Gut microbiota and diet matrix modulate the effects of the flavonoid quercetin on atherosclerosis

Abstract

Gut bacterial metabolism of dietary flavonoids results in the production of a variety of phenolic acids, whose contributions to health remain poorly understood. Here, we show that supplementation with the commonly consumed flavonoid quercetin impacted gut microbiome composition and resulted in a significant reduction in atherosclerosis burden in conventionally-raised (ConvR) Apolipoprotein E (ApoE) knockout (KO) mice fed a high-MAC (microbiota-accessible carbohydrates) diet. However, this effect was not observed in animals consuming a defined diet containing low levels of MAC. Furthermore, we found that the effect of quercetin on atherosclerosis required gut microbes, as supplementation of this flavonoid to germ-free (GF) ApoE KO mice consuming the high-MAC diet did not affect the development of atherosclerosis. Metabolomic analysis revealed that consumption of quercetin significantly increased plasma levels of benzoylglutamic acid and protocatechuic acid in ConvR mice exposed to the high-MAC diet, while these increases were not observed in GF mice or conventional animals consuming the low-MAC diet supplemented with the flavonoid. Furthermore, levels of these metabolites were negatively associated with atherosclerosis burden. Altogether, these results suggest that the beneficial effects of quercetin on atherosclerosis are influenced by gut microbes and dietary MAC.

Figure 1.. Dietary quercetin does not affect atherosclerosis development in mice fed a low-MAC diet.

A) Experimental design. B-E) Representative sections and quantitative analyses of Oil Red O staining (B,C; n=11 in the ConvR/low-MAC group and n=10 in the ConvR/low-MAC+Q group), MOMA-2 staining (B,D; n=11 in the ConvR/low-MAC group and n=10 in the ConvR/low-MAC+Q group), and Masson’s trichrome staining (B,E; n=11 in the ConvR/low-MAC group and n=10 in the ConvR/low-MAC+Q group) in the aortic sinus. The data were expressed as box-and-whisker plots, where the boxes indicate the median values and the interquartile ranges, and the whiskers represent the minimum and maximum values. F) Plasma lipid profiles (n=7 in the ConvR/low-MAC group and n=7 in the ConvR/low-MAC+Q group). Unpaired two-tailed Student’s t-test were performed. MAC; microbiota-accessible carbohydrates, ApoE; Apolipoprotein E, ConvR; conventionally-raised, Q; quercetin, MOMA; monocytes and macrophages.

Figure 2.. The gut microbiota mediates the athero-protective effect of quercetin in mice fed a high-MAC diet.

A) Experimental design. B) Plasma lipid profiles (n=17 in the ConvR/high-MAC group, n=12 in the ConvR/high-MAC+Q group, n=11 in the GF/high-MAC group, and n=8 in the GF/high-MAC+Q group). C-F) Representative sections and quantitative analyses of Oil Red O staining (C,D; n=17 in the ConvR/high-MAC group, n=12 in the ConvR/high-MAC+Q group, n=11 in the GF/high-MAC group, and n=8 in the GF/high-MAC+Q group), MOMA-2 staining (C,E; n=8 in the ConvR/high-MAC group, n=12 in the ConvR/high-MAC+Q group, n=11 in the GF/high-MAC group, and n=8 in the GF/high-MAC+Q group), and Masson’s trichrome staining (C,F; n=8 in the ConvR/high-MAC group, n=12 in the ConvR/high-MAC+Q group, n=11 in the GF/high-MAC group, and n=8 in the GF/high-MAC+Q group) in the aortic sinus. The data were expressed as box-and-whisker plots, where the boxes indicate the median values and the interquartile ranges and the whiskers represent the minimum and maximum values. Significance was calculated by two-way ANOVA with Bonferroni post-tests as follows: *, P value of <0.05; ***, P value of <0.001. MAC; microbiota-accessible carbohydrates, ApoE; Apolipoprotein E, ConvR; conventionally-raised, GF; germ-free, Q; quercetin, MONA; monocytes and macrophages.

Fig. 3. Supplementation of quercetin modulates gut microbiota composition in ConvR mice fed a high-MAC diet.

A) Alpha diversity of gut microbial communities assessed by Chao1 and the Shannon index (t-test; **, P value of <0.01; ***, P value of <0.001). B) Non-metric multidimensional scaling (NMDS) plot of weighted UniFrac analysis of relative sample ASV composition with the PERMANOVA test showing a significant influence of quercetin on microbial community composition. C) Cladogram generated from LEfSe analysis showing the relationship between taxa (the levels represent, from the inner to outer rings, phylum, class, order, family, and genus). D) Linear discriminant analysis (LDA) scores derived from LEfSe analysis, showing the biomarker taxa (LDA score [log 10] of >3 and a significance of P < 0.05 determined by the Wilcoxon signed-rank test). E) Bacterial families differentially represented in cecal contents from the high-MAC+Q mice compared to the control group (P value of <0.05, FDR-corrected). F) Correlation of bacterial families with atherosclerotic plaque area. Pearson’s rho and P values were calculated by Pearson correlation coefficient. n=17 in the ConvR/high-MAC group and n=12 in the ConvR/high-MAC+Q group. MAC; microbiota-accessible carbohydrates, ConvR; conventionally-raised, Q; quercetin, ASV; amplicon sequence variant.

Figure 4.. Plasma metabolites derived from quercetin are associated with athero-protective effects.

A) Partial Least Squares Discriminant Analysis (PLS-DA) plot based on the data derived from the targeted metabolomics of plasma in the ConvR mice and the GF mice. (n=8 in the ConvR/HPP high-MAC group, n=8 in the ConvR/high-MAC+Q group, n=7 in the GF/high-MAC group, n=7 in the GF/high-MAC+Q group, n=6 in the ConvR/low-MAC group, n=6 in the ConvR/low-MAC+Q group). B) Volcano plot of metabolites in the ConvR/high-MAC vs ConvR/high-MAC+Q group, with log-transformed adjusted P values and fold changes. Red circles; increased in the ConvR/high-MAC+Q group. Blue circles; increased in the ConvR/high-MAC group. C) The values for 3,4-dihydroxybenzoic acid and its sulfate, N-benzoylglutamic acid, and phenylacetic acid were expressed as box-and-whisker plots. Significance was calculated by two-way ANOVA (high-MAC) and unpaired two-tailed Student’s t-test (low-MAC) with the Benjamini-Hochberg correction as follows; *, P value of <0.05; **, P value of <0.01. D) Correlation of phenols with atherosclerotic plaque area. Pearson’s rho and P values were calculated by Pearson correlation coefficient. MAC; microbiota-accessible carbohydrates, ConvR; conventionally-raised, GF; germ-free, Q; quercetin.