Resveratrol Attenuates Trimethylamine-N-Oxide (TMAO)-Induced Atherosclerosis by Regulating TMAO Synthesis and Bile Acid Metabolism via Remodeling of the Gut Microbiota

Abstract

The gut microbiota is found to be strongly associated with atherosclerosis (AS). Resveratrol (RSV) is a natural phytoalexin with anti-AS effects; however, its mechanisms of action remain unclear. Therefore, we sought to determine whether the anti-AS effects of RSV were related to changes in the gut microbiota. We found that RSV attenuated trimethylamine-N-oxide (TMAO)-induced AS in ApoE(-/-) mice. Meanwhile, RSV decreased TMAO levels by inhibiting commensal microbial trimethylamine (TMA) production via gut microbiota remodeling in mice. Moreover, RSV increased levels of the genera Lactobacillus and Bifidobacterium, which increased the bile salt hydrolase activity, thereby enhancing bile acid (BA) deconjugation and fecal excretion in C57BL/6J and ApoE(-/-) mice. This was associated with a decrease in ileal BA content, repression of the enterohepatic farnesoid X receptor (FXR)-fibroblast growth factor 15 (FGF15) axis, and increased cholesterol 7a-hydroxylase (CYP7A1) expression and hepatic BA neosynthesis. An FXR antagonist had the same effect on FGF15 and CYP7A1 expression as RSV, while an FXR agonist abolished RSV-induced alterations in FGF15 and CYP7A1 expression. In mice treated with antibiotics, RSV neither decreased TMAO levels nor increased hepatic BA synthesis. Additionally, RSV-induced inhibition of TMAO-caused AS was also markedly abolished by antibiotics. In conclusion, RSV attenuated TMAO-induced AS by decreasing TMAO levels and increasing hepatic BA neosynthesis via gut microbiota remodeling, and the BA neosynthesis was partially mediated through the enterohepatic FXR-FGF15 axis.

Importance: Recently, trimethylamine-N-oxide (TMAO) has been identified as a novel and independent risk factor for promoting atherosclerosis (AS) partially through inhibiting hepatic bile acid (BA) synthesis. The gut microbiota plays a key role in the pathophysiology of TMAO-induced AS. Resveratrol (RSV) is a natural phytoalexin with prebiotic benefits. A growing body of evidence supports the hypothesis that phenolic phytochemicals with poor bioavailability are possibly acting primarily through remodeling of the gut microbiota. The current study showed that RSV attenuated TMAO-induced AS by decreasing TMAO levels and increasing hepatic BA neosynthesis via gut microbiota remodeling. And RSV-induced hepatic BA neosynthesis was partially mediated through downregulating the enterohepatic farnesoid X receptor-fibroblast growth factor 15 axis. These results offer new insights into the mechanisms responsible for RSV's anti-AS effects and indicate that the gut microbiota may become an interesting target for pharmacological or dietary interventions to decrease the risk of developing cardiovascular diseases.

FIG 1

RSV inhibited TMAO synthesis in C57BL/6J mice. (A and B) Eight-week-old female C57BL/6J mice were administered choline (400 mg/kg of body weight, n = 10) (A) or TMA (40 mg/kg, n = 10) (B). Blood samples were collected at the indicated times. Serum TMA and TMAO levels were determined by LC/MS. (C and D) Eight-week-old female C57BL/6J mice were fed a chow diet with or without RSV (0.4%) in the presence or absence of choline (1%) or Abs. After 30 days, several mice of the chow-and-RSV-fed group were administered choline (400 mg/kg, n = 10) (C) or TMA (40 mg/kg, n = 10) (D). At 4 h after choline was given, or 1 h after TMA was given, the mice were euthanized and blood was collected. Serum TMA and TMAO levels were determined by LC/MS. (E and F) The other mice were also euthanized, and blood was collected. Serum TMA and TMAO levels (E) and RSV levels (F) were determined by LC/MS. Values are presented as means ± SD (n = 10). a, P < 0.05; b, P < 0.01 (versus vehicle-treated control group); d, P < 0.01 (versus choline-treated group); #, P < 0.05 (versus group cotreated with choline and RSV).

