Nutr Res Pract.  2015 Aug;9(4):343-349. 10.4162/nrp.2015.9.4.343.

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

• Jung TH
• Park JH
• Jeon WM
• Han KS

• KMID: 2313849
• DOI: http://doi.org/10.4162/nrp.2015.9.4.343

Abstract

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.

Keyword

Butyrate; LS174T cell; mucin protein; MUC gene; MAPK

MeSH Terms

Bacteria
Bifidobacterium
Butyrates*
Colon
Dietary Fiber
Fatty Acids
Fermentation
Gene Expression
Goblet Cells
Humans
Lactobacillus
Mucins*
Mucous Membrane
Phosphotransferases
Probiotics
Protein Kinases
Real-Time Polymerase Chain Reaction
Transcription Factors
Butyrates
Fatty Acids
Mucins
Phosphotransferases
Protein Kinases
Transcription Factors

Figure

• 

  Fig. 1 Effect of sodium butyrate treatment on mucin protein production in LS174T cells. (A) Cells were treated with sodium butyrate at final concentrations of 0, 3, 6 and 9 mM. Mucin protein contents were measured by enzyme-linked immunosorbent assay. Values are expressed as mean ± SEM. Means without a common letter differ, P < 0.05. Con, control. (B) Periodic acid-Schiff staining of cells treated with sodium butyrate at final concentrations of 0 (I), 3 (II), 6 (III), and 9 mM (IV).

• 

  Fig. 2 Adherence of Lactobacillus acidophilus ATCC 4356 to LS174T cells and its inhibitory effect on Escherichia coli ATCC 43896. LS174T cells were treated with sodium butyrate at final concentrations of 0, 3, 6, and 9 mM. Values are expressed as mean ± SEM. Means without a common letter differ, P < 0.01 for L. acidophilus (black bar) and P < 0.05 for E. coli (grey bar).

• 

  Fig. 3 Adherence of Bifidobacterum longum ATCC 15707 to LS174T cells and its inhibitory effect on Escherichia coli ATCC 43896. LS174T cells were treated with sodium butyrate at final concentrations of 0, 3, 6, and 9 mM. Values are expressed as mean ± SEM. Means without a common letter differ, P < 0.01 for B. longum (black bar) and P < 0.05 for E. coli (grey bar).

• 

  Fig. 4 Effect of 6 mM sodium butyrate treatment on mRNA expression levels of four mucin genes (MUC2, MUC3, MUC4, and MUC12) in LS174T cells. The mRNA expression of mucin genes was normalized to the GAPDH mRNA level in each sample. Real time PCR was performed in triplicate at each concentration. The results are expressed as percentage increases as compared to the control (0 mM). Graphs represent mean ± SEM of three separate experiments; * P < 0.05 and ** P < 0.01 relative to control (CON) cells.

• 

  Fig. 5 Analysis of signaling pathways in 6 mM butyrate-treated LS174T cells. The synthesized cDNA was applied to a TaqMan array 96-well fast plate, and comparative Ct method for relative quantification was performed. The delta delta Ct value was determined by subtracting the delta Ct of the control sample from the individual delta Ct of the test sample. Significant changes in butyrate-induced gene expression were analyzed based on more than 5-fold changes compared to the control group. MAPKAPK, mitogen-activated protein kinase activated protein kinase; P, protein

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