Tetraose (DSLNT) was reported as an effective strategy to protect infants against IBD with unclear mechanism. OBJECTIVE: This study was to examine the distribution of chymase-positive mast cells in the intestinal-epithelium-tissue of IBD infants, to explore the MC-chymase function on intestinal-epithelium, and to investigate the influences of DSLNT against MC-chymase-induced disruptions. MATERIALS AND METHODS: The intestinal-biopsies (surgical-waste) of the infants with IBD or with intestinal-atresia (non-IBD) were paraffin-embedded for immunohistochemistry. In-situ intestinal-tissue model and in-vitro human-intestinal-epithelial-cell (Caco-2) model were established with or without the treatments of MC-chymase (50mU/mL), DSLNT (600 µM) and DSLNT + MC-chymase respectively. The tissue morphology analysis, cell proliferation assay, cell-gap-closure assessment, fluorescence-immunocytochemistry, western blot, trans-epithelial-electrical-resistance, cell-cycle and statistical analysis were applied. RESULTS: There was an increased number of MCTC subset around the inflamed intestinal area in-vivo; MC-chymase caused intestinal-epithelial-barrier damage in-situ, decreased trans-epithelial-electrical-resistance of caco-2 cell monolayer in-vitro; while DSLNT protected epithelium against MC-chymase induced disruptions. MC-chymase reduced cell-viability, proliferation and migration, altered cell-cycle, down-regulated ZO-1, FAK, and P38 expressions, while DSLNT protected cells by impairing MC-chymase-induced interruptions. DSLNT can rescue ZO-1, FAK and P38 expressions and restore epithelial-cell integrity and cell cycle. CONCLUSIONS: Chymase-positive MCs are involved in IBD progress. MC-chymase disrupts intracellular ZO-1/FAK/P38 signal pathway and cell-cell/cell-matrix contacts, while DSLNT protects intestinal-epithelium against MC-chymase to maintain the intestinal epithelium integrity.

Causative role of mast cell and mast cell-regulatory function of disialyllacto-N-tetraose in necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) remains a fatal gastrointestinal disorder in neonates. Disialyllacto-N-tetraose (DSLNT), a function-unclear human milk-derived hexasaccharide, shows anti-NEC potential in previous animal studies. This study is aimed to explore the role of mast cell (MC), a fundamental cell type of mucosal immune system and protective DSLNT in regulating pathological process of NEC. For this purpose, infantile intestinal-tissues were collected from NEC neonates for examination of MCs and its proteases-positive cells. MC accumulation and MC-specific proteases (chymase, tryptase and dipeptidyl peptidase I) were firstly found in lesioned area of NEC infants in-vivo. Subsequent in-situ experiments on neonatal ileum segments showed that purified MC-chymase induced a destructive epithelial layer shedding from basement and microvascular endothelium damage in infantile intestinal segments. Human foreskin MC-activation model was established and DSLNT were applied; MC products (histamine and MC-proteases) were used as MC activation/degranulation indicators. In this in-vitro model, DSLNT pretreatment suppressed release of histamine, chymase and tryptase by MC to the tissue supernatants during lipopolysaccharide or complement C5a stimulation. Newborn rats were formula-hand-fed with or without DSLNT supplement and exposed to hypoxia/cold-stress to induce experimental-NEC-model. In NEC rats, DSLNT supplementation reduced the incidence and pathological scores of NEC, inhibited local accumulation of MC and reduced cytokines (IL-1ß, IL-6 and TNF-α) levels in the ileum of rats. In conclusion, MC was causally implicated in epithelium barrier failure in pathogenesis of NEC. DSLNT favorably modulated MC homeostasis by regulating MC degranulation/accumulation, contributing to attenuated NEC. This indicated novel pathomechanisms and potential targets of NEC.

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-1ß, 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-1ß, 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.

Human breast milk oligosaccharides attenuate necrotizing enterocolitis in rats by suppressing mast cell accumulation, DPPI activity and TLR4 expression in ileum tissue, and regulating mitochondrial damage of Caco-2 cells.

Necrotizing enterocolitis (NEC), a devastating infant disease characterized by severe intestinal necrosis, its pathogenesis is poorly understood, but appears to be multifactorial and highly associated with immaturity of gastrointestinal tract and immature innate-immune system. Breast-milk is effective strategy to protect infants against NEC. This study is using a NEC rat model to investigate the pathological mechanism of NEC involved intestinal-damages, and the therapeutic mechanism of sialylated human milk oligosaccharides (SHMOs) on NEC rats; also using cell model to investigate the effects of SHMOs on colon-epithelial cells (Caco-2) in-vitro. Extraction and characterization of SHMOs from breast milk, establishment of a NEC rat model, histopathological analysis and mast cell accounting of the terminal ileum were taken; The levels of DPPI, TLR4, IL-6, TNF-α, MMP-2/9 and glutathione were measured using various methods. Caco-2 cells were pre-treated with SHMOs and cultured with LPS, histamine, chymase or DPPI, cell viabilities and mitochondrial membrane potential were examined; flow cytometry was used to detect cell cycle. The accumulation of mast cells was found in the ileum of NEC rats, but prohibited by SHMOs treatment; the increased levels of TLR4, DPPI, IL-6, TNF-α, MMP-2/9 in NEC ileum were suppressed by SHMOs in-vivo. SHMOs prevented Caco-2 cells from LPS, histamine, chymase induced damages by surviving cell viability, regulating G0/G1 and S phase in cell cycles, and increasing mitochondrial membrane potential. These findings provide a new insight into the pharmacological mechanism of SHMOs treatment for NEC and suggest that SHMOs needs well attention for therapeutic aims.