Butyrate Treatment of DSS-Induced Ulcerative Colitis Affects the Hepatic Drug Metabolism in Mice

Abstract

The development of inflammatory bowel disease (IBD) is associated with alterations in the gut microbiota. There is currently no universal treatment for this disease, thus emphasizing the importance of developing innovative therapeutic approaches. Gut microbiome-derived metabolite butyrate with its well-known anti-inflammatory effect in the gut is a promising candidate. Due to increased intestinal permeability during IBD, butyrate may also reach the liver and influence liver physiology, including hepatic drug metabolism. To get an insight into this reason, the aim of this study was set to clarify not only the protective effects of the sodium butyrate (SB) administration on colonic inflammation but also the effects of SB on hepatic drug metabolism in experimental colitis induced by dextran sodium sulfate (DSS) in mice. It has been shown here that the butyrate pre-treatment can alleviate gut inflammation and reduce the leakiness of colonic epithelium by restoration of the assembly of tight-junction protein Zonula occludens-1 (ZO-1) in mice with DSS-induced colitis. In this article, butyrate along with inflammation has also been shown to affect the expression and enzyme activity of selected cytochromes P450 (CYPs) in the liver of mice. In this respect, CYP3A enzymes may be very sensitive to gut microbiome-targeted interventions, as significant changes in CYP3A expression and activity in response to DSS-induced colitis and/or butyrate treatment have also been observed. With regard to medications used in IBD and microbiota-targeted therapeutic approaches, it is important to deepen our knowledge of the effect of gut inflammation, and therapeutic interventions were followed concerning the ability of the organism to metabolize drugs. This gut-liver axis, mediated through inflammation as well as microbiome-derived metabolites, may affect the response to IBD therapy.

Keywords: butyrate; cytochromes P450; drug metabolism; gut inflammation; gut–liver axis.

FIGURE 1

Impact of butyrate pre-treatment on clinical symptoms and histopathological changes in the dextran sodium sulfate mouse model of ulcerative colitis. Altogether 25 mice were equally separated into five groups according to sodium butyrate (SB) and DSS treatment. (A) Occurrence of diarrhea and rectal bleeding was summarized in the clinical score. (B) Shortening of colon length and (C) histopathological changes in colonic mucosa after DSS treatment summarized as the histological score was analyzed at the end of experiment. Changes in colonic mucosa after DSS-treatment are shown on representative histological sections stained by hematoxylin/eosin. (D) Occurrence of goblet cells in mucosa of representative colon sections was visualized by Alcian blue/nuclear fast red staining. Data in graphs are expressed as mean ± SD and represent one out of two experiments. One-way ANOVA with Tukey’s multiple post hoc test was used for comparison of experimental groups to CT controls (group 1: *p < 0.05, ***p < 0.001, ****p < 0.0001) or to DSS-controls (group 5: ♯p < 0.05, ♯♯p < 0.001, ♯♯♯♯p < 0.0001). CT: drinking water; SB: 0.5% sodium butyrate in drinking water for 2 weeks; SB + DSS: 0.5% sodium butyrate in drinking water 1 week before and the next week together with 2.5% DSS in drinking water; 2SB + DSS: 0.5% sodium butyrate in drinking water 2 weeks before 2.5% DSS in drinking water; DSS: 1 week 2.5% DSS in drinking water.

FIGURE 2

Sodium butyrate administration restored zonula occludens-1 (ZO-1) production in the colon of DSS-treated mice. Effect of butyrate and DSS treatment on production of tight-junction protein ZO-1 was evaluated in cryosections of colonic mucosa of experimental mice. (A) Immunohistochemical evaluation was detected using monoclonal antibody ZO-1/CY3 and representative images are shown. (B) Representative out of three separated Western blotting assays of ZO-1 protein in tissue of colon is shown. Expression of β-actin was used as an internal control. (C) Quantification of the signals was performed using ImageJ, and data are expressed as the mean ± SD from two separated measurements (2 mice per each group), Student’s paired two-tailed t-test was used for comparison of experimental groups to CT controls (group 1: *p < 0.05) or to DSS-controls (group 5: ♯p < 0.05). CT: drinking water for 1 week; SB: 0.5% sodium butyrate in drinking water for 2 weeks; SB + DSS: 0.5% sodium butyrate in drinking water 1 week before and next week together with 2.5% DSS in drinking water; 2SB + DSS: 0.5% sodium butyrate in drinking water 2 weeks before and the next week together with 2.5% DSS in drinking water; DSS: 1 week 2.5% DSS in drinking water.

FIGURE 3

Sodium butyrate administration influence the level of DSS-induced cytokine response in plasma and liver of experimental mice. (A) IFN-γ, (B) IL-6, and (C) IL-10 cytokine levels were measured by ELISA in plasma of experimental mice and are expressed as pg/ml. (D) Quantitative PCR analysis of mRNA expression of IL-1β was performed in the liver of mice. Values are expressed as mean ± SD (n = 5) and represent one out of two experiments. One-way ANOVA with Tukey’s multiple post hoc test was used for comparison of experimental groups to CT controls (group 1: *p < 0.05). CT: drinking water for 1 week; SB: 0.5% sodium butyrate in drinking water for 2 weeks; SB + DSS: 0.5% sodium butyrate in drinking water 1 week before and the next week together with 2.5% DSS in drinking water; 2SB + DSS: 0.5% sodium butyrate in drinking water 2 weeks before 2.5% DSS treatment in drinking water; DSS: 1 week 2.5% DSS in drinking water.

FIGURE 4

Quantitative PCR analysis of selected hepatic cytochromes P450 (A) Cyp1a1, (B) Cyp1a2, (C) Cyp2b10, (D) Cyp2c38, (E) Cyp3a11, and (F) Cyp3a13 mRNA expressions in the liver of mice. Values are expressed as mean ± SD (n = 5). One-way ANOVA with Tukey’s multiple post hoc test was used for comparison of experimental groups to controls; * significance to CT (*p < 0.05, **p < 0.01, ***p < 0.001) and # significance to DSS (#p < 0.05). CT: drinking water for 1 week; SB: 0.5% sodium butyrate in drinking water for 2 weeks; SB + DSS: 0.5% sodium butyrate in drinking water 1 week before, and the next week together with 2.5% DSS in drinking water; 2SB + DSS: 0.5% sodium butyrate in drinking water 2 weeks before, and the next week together with 2.5% DSS in drinking water; DSS: 1 week 2.5% DSS in drinking water.

FIGURE 5

Enzyme activity of selected hepatic cytochromes P450. Enzyme activities of cytochromes P450 (A) CYP1A1/2, (B) CYP2B, (C) CYP2C, and (D) CYP3A were measured in the mouse hepatic microsomal fractions using the HPLC system with UV or fluorescence detection. Data represent the mean ± SD from three separated assays measured in pooled microsomal samples of five mice from each group. One-way ANOVA with Tukey’s multiple post hoc test was used for comparison of experimental groups to controls; * significance to CT (*p < .05), # significance to DSS (#p < .05), and ∆ significance of group 3 to group 4 (∆p < .05). CT: drinking water for 1 week; SB: 0.5% sodium butyrate in drinking water for 2 weeks; SB + DSS: 0.5% sodium butyrate in drinking water 1 week before and the next week together with 2.5% DSS in drinking water; 2SB + DSS: 0.5% sodium butyrate in drinking water 2 weeks before and the next week together with 2.5% DSS in drinking water; DSS: 1 week 2.5% DSS in drinking water.