Gut microbes 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 but not in germ-free (GF) ApoE KO mice. Metabolomic analysis revealed that consumption of quercetin significantly increased plasma levels of benzoylglutamic acid, 3,4 dihydroxybenzoic acid (3,4-DHBA) and its sulfate-conjugated form in ConvR mice, but not in GF mice supplemented with the flavonoid. Levels of these metabolites were negatively associated with atherosclerosis burden. Furthermore, we show that 3,4-DHBA prevented lipopolysaccharide (LPS)-induced decrease in transendothelial electrical resistance (TEER). These results suggest that the effects of quercetin on atherosclerosis are influenced by gut microbes and are potentially mediated by bacterial metabolites derived from the flavonoid.

Fig. 1. The beneficial effects of quercetin on atherosclerosis are microbiome-dependent.

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, MOMA monocytes and macrophages.

Fig. 2. 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 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 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, NMDS non-metric multidimensional scaling, LDA linear discriminant analysis.

Fig. 3. Plasma metabolites derived from quercetin are associated with athero-protective effects.

A Sparse partial least squares discriminant analysis (sPLS-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-DHBA and its sulfate, benzoylglutamic acid, and phenylacetic acid were expressed as box-and-whisker plots. Significance was calculated by two-way ANOVA with the Benjamini–Hochberg correction as follows: *P value of <0.05; **P value of <0.01; ***P value of <0.001. 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, 3,4-DHBA 3,4-dihydroxybenzoic acid.

Fig. 4. 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.

Fig. 5. In vitro effects of 3,4-dihydroxybenzoic acid (3,4-DHBA).

A 3,4-DHBA does not affect LPS-mediated activation of inflammation in macrophages. B The detrimental effect of LPS on endothelial cells monolayer integrity is ameliorated by 3,4-DHBA. Human aortic endothelial cells were grown to confluence on transwell inserts (12 well plates) and exposed to LPS (100 ng/ml) and LPS together with two different BA concentrations for 24 h. After that time, the endothelial monolayer integrity was evaluated using a voltohmmeter. Cells were equilibrated and transendothelial electrical resistance (TEER) was measured at room temperature. Data are presented as mean ± SEM (n = 3). LPS lipopolysaccharide, 3,4-DHBA 3,4-dihydroxybenzoic acid.