Fructo-oligosaccharide-mediated alteration in claudin expression in small intestinal absorptive Caco-2 cells is positively associated with the induction of inflammatory genes and the glucan receptor gene CLEC7A

Aya Harasawa; Shiori Ishiyama; Kazuki Mochizuki

doi:10.1016/j.nut.2023.112140

Fructo-oligosaccharide-mediated alteration in claudin expression in small intestinal absorptive Caco-2 cells is positively associated with the induction of inflammatory genes and the glucan receptor gene CLEC7A

Nutrition. 2023 Nov:115:112140. doi: 10.1016/j.nut.2023.112140. Epub 2023 Jun 20.

Authors

Aya Harasawa¹, Shiori Ishiyama², Kazuki Mochizuki³

Affiliations

¹ Department of Integrated Applied Life Science, Integrated Graduate School of Medicine, Engineering, and Agricultural Sciences, University of Yamanashi, Kofu, Japan.
² Laboratory of Food and Nutritional Sciences, Department of Local Produce and Food Sciences, Faculty of Life and Environmental Sciences, University of Yamanashi, Kofu, Japan.
³ Laboratory of Food and Nutritional Sciences, Department of Local Produce and Food Sciences, Faculty of Life and Environmental Sciences, University of Yamanashi, Kofu, Japan. Electronic address: mochizukik@yamanashi.ac.jp.

PMID: 37481839
DOI: 10.1016/j.nut.2023.112140

Abstract

Objectives: Indigestible carbohydrates may strengthen tight junctions (TJs) independently of intestinal bacteria. This study investigated whether indigestible carbohydrates (i.e., fructo-oligosaccharides [FOS]) promote TJs directly to intestinal absorptive Caco-2 cells and examined the association between the expression of genes constructing TJs and other genes using mRNA microarray analysis.

Methods: Caco-2 cells at 1.0 × 10⁵/mL were seeded in a type I collagen plate and cultured in high-glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS); the cells reached confluence at 7 d after seeding. Ten days after the cells reached confluency, they were cultured for 24 h in 10% FCS-containing DMEM medium supplemented with 0%, 5%, or 10% FOS. We performed mRNA microarray to identify the genes whose expression was altered by FOS. Subsequently, quantitative reverse transcription polymerase chain reaction was performed for these altered genes, including CLEC7A encoding the glucan receptor, and for the claudin (CLDN) family genes. The expression of CLDN2, CLDN4, and CLEC7A proteins was assessed using western blot analysis.

Results: FOS decreased the mRNA and protein expression of CLDN2, which weakens TJs, and increased those of CLDN4, which strengthens TJs, in Caco-2 cells. FOS treatment (10%) reduced the mRNA expression of antioxidative genes and induced the expression of immune response-related genes, including CLEC7A, CCL2, and ITGA2. Furthermore, the expression of CLEC7A protein was enhanced by FOS.

Conclusions: Induction of TJ-strengthening CLDN4 and reduction of TJ-weakening CLDN2 by FOS treatment in small intestinal absorptive Caco-2 cells is positively associated with the induction of inflammatory genes, including CLEC7A.

Keywords: CLEC7A; Claudin; Fructo-oligosaccharides; Immune response; Tight junction.