The Combination of Berberine, Tocotrienols and Coffee Extracts Improves Metabolic Profile and Liver Steatosis by the Modulation of Gut Microbiota and Hepatic miR-122 and miR-34a Expression in Mice

Abstract

Non-alcoholic-fatty liver disease (NAFLD) is spreading worldwide. Specific drugs for NAFLD are not yet available, even if some plant extracts show beneficial properties. We evaluated the effects of a combination, composed by Berberis Aristata, Elaeis Guineensis and Coffea Canephora, on the development of obesity, hepatic steatosis, insulin-resistance and on the modulation of hepatic microRNAs (miRNA) levels and microbiota composition in a mouse model of liver damage. C57BL/6 mice were fed with standard diet (SD, n = 8), high fat diet (HFD, n = 8) or HFD plus plant extracts (HFD+E, n = 8) for 24 weeks. Liver expression of miR-122 and miR-34a was evaluated by quantitativePCR. Microbiome analysis was performed on cecal content by 16S rRNA sequencing. HFD+E-mice showed lower body weight (p < 0.01), amelioration of insulin-sensitivity (p = 0.021), total cholesterol (p = 0.014), low-density-lipoprotein-cholesterol (p < 0.001), alanine-aminotransferase (p = 0.038) and hepatic steatosis compared to HFD-mice. While a decrease of hepatic miR-122 and increase of miR-34a were observed in HFD-mice compared to SD-mice, both these miRNAs had similar levels to SD-mice in HFD+E-mice. Moreover, a different microbial composition was found between SD- and HFD-mice, with a partial rescue of dysbiosis in HFD+E-mice. This combination of plant extracts had a beneficial effect on HFD-induced NAFLD by the modulation of miR-122, miR-34a and gut microbiome.

Keywords: NAFLD; gut microbiome; insulin resistance; metabolic syndrome; miR-122; miR-34a.

Figure 1

Effect of plant extracts on weight gain. Body weight was monitored every week (a), food intake every 4 weeks (b) during the diet protocol. SD: standard diet (black circles); HFD: high fat diet (white circles); HFD+E: high fat diet plus plant extracts (crosses); n = 8 per group. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 HFD vs. SD, # p < 0.05, ## p < 0.01, ### p < 0.001 HFD+E vs. SD, $ p < 0.05, $$ p < 0.01 HFD vs. HFD+E.

Figure 2

Effect of plant extracts on insulin sensitivity in HFD-fed mice. At 24 weeks of diet protocol, mice were subjected to insulin tolerance test. (a) Curves show the glycemic trend over 120 min following insulin bolus in SD (circles), HFD (squares) and HFD+E (triangles)-fed C57BL/6. (b) Bars show the inverse calculation of the area under the curves (iAUC) of the three groups of mice. Values are expressed as means ± SD of determinations in 8 mice per group. Statistical significance was evaluated using one-way ANOVA.

Figure 3

Effect of plant extracts on the development of hepatic steatosis. Representative images of Hematoxylin-Eosin staining of liver sections (10× magnification) from SD (A), HFD (B), and HFD+E mice (C).

Figure 4

HFD and plant extracts effect on hepatic miRNAs expression. The expression of miR-122 (a) and miR-34a (b) was evaluated in hepatic tissue from SD, HFD and HFD+E-fed C57BL/6 mice by qRT-PCR. Data are shown as expression units relative to U6 snRNA levels and reported in box plots, where center lines show the medians, “+” symbols the means, box limits the 25th and 75th percentiles and whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Statistical significance was evaluated using one-way ANOVA.

Figure 5

Correlation of hepatic miRNAs expression and metabolic parameters. Correlation of miR-122 (a,b) and miR-34a (c,d) expression with total cholesterol (a,c) and LDL-cholesterol (b,d) was assessed by linear regression analysis and calculated using Pearson’s correlation coefficient. Correlations with a p value < 0.05 was considered statistically significant.

Figure 6

Bacterial communities profiling between SD, HFD and HFD+E treated mice. Alpha diversity was measured to assess any difference in the structure of bacterial communities among the study groups. Several metrics have been used to test the within-group diversity, Shannon index showing a significant difference (p = 0.003, ANOVA) between the HFD+E group and the others (A). To verify differences in the microbial composition between the three study groups, beta diversity has been also evaluated by Bray–Curtis metric. Statistical significance (p < 0.001) of grouping was assessed by ANOSIM (B). Taxonomic assignment highlights a different microbial composition between the three groups. Phyla variation into three groups (C).

Figure 7

Microbial communities cluster analyses. Heatmap of variance was obtained by grouping the reads according to the observed taxa to evaluate abundance patterns. A clear cluster was obtained between the three groups at phylum level (A). This separation is observed also in the dendrogram analyses (B).

Figure 8

Significantly different taxa identified at phylum level between SD, HFD and HFD+E treated mice. Differential abundance analyses allowed to identify significantly different taxa between the three study groups. t-Test/ANOVA were used to assess significant results (adjusted p-value < 0.05, specific p-values are detailed in Supplementary Table S2). At phylum level, Actinobacteria (A) and Firmicutes (B) phyla are highly represented in the HFD mice respect to the other two groups. On the contrary, the Bacteroidetes phylum (C) is less abundant in the HFD than in the SD and in the HFD+E mice. Interestingly, the Deferribacteres (D) and the Verrucomicrobia (E) phyla are respectively high and low expressed in the HFD+E mice respect to both SD and HFD.

Figure 9

Significantly different taxa identified at genus level between SD, HFD and HFD+E treated mice. Significantly different taxa have been identified using differential abundance analyses coupled with t-test/ANOVA (adjusted p-value < 0.05). At genus level, the Bacteroides (A) was highly represented in the HFD+E treated mice than in the other two groups, as well as Parabacteroides (C), Anaerotruncus (D) and Mucispirillum (E) genera. On the other side, the genera Desulfovibrio (B) and Olsenella (F) were more abundant in the HFD than in the other groups.