Butyrate modulates bacterial adherence on LS174T human colorectal cells by stimulating mucin secretion and MAPK signaling pathway

BACKGROUND/OBJECTIVES: Fermentation of dietary fiber results in production of various short chain fatty acids in the colon. In particular, butyrate is reported to regulate the physical and functional integrity of the normal colonic mucosa by altering mucin gene expression or the number of goblet cells. The objective of this study was to investigate whether butyrate modulates mucin secretion in LS174T human colorectal cells, thereby influencing the adhesion of probiotics such as Lactobacillus and Bifidobacterium strains and subsequently inhibiting pathogenic bacteria such as E. coli. In addition, possible signaling pathways involved in mucin gene regulation induced by butyrate treatment were also investigated. MATERIALS/METHODS: Mucin protein content assay and periodic acid-Schiff (PAS) staining were performed in LS174T cells treated with butyrate at various concentrations. Effects of butyrate on the ability of probiotics to adhere to LS174T cells and their competition with E. coli strains were examined. Real time polymerase chain reaction for mucin gene expression and Taqman array 96-well fast plate-based pathway analysis were performed on butyrate-treated LS174T cells. RESULTS: Treatment with butyrate resulted in a dose-dependent increase in mucin protein contents in LS174T cells with peak effects at 6 or 9 mM, which was further confirmed by PAS staining. Increase in mucin protein contents resulted in elevated adherence of probiotics, which subsequently reduced the adherent ability of E. coli. Treatment with butyrate also increased transcriptional levels of MUC3, MUC4, and MUC12, which was accompanied by higher gene expressions of signaling kinases and transcription factors involved in mitogen-activated protein kinase (MAPK) signaling pathways. CONCLUSIONS: Based on our results, butyrate is an effective regulator of modulation of mucin protein production at the transcriptional and translational levels, resulting in changes in the adherence of gut microflora. Butyrate potentially stimulates the MAPK signaling pathway in intestinal cells, which is positively correlated with gut defense.

Application of Method to Measure Calcineurin Activity in Healthy Human Volunteers after Cyclosporine Administration

BACKGROUND: Calcineurin-inhibitors have wide inter-individual variation in drug response. Although therapeutic drug monitoring has been conducted to optimize personalized regimen, toxicity or rejection may occur. In this study, pharmacologic effect was evaluated by measuring calcineurin activity in peripheral blood after administration of a single dose of cyclosporine in healthy volunteers. METHODS: 7 healthy Korean male subjects received cyclosporine 200 mg and blood samples were drawn immediately before and at 1, 1.5, 4, 6, 12 h after dosing to measure calcineurin activity. The blood concentrations of cyclosporine were determined for 24 hours. Calcineurin activity assay was done with Calcineurin cellular activity assay kit (Calbiochem, USA). Frozen whole blood samples in liquid N2 were thawed and lysed with lysis buffer. 50 microL of phosphate standard curve samples were added to each well of a 96-well plate and 10 microL of diluted lysate were added to the well with RII phosphopeptide substrate. After incubating for 30 min, reaction was terminated by adding 100 microL GREEN(TM) reagent. Absorbance was read at 620 nm using spectrophotometer. We evaluated percent change in calcineurin activity from baseline level in relation to the lowest level. RESULTS: Decrease of calcineurin activity was confirmed after cyclosporine administration (mean +/- SD: 58.9 +/- 48.6 (%)). Significant correlation was shown between calcineurin activity change and pharmacokinetic parameters (AUClast: r = 0.834, p value = 0.01, Cmax: r = 0.774, p value = 0.02). CONCLUSION: In this study, we confirmed the pharmacologic effect and its correlation with pharmacokinetics after administration of a single dose of cyclosporine by measuring calcineurin activity in peripheral blood in healthy volunteers.