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. 2000 Sep;47(3):397–403. doi: 10.1136/gut.47.3.397

Butyrate inhibits inflammatory responses through NFκB inhibition: implications for Crohn's disease

J Segain 1, D R de la Bletiere 1, A Bourreille 1, V Leray 1, N Gervois 1, C Rosales 1, L Ferrier 1, C Bonnet 1, H Blottiere 1, J Galmiche 1
PMCID: PMC1728045  PMID: 10940278

Abstract

BACKGROUND/AIM—Proinflammatory cytokines are key factors in the pathogenesis of Crohn's disease (CD). Activation of nuclear factor kappa B (NFκB), which is involved in their gene transcription, is increased in the intestinal mucosa of CD patients. As butyrate enemas may be beneficial in treating colonic inflammation, we investigated if butyrate promotes this effect by acting on proinflammatory cytokine expression.
METHODS—Intestinal biopsy specimens, isolated lamina propria cells (LPMC), and peripheral blood mononuclear cells (PBMC) were cultured with or without butyrate for assessment of secretion of tumour necrosis factor (TNF) and mRNA levels. NFκB p65 activation was determined by immunofluorescence and gene reporter experiments. Levels of NFκB inhibitory protein (IκBα) were analysed by western blotting. The in vivo efficacy of butyrate was assessed in rats with trinitrobenzene sulphonic acid (TNBS) induced colitis.
RESULTS—Butyrate decreased TNF production and proinflammatory cytokine mRNA expression by intestinal biopsies and LPMC from CD patients. Butyrate abolished lipopolysaccharide (LPS) induced expression of cytokines by PBMC and transmigration of NFκB from the cytoplasm to the nucleus. LPS induced NFκB transcriptional activity was decreased by butyrate while IκBα levels were stable. Butyrate treatment also improved TNBS induced colitis.
CONCLUSIONS—Butyrate decreases proinflammatory cytokine expression via inhibition of NFκB activation and IκBα degradation. These anti-inflammatory properties provide a rationale for assessing butyrate in the treatment of CD.


Keywords: inflammation; butyrate; Crohn's disease; nuclear factor kappa B; cytokines

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Figure 1  .

Figure 1  

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Effect of butyrate on tumour necrosis factor (TNF). Colonic biopsies (A) from inflamed (n=14) or non-inflamed (n=15) mucosa of patients with Crohn's disease or from normal (n=6) mucosa of healthy controls were cultured for 24 hours with or without 2 or 10 mM butyrate. **p<0.01,***p<0.001 v 0 mM. (B) Peripheral blood mononuclear cells (PBMC) (n=7) were cultured for 24 hours alone or in the presence of 2.5 µg/ml lipopolysaccharide (LPS) followed by 20 hours under the same conditions with or without 2 mM butyrate. Supernatant concentrations of total TNF were assessed by WEHI cell bioassay, and results are expressed as mean (SEM). **p<0.01 v LPS.

Figure 2  .

Figure 2  

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Effect of butyrate on proinflammatory cytokine mRNA. Biopsies and isolated lamina propria mononuclear cells (LPMC) (A) from inflamed mucosa of patients with Crohn's disease were cultured for 24 hours with or without 2 or 10 mM butyrate. Peripheral blood mononuclear cells (PBMC) (B) were cultured for 24 hours alone or in the presence of 2.5 µg/ml lipopolysaccharide (LPS) and then for another 20 hour period under the same conditions with or without 2 mM butyrate. RNA was extracted and RT-PCR was performed using primers designed for interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-β, TNF-α, and control β-actin.

Figure 3  .

Figure 3  

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Effect of butyrate on nuclear translocation of nuclear factor kappa B (NFκB). Peripheral blood mononuclear cells (PBMC) (A) unstimulated, (B) lipopolysaccharide (LPS) stimulated, or (C) LPS stimulated and treated with 2 mM butyrate. The intracellular location of NFκB p65 (RelA) was determined by immunofluorescence using an anti-NFκB p65 polyclonal antibody. Magnification, ×400.

Figure 4  .

Figure 4  

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Effect of butyrate on nuclear factor kappa B (NFκB) transcriptional activity. THP-1 cells were transiently transfected with the NFκB reporter plasmid 3XMHC-luc and cultured alone or in the presence of 2.5 µg/ml lipopolysaccharide (LPS), with or without 2 mM butyrate for four hours. Luciferase activity was then determined. Data are mean (SEM) of four experiments.

Figure 5  .

Figure 5  

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Effect of butyrate on inhibitor protein kappa Bα (IκBα) expression. THP-1 cells were stimulated for various periods of time with 2.5 µg/ml lipopolysaccharide (LPS), in the presence or absence of 2 mM butyrate. Cell lysates were subjected to western blot analysis of IκBα.

Figure 6  .

Figure 6  

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Comparison of the effects of butyrate and trichostatin A (TSA) on production of tumour necrosis factor (TNF). Peripheral blood mononuclear cells were cultured for 24 hours alone or in the presence of 2.5 µg/ml lipopolysaccharide (LPS) followed by 20 hours under the same conditions with or without 2 mM butyrate or 0.5 µM/ml of the histone deacetylase inhibitor TSA. Supernatant concentrations of total TNF were assessed by WEHI cell bioassay, and the results are expressed as mean (SEM) of three experiments.

Figure 7  .

Figure 7  

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Effects of butyrate treatment on established colitis. Haematoxylin and eosin stained (A, B) paraffin sections of colon of rats with trinitrobenzene sulphonic acid (TNBS) induced colitis receiving isotonic saline (A) or 100 mM butyrate (B) enemas for two weeks. Magnification, ×200. Immunofluorescence staining (C, D) of paraffin sections of colon of rats with TNBS induced colitis receiving isotonic saline (C) or 100 mM butyrate (D) with a monoclonal antibody specific to the activated form of nuclear factor kappa B. Magnification, ×300.

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