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

Abstract

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 MC_TC (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 MC_TC 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.

Keywords:

• Mast cell chymase
• inflammatory bowel disease
• intestinal epithelium
• human milk disialyllacto-N-tetraose
• ZO-1

Acknowledgments

Thanks to Changzhou University, Jiangsu Education Department and National Natural Science Foundation of China for the financial supports; also thanks to the Changzhou Children’s Hospital for the clinical research supports.

Author contributions

Correspondent author, Prof. and Dr. Xiaoying Zhou has contributed to the funding, organizations, conception, designing, interpretation of data, drafted/edited and corrected the paperwork critically for intellectual content; approved the final version. First author: Miss Xuejiao Lian has contributed to the experimental works, technique operation, involved in cell model, data analysis, preparing the first draft and figures and corrections, and approved the final version for submission. Co-First author: Miss Jingqiu He-Yang has contributed to the experimental works, involved in cell model, data analysis, preparing the first draft and figures and corrections, and approved the final version for submission. Dr. Wenting Zhang has contributed to the design, technique operation, data analysis, funding, and draft corrections, and approved the final version for submission. Everyone agrees to be accountable for all aspects of this work.

Author statement

Prof. Xiaoying Zhou, I have made substantial contributions to the funding, organizations, conception, design, interpretation of data, drafted/edited and corrected the paper work critically for intellectual content; approved the final version.

Xuejiao Lian, I have made substantial contributions to the experimental works, technique operation, involved in cell model, data analysis, preparing the first draft and figures and corrections, and approved the final version for submission.

Jingqiu He-Yang: I have made substantial contributions to the funding, experimental works, data analysis, and approved the final version for submission.

Wenting Zhang: I have made substantial contributions to the technique operation, data analysis, funding, draft corrections and approved the final version for submission.

All: everyone agrees to be accountable for all aspects of this work.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by Changzhou University Life Science Research Fund, China [ZMF14020066]; Start-up Research Laboratory for Over-sea Talent Fund, China [Z391405]; National Natural Science Foundation of China [NO.81801493]; Jiangsu Province Graduate Student Scientific Research Innovation Scheme, China [2019-02-C-103]; Postgraduate Research & Practice Innovation Program of Jiangsu Province, China [SJCX22_1317].