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

Abstract

AimsRegulation 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 methodsWhole 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 findingsCldn23 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. Significancen-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.