Xylobiose treatment strengthens intestinal barrier function by regulating claudin 2 and heat shock protein 27 expression in human Caco-2 cells.

BACKGROUND: Xylobiose, a non-digestible disaccharide, largely contributes to the beneficial physiological effects of xylooligosaccharides. However, there is insufficient evidence to assess the direct effect of xylobiose on intestinal barrier function. Here, we investigated the intestinal barrier function in human intestinal Caco-2 cells treated with xylobiose. RESULTS: In total, 283 genes were upregulated and 256 genes were downregulated in xylobiose-treated Caco-2 cells relative to the controls. We focused on genes related to intestinal barrier function, such as tight junction (TJ) and heat shock protein (HSP). Xylobiose decreased the expression of the TJ gene Claudin 2 (CLDN2) and increased the expression of the cytoprotective HSP genes HSPB1 and HSPA1A, which encode HSP27 and HSP70, respectively. Immunoblot analysis confirmed that xylobiose suppressed CLDN2 expression and enhanced HSP27 and HSP70 expression. A quantitative reverse transcription-PCR and promoter assays indicated that xylobiose post-transcriptionally regulated CLDN2 and HSPB1 levels. Additionally, selective inhibition of phosphatidyl-3-inositol kinase (PI3K) inhibited xylobiose-mediated CLDN2 expression, whereas HSP27 expression induced by xylobiose was sensitive to the inhibition of PI3K, mitogen-activated protein kinase kinase and Src. CONCLUSION: The results of the present study reveal that xylobiose suppresses CLDN2 and increases HSP27 expression in intestinal Caco-2 cells via post-transcriptional regulation, potentially strengthening intestinal barrier integrity; however, these effects seem to occur via different signaling pathways. Our findings may help to assess the physiological role of xylobiose. © 2023 Society of Chemical Industry.

Microbial metabolite n-butyrate upregulates intestinal claudin-23 expression through SP1 and AMPK pathways in mouse colon and human intestinal Caco-2 cells.

AIMS: Regulation of the intestinal barrier is closely related to intestinal microbial metabolism. This study investigated the role of intestinal microflora in the regulation of the tight junction (TJ) barrier in epithelial cells, focusing on the microbial metabolite n-butyrate, a major short-chain fatty acid, using mice and human intestinal Caco-2 cells. MATERIALS AND METHODS: Whole transcriptome analysis with RNA sequencing and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were performed in the colon of germ-free (GF) and specific pathogen-free (SPF) mice. Claudin-23 expression was examined by qRT-PCR, immunoblotting, and immunofluorescence in Caco-2 cells treated with n-butyrate. Luciferase reporter assay was performed to examine the effect of n-butyrate on claudin-23 transcriptional activity. The siRNA targeting the transcription factor SP1 and pharmacological inhibitor of AMPK were used in combination. TJ permeability was examined in canine kidney MDCKII cells stably expressing human claudin-23. KEY FINDINGS: Cldn23 mRNA expression was downregulated in the colon of GF mice (0.6-fold) compared to that in SPF mice. n-Butyrate upregulated claudin-23 mRNA (1.7-fold) and protein (2.1-fold) expression as well as increased the transcriptional activity (15-fold) of CLDN23 in Caco-2 cells. The n-butyrate-mediated increase in claudin-23 expression and transcriptional activity was reduced by inhibition of SP1 and AMPK. Exogenously expressed human claudin-23 in MDCKII cells did not affect TJ permeability to ions and macromolecules. SIGNIFICANCE: n-Butyrate regulates intestinal claudin-23 expression through the SP1 and AMPK pathways. This mechanism may be involved in the beneficial effects of n-butyrate-mediated intestinal homeostasis.

Partially hydrolyzed guar gum upregulates heat shock protein 27 in intestinal Caco-2 cells and mouse intestine via mTOR and ERK signaling.

BACKGROUND: The intestinal epithelium acts as a barrier against harmful luminal materials, thus preventing intestinal diseases and maintaining intestinal health. Heat shock protein 27 (HSP27) promotes intestinal epithelial integrity under both physiological and stressed conditions. The effects of partially hydrolyzed guar gum (PHGG) on HSP27 expression in intestinal Caco-2 cells and mouse intestines were investigated. RESULTS: The present study showed that PHGG upregulated HSP27 expression in Caco-2 cells without upregulating Hspb1, the gene encoding HSP27. Feeding PHGG increased HSP25 expression in epithelial cells of the small intestine of mice. Inhibition of protein translation using cycloheximide suppressed PHGG-mediated HSP27 expression, indicating that PHGG upregulated HSP27 via translational modulation. Signaling inhibition of the mechanistic target of rapamycin (mTOR) and phosphatidyl 3-inositol kinase reduced PHGG-mediated HSP27 expression, whereas mitogen-activated protein kinase kinase inhibition by U0126 increased HSP27 expression, irrespective of PHGG administration. PHGG increases mTOR phosphorylation and reduces extracellular signal-regulated protein kinase (ERK) phosphorylation. CONCLUSION: PHGG-mediated translation of HSP27 in intestinal Caco-2 cells and mouse intestine via the mTOR and ERK signaling pathways may promote intestinal epithelial integrity. These findings help us better understand how dietary fibers regulate the physiological function of the intestines. © 2023 Society of Chemical Industry.

Propionate regulates tight junction barrier by increasing endothelial-cell selective adhesion molecule in human intestinal Caco-2 cells.

Regulation of the intestinal barrier is closely associated with intestinal microbial metabolism. This study investigated the role of propionate, a major short-chain fatty acid produced by intestinal microorganisms, in the regulation of the tight junction (TJ) barrier in human intestinal Caco-2 cells. Propionate strengthened TJ barrier integrity, as indicated by decreased permeability to macromolecules and increased transepithelial electrical resistance in Caco-2 cells. DNA microarray analysis revealed that propionate upregulated endothelial cell-selective adhesion molecule (ESAM), a TJ-associated protein, without any increase in other TJ proteins. The upregulation of ESAM was confirmed using quantitative reverse transcription-PCR, immunoblotting, and immunofluorescence analyses. Luciferase promoter analysis demonstrated that propionate induced the transcriptional activation of ESAM. The effects of propionate were sensitive to nilotinib inhibition of NR2C2. Overexpression of human ESAM (hESAM) in canine kidney epithelial MDCK-II cells lowered the permeability to macromolecules in a manner similar to that of propionate-treated Caco-2 cells. hESAM overexpression facilitated calcium-induced assembly of the TJ complex in MDCK-II cells. Taken together, propionate strengthened the intestinal TJ barrier by increasing ESAM levels in Caco-2 cells.