FIG 2

RSV inhibited TMAO synthesis by decreasing TMA generation via remodeling microbiota in C57BL/6J mice. Eight-week-old female C57BL/6J mice (n = 5 per group) were fed chow with or without RSV (0.4%) for 30 days. ACTB, β-actin. (A) Western blotting detection of FMO3 in the liver. (B) Expression levels of FMO3 gene mRNAs were quantified using qPCR assays. (C) Liver FMO activity was assessed as described in Materials and Methods. (D) 16S rRNA gene sequencing analysis of cecal content at the phylum level. (E) Heat map of 16S rRNA gene sequencing analysis of cecal content at the genus level. The scale reflects the data as follows: red indicates high values whereas blue indicates low values for the percentages of reads that were classified at that rank. (F) Linear discriminant analysis (LDA) coupled with effect size measurements identifies the taxons most differentially abundant between the chow and RSV diets at the genus level. RSV-diet-enriched taxa are indicated with a positive LDA score (green), and taxa enriched in the normal chow diet have a negative score (red). Only taxa meeting an LDA significant threshold value of >2 are shown. (G) Correlation heat map demonstrating the association between the indicated microbiota taxonomic genera and TMA and TMAO concentrations (all reported as means ± SD in micromoles) from mice grouped by dietary status (chow and RSV). Red denotes a positive association, blue a negative association, and white no association. A single asterisk indicates a significant FDR-adjusted association at P values of ≤0.05, and a double asterisk indicates a significant FDR-adjusted association at P values of ≤0.01. (H) The production of TMA from choline by cecal content in vitro. (I) Cecal RSV content. (J) The same 8-week-old female C57BL/6J mice (n = 5 per group) were administered choline (400 mg/kg) at visit 1 (baseline), visit 2 (RSV treatment for 1 month), and visit 3 (washout for 1 month). Four hours after choline was given at each visit, blood samples were obtained from the tail veins. Serum TMA and TMAO levels were determined by LC/MS. Values are presented as means ± SD (n = 5). a, P < 0.05; b, P < 0.01 (versus vehicle-treated control group); &, P < 0.01 (versus RSV-treated group).

FIG 3

RSV enhanced BA deconjugation and fecal excretion in C57BL/6J mice. Eight-week-old female C57BL/6J mice (n = 10 per group) were fed chow with or without RSV (0.4%) in the presence or absence of Abs for 30 days. Fecal samples were collected. (A) Fecal BA excretion was measured by a total BA test (Wako). Fecal BA composition was determined by LC/MS as described in Materials and Methods. (B) CA/DCA ratio. (C) Conjugated/unconjugated (conj/unconj) BA ratio in fecal samples. (D) BSH activity. Values are expressed as means ± SD (n = 10). b, P < 0.01 (versus vehicle-treated control group); &, P < 0.01 (versus RSV-treated group).

FIG 4

RSV induced hepatic BA synthesis in C57BL/6J mice. Eight-week-old female C57BL/6J mice (n = 10 per group) were fed chow with or without RSV (0.4%) in the presence or absence of Abs for 30 days. (A) Liver cholesterol content. (B) The BA pool size was determined for the total BA content of gallbladder bile, liver, and the SI lumenal. (C to F) Total biliary BA content (C) and total BA content in SI lumenal (D), serum (E), and liver (F) were detected. (G) Gallbladder bile samples were subjected to LC/MS, and TCA/TβMCA ratios were calculated. (H) Relative expression levels of the indicated mRNAs in the liver were determined by qPCR. (I) Expression of CYP7A1 was analyzed by Western blotting. Values are expressed as means ± SD (n = 10). a, P < 0.05; b, P < 0.01 (versus vehicle-treated control group); &, P < 0.01 (versus RSV-treated group).

FIG 5

The enterohepatic FXR-FGF15 axis played a key role in RSV-induced BA synthesis. Eight-week-old female C57BL/6J mice (n = 10 per group) were fed chow with or without RSV (0.4%) for 30 days. Z-Gug (100 mg/kg body weight) or GW4064 (75 mg/kg body weight) was given 7 days prior to surgical procedures. (A) Relative expression levels of ileal FXR and FGF15 gene mRNAs. (B) Western blotting was used to detect ileal FXR and FGF15 expression. (C) Expression of the indicated proteins in ileal tissues. (D) Liver CYP7A1 expression was detected by Western blotting. (E) FGF15 expression in ileal tissues. (F) CYP7A1 expression in liver samples. Values are expressed as means ± SD (n = 10). b, P < 0.01 (versus vehicle-treated control group); &, P < 0.01 (versus RSV-treated group).

FIG 6

RSV protected ApoE^−/− mice from TMAO-induced AS. Eight-week-old female ApoE^−/− mice (n = 10 per group) were given RSV (0.4%) with or without choline (1%) in the absence or presence of Abs for 4 months. The control group was fed with a chow diet. (A) Ultrasound B-mode images of the aortic sinus and quantification. Arrows indicate the regions of interest. RV, right ventricle; RA, right atrium; LA, left atrium. (B) Oil red O-stained aortic roots (counterstained with hematoxylin) and quantification. (C) Oil red O staining of whole aortas, including the aortic arch, thoracic, and abdominal regions, and their quantitation. (D) Total cholesterol content in the thoracic and abdominal aorta. Values are expressed as means ± SD (n = 10). a, P < 0.05; b, P < 0.01 (versus vehicle-treated control group); c, P < 0.05; d, P < 0.01 (versus choline-treated group); #, P < 0.05 (versus group cotreated with RSV and choline).

