Oral Supplementation of Sodium Butyrate Attenuates the Progression of Non-Alcoholic Steatohepatitis - PubMed (original) (raw)
Oral Supplementation of Sodium Butyrate Attenuates the Progression of Non-Alcoholic Steatohepatitis
Anja Baumann et al. Nutrients. 2020.
Abstract
Sodium butyrate (SoB) supplementation has been suggested to attenuate the development of non-alcoholic fatty liver disease (NAFLD). Here, we determined the therapeutic potential of SoB on NAFLD progression and molecular mechanism involved. Eight-week old C57BL/6J mice were pair-fed a fat-, fructose- and cholesterol-rich diet (FFC) or control diet (C). After 8 weeks, some mice received 0.6g SoB/kg bw in their respective diets (C+SoB; FFC+SoB) or were maintained on C or FFC for the next 5 weeks of feeding. Liver damage, markers of glucose metabolism, inflammation, intestinal barrier function and melatonin metabolism were determined. FFC-fed mice progressed from simple steatosis to early non-alcoholic steatohepatitis, along with significantly higher TNFα and IL-6 protein levels in the liver and impaired glucose tolerance. In FFC+SoB-fed mice, disease was limited to steatosis associated with protection against the induction of Tlr4 mRNA and iNOS protein levels in livers. SoB supplementation had no effect on FFC-induced loss of tight junction proteins in the small intestine but was associated with protection against alterations in melatonin synthesis and receptor expression in the small intestine and livers of FFC-fed animals. Our results suggest that the oral supplementation of SoB may attenuate the progression of simple steatosis to steatohepatitis.
Keywords: inducible nitric oxide synthase; melatonin synthesis; non-alcoholic steatohepatitis; sodium butyrate; toll-like receptor 4.
Conflict of interest statement
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
Figures
Figure 1
Study design and treatment groups. After an adaption phase during which mice were adapted to consuming a liquid diet, animals were either fed a C or an FFC diet. After 8 weeks, feeding of C and FFC was either sustained or animals were fed the different diets enriched with 0.6 g SoB/kg bw for 5 weeks. In week 11, all animals underwent a GTT. C, control diet; FFC, fat-, fructose-, and cholesterol-rich diet; GTT, glucose tolerance test; NASH, non-alcoholic steatohepatitis; SoB, sodium butyrate.
Figure 2
Effect of supplementation of SoB on liver status in mice with FFC-induced NASH. (a) Representative photomicrographs of hematoxylin and eosin staining of liver sections (magnification 200× and 400×), (b) evaluation of liver damage using a non-alcoholic fatty liver disease activity score (NAS), number of (c) F4/80-positive cells and (d) neutrophils per microscopic field in the livers. Data are expressed as means ± SEM, n = 8. Unpaired Student´s _t_-test was used to compare C and FFC after 8 weeks of feeding, * p < 0.05 compared with mice fed a C diet for 8 weeks. Two-way ANOVA was used to compare C, FFC, C+SoB and FFC+SoB after 13 weeks of feeding. Data with different letters are significantly different, p < 0.05. C, control diet; DE, diet effect; DExSoBE, interaction between diet and SoB; FFC, fat-, fructose-, and cholesterol-rich diet; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; SoB, sodium butyrate; SoBE, sodium butyrate effect.
Figure 3
Effect of supplementation of SoB on glucose metabolism in mice with FFC-induced NASH. (a) Blood glucose levels during glucose tolerance test (GTT) and, (b) quantitative analysis of area under the curve of GTT (0-120 min). Data are expressed as means ± SEM, n = 8. Data with different letters are significantly different, p < 0.05. C, control diet; DE, diet effect; DExSoBE, interaction between diet and SoB; FFC, fat-, fructose-, and cholesterol-rich diet; NASH, non-alcoholic steatohepatitis; SoB, sodium butyrate; SoBE; sodium butyrate effect.
Figure 4
Effect of supplementation of SoB on lipid peroxidation in livers of mice with FFC-induced NASH. Representative photomicrographs of (a) inducible nitric oxide synthase (iNOS) and (b) 4-hydroxynonenal (4-HNE) protein adducts staining in paraffin embedded tissue (magnification 200×) as well as densitometric analysis of (c) iNOS and (d) 4-HNE protein adducts staining in liver tissue. Data are expressed as means ± SEM, n = 8. Data with different letters are significantly different, p < 0.05. C, control diet; DE, diet effect; DExSoBE, interaction between diet and SoB; FFC, fat-, fructose-, and cholesterol-rich diet; NASH, non-alcoholic steatohepatitis; SoB, sodium butyrate; SoBE; sodium butyrate effect.
Figure 5
Effect of supplementation of SoB on tight junction proteins in upper parts of the small intestine, bacterial endotoxin levels and on markers of the toll-like receptor 4 (Tlr4) signaling cascade of mice with FFC-induced NASH. Qualitative analysis of (a) occludin, (b) ZO-1 protein staining in proximal small intestine, (c) bacterial endotoxin concentration in portal plasma as well as expression of (d) Tlr4 and (e) myeloid differentiation primary response gene 88 (Myd88) mRNA in liver tissue. Data are expressed as means ± SEM, n = 6–8. Data with different letters are significantly different, p < 0.05. C, control diet; DE, diet effect; DExSoBE, interaction between diet and SoB; FFC, fat-, fructose-, and cholesterol-rich diet; NASH, non-alcoholic steatohepatitis; SoB, sodium butyrate; SoBE; sodium butyrate effect, ZO-1, zona occludens 1.
Figure 6
Effect of supplementation of SoB on enzymes involved in melatonin synthesis and expression of melatonin receptor in livers of mice with FFC-induced NASH. (a) Representative photomicrographs of HIOMT staining, (b) densitometric analysis of HIOMT protein concentration, (c) melatonin concentration in the upper part of the small intestine and (d) mRNA expression of Mtr1a in liver tissue. Data are expressed as means ± SEM, n = 8. Data with different letters are significantly different, p < 0.05. C, control diet; DE, diet effect; DExSoBE, interaction between diet and SoB; FFC, fat-, fructose-, and cholesterol-rich diet; HIOMT, hydroxyindole-O-methyltransferase; Mtr1a, melatonin receptor 1a; NASH, non-alcoholic steatohepatitis; SoB, sodium butyrate; SoBE, sodium butyrate effect.
Figure 7
Effect of SoB on melatonin and serotonin concentration, protein expression of AANAT and activity of histone deacetylases enzymes in everted small intestinal sacs. (a) Melatonin concentration in whole intestinal tissue specimen, (b) activity of HDAC enzymes, and (c) representative blot of acetylated histone complex H3 protein expression treated with SoB. (d) Phosphorylated AANAT in mucosa of intestinal tissue obtained of everted sacs of naïve mice challenged with 6 mM SoB, (e,g) melatonin concentration and (f,h) phosphorylated AANAT in whole intestinal tissue obtained of everted sacs treated with 6 mM NADPH and/or 6 mM SoB as well as 10 mM fructose and/or 6 mM SoB. (i) Serotonin concentration in whole intestinal tissue specimen treated with 10 mM fructose. Data are expressed as means ± SEM, n = 3–6. * p < 0.05. Data with different letters are significantly different, p < 0.05. Aanat, serotonin N-acetyltransferase; Ac-H3 (Lys9), acetylated histone complex H3; C, everted gut sacs incubated only in 1 × Krebs–Henseleit-bicarbonate-buffer; F, fructose; HDAC, histone deacetylases; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); SoB, sodium butyrate.
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