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.