FIG 7

RSV reduced TMAO levels by decreasing TMA generation via remodeling microbiota in ApoE^−/− mice. Eight-week-old female ApoE^−/− mice (n = 5 per group) were fed chow, chow with RSV (0.4%), chow with choline (1%), or chow with choline (1%) plus RSV (0.4%) in the absence or presence of Abs for 4 months. (A) Serum TMA and TMAO levels were measured by LC/MS. (B) Western blotting detection of FMO3 expression in the liver. (C) Expression levels of FMO3 gene mRNAs were quantified using qPCR assays. (D) Liver FMO activity was assessed as described in Materials and Methods. (E) 16S rRNA gene sequencing analysis of cecal content at the phylum level. (F) Heat map of 16S rRNA gene sequencing analysis of cecal content at the genus level. The scale reflects the data as follows: red indicates high values whereas blue indicates low values for the spercentage of reads that were classified at that rank. (G) Linear discriminant analysis (LDA) coupled with effect size measurements identifies the most differentially abundant taxons at the genus level between the choline diet and the RSV-plus-choline diet. RSV-plus-choline-diet-enriched taxa are indicated with a positive LDA score (green), and taxa enriched in the choline diet have a negative score (red). Only taxa meeting an LDA significance threshold of >2 are shown. (H) Correlation heat map demonstrating the association between the indicated microbiota taxonomic genera and TMA and TMAO concentrations (all reported as means ± SD in micromoles) from mice grouped by dietary status (choline and RSV plus choline). Red denotes a positive association, blue a negative association, and white no association. A single asterisk indicates a significant FDR-adjusted association of P values of ≤0.05, and a double asterisk indicates a significant FDR-adjusted association of P values of ≤0.01. (I) Production of TMA from choline by cecal content in vitro. Values are expressed as means ± SD (n = 5). a, P < 0.05; b, P < 0.01 (versus vehicle-treated control group); c, P < 0.05; d, P < 0.01 (versus choline-treated group); #, P < 0.05 (versus group cotreated with RSV and choline).

FIG 8

RSV induced hepatic BA synthesis in choline-treated ApoE^−/− mice. Eight-week-old female ApoE^−/− mice (n = 10 per group) were fed chow, chow with RSV (0.4%), chow with choline (1%), or chow with choline (1%) plus RSV (0.4%) in the absence or presence of Abs for 4 months. (A) Serum TC levels were measured enzymatically using commercially available kits. (B) Liver cholesterol content. (C) The BA pool size was determined for the total BA content of gallbladder bile, liver, and the SI lumenal. BW, body weight. (D to F) Total biliary BA content (D) and total BA content in SI lumenal (E) and liver (F) were detected. (G) Relative expression levels of the indicated mRNAs in the liver were determined by qPCR. (H) Expression of CYP7A1 was analyzed by Western blotting. Values are expressed as means ± SD (n = 10). a, P < 0.05; b, P < 0.01 (versus vehicle-treated control group); c, P < 0.05; d, P < 0.01 (versus choline-treated group); #, P < 0.05 (versus choline and RSV-cotreated group).

FIG 9

RSV downregulated the enterohepatic FXR-FGF15 axis in choline-treated ApoE^−/− mice. Eight-week-old female ApoE^−/− mice (n = 10 per group) were fed chow, chow with RSV (0.4%), chow with choline (1%), or chow with choline (1%) plus RSV (0.4%) in the absence or presence of Abs for 4 months. (A) Expression of CYP7A1 was analyzed by Western blotting. (B and C) Relative expression levels of the indicated mRNAs in the liver (B) and ileal tissue (C) were determined by qPCR. (D) Western blotting was used to detect ileal FXR and FGF15 expression. (E and F) BA content in SI tissue (E) and fecal samples (F). b, P < 0.01 (versus vehicle-treated control group); d, P < 0.01 (versus choline-treated group); #, P < 0.05 (versus group cotreated with choline and RSV).

FIG 10

A model depicting use of RSV to attenuate AS by targeting gut microbiota. Dietary RSV can alter the composition of gut flora. On the one hand, this can result in reduced levels of gut microbial TMA production, subsequently leading to decreased TMAO synthesis in the liver and, ultimately, to inhibition of AS. On the other hand, RSV increases BSH activity by gut microbiota remodeling, which promotes the generation of unconjugated BAs from conjugated BAs and enhances fecal BA loss. RSV-induced fecal BA loss leads to a decrease in ileal BA content, thereby inhibiting the ileal FXR-FGF15 axis, and then increases the expression levels of CYP7A1 in the liver, subsequently inducing hepatic BA neosynthesis which contributes to cholesterol homeostasis, finally attenuating AS. Red indicates the effect of RSV, purple indicates downregulation by RSV, green indicates upregulation by RSV, and blue indicates the gut flora. LCA, lithocholic acid.