Theabrownin from Pu-erh tea attenuates hypercholesterolemia via modulation of gut microbiota and bile acid metabolism

Abstract

Pu-erh tea displays cholesterol-lowering properties, but the underlying mechanism has not been elucidated. Theabrownin is one of the most active and abundant pigments in Pu-erh tea. Here, we show that theabrownin alters the gut microbiota in mice and humans, predominantly suppressing microbes associated with bile-salt hydrolase (BSH) activity. Theabrownin increases the levels of ileal conjugated bile acids (BAs) which, in turn, inhibit the intestinal FXR-FGF15 signaling pathway, resulting in increased hepatic production and fecal excretion of BAs, reduced hepatic cholesterol, and decreased lipogenesis. The inhibition of intestinal FXR-FGF15 signaling is accompanied by increased gene expression of enzymes in the alternative BA synthetic pathway, production of hepatic chenodeoxycholic acid, activation of hepatic FXR, and hepatic lipolysis. Our results shed light into the mechanisms behind the cholesterol- and lipid-lowering effects of Pu-erh tea, and suggest that decreased intestinal BSH microbes and/or decreased FXR-FGF15 signaling may be potential anti-hypercholesterolemia and anti-hyperlipidemia therapies.

Fig. 1

Body weight and lipid lowering effects of Pu-erh tea. a Pu-erh tea reduced the body weight of mice with either normal diet or HFD for 26 weeks. n = 8 individuals/group. Data in ND and HFD groups were compared to ND + PTea and HFD + PTea groups, respectively. b The total energy intake of normal diet or HFD fed mice after 26 weeks of tea intervention. n = 8 individuals/group. c Pu-erh tea reduced serum lipids of mice with either normal diet or HFD for 26 weeks. n = 8 individuals/group. d Pu-erh tea reduced hepatic TG and TC contents of mice with either normal diet or HFD. n = 8 individuals/group. e Pu-erh tea reduced serum lipids of human subjects after 4 weeks of consumption. n = 13 individuals/group. Data were expressed as mean ± SEM. Differences of data in mice and human subjects were assessed by the Mann–Whitney U test and Wilcoxon rank-sum test, respectively, *p < 0.05, ^#p < 0.005

Fig. 2

Pu-erh tea reshaped the gut microbiota in mice and human. a Principle coordinate analysis (PCoA) plot based on the OTU matrix of mouse ileal microbiota in ND, ND + PTea, HFD and HT + PTea groups. n = 5 individuals/group. b PCoA plot based on the OTU matrix of human fecal microbiota. n = 13 individuals/group. c Heatmap of the relative abundance of representative OTUs at the genus and species levels in mice. Part of the OTUs which were simultaneously reduced by Pu-erh tea in both normal diet and HFD are shown, which can be referenced to the OTUs in blue outline of the full figure (Supplemantary Fig. 6a). n = 5 individuals/group. d Heatmap of the relative abundance of representative OTUs at the genus and species levels in humans. n = 13 individuals/group. Part of the OTUs which were reduced by Pu-erh tea similar to mice are shown, which can be referenced to the OTUs in blue outline of the full figure (Supplemantary Fig. 6b). The color of each spot in the heatmap corresponds to the normalized and log-transformed raw abundance of the OTUs in each sample. The OTUs were organized according to their order in the phylogenetic tree generated by their representative sequences

Fig. 3

Pu-erh tea reduced the abundance of BSH microbes and BSH activity. a Abundance of BSH enriched microbes in ileum of mice undergoing Pu-erh tea consumption. n = 5 individuals/group. b Abundance of BSH enriched microbes in feces of humans consuming Pu-erh tea. n = 13 individuals/group. c The microbial BSH enzyme activity in the ileal contents of mice treated with Pu-erh tea. d The microbial BSH activity in human feces from subjects on Pu-erh tea consumption. The functional profiles were identified by metagenomic analysis utilizing both KEGG and eggNOG databases. BSH related proteins ranked in the top ten are shown and the mean abundances are illustrated as the horizontal line in each group. e Ileal BSH activity in mice on Pu-erh tea consumption. n = 8 individuals/group. f Fecal BSH activity inhuman subjects on Pu-erh tea consumption. n = 13 individuals/group. Data were expressed as mean ± SEM. Differences in the data for mice and human subjects were assessed by the Mann–Whitney U test and Wilcoxon rank-sum test, respectively, *p < 0.05, ^#p < 0.005

Fig. 4

Pu-erh tea elevated conjugated BAs in serum and ileum. a Serum BA classes and BA profile of mice after 450 mg/Kg/day Pu-erh tea consumption for 26 weeks. n = 8 individuals/group. b Serum BA classes and BA profile of human subjects after 50 mg/Kg/day Pu-erh tea consumption for 4 weeks. n = 13 individuals/group. c Ileal BA classes and BA profile of mice after 450 mg/Kg/day Pu-erh tea consumption for 26 weeks. n = 8 individuals/group. Data were expressed as mean ± SEM. Differences of data for mice and human subjects were assessed using the Mann–Whitney U test and Wilcoxon rank-sum test, respectively, *p < 0.05, ^#p < 0.005

Fig. 5

Theabrownin directly reduced BSH activity to elevate ileal conjugated BAs. a Spearman correlation between Pu-erh tea constituents and OTUs of BSH-producing microbes in mice fed normal diet and HFD supplied with 450 mg/Kg/day Pu-erh tea for 26 weeks. n = 8 individuals/group. The color of each spot in the heatmap corresponds to the R value of the spearman correlation analysis between microbial abundance and tea components concentration, and the spot with asterisk in magenta color refers to the significant positive correlation with R > 0.3 and P < 0.05 while the spot with asterisk in green color refers to the significant negative correlation with R < −0.3 and P < 0.05. b Pu-erh tea and theabrownin reduced serum and hepatic TC and TG of mice. n = 8 individuals/group. c Ileal BAs changes in Pu-erh tea and theabrownin intervention in vivo. n = 8 individuals/group. d BSH-producing microbe changes induced by Pu-erh tea and theabrownin in vivo. n = 8 individuals/group. e Ileal BSH activity changes induced by Pu-erh tea and theabrownin in vivo. n = 8 individuals/group. f Theabrownin and Pu-erh tea reduced BSH activity in cultured ileal microbes. n = 8 individuals/group. Data were expressed as mean ± SEM. Differences between data were assessed using the Mann–Whitney U test, *p < 0.05, ^#p < 0.005

Fig. 6

Conjugated BAs inhibited the FXR-FGF15 to induce hepatic BA synthesis. a Gene expression of FXR and FGF15 in ileum of mice fed HFD and 450 mg/Kg/day Pu-erh tea for 26 weeks. n = 8 individuals/group. b FXR and FGF15 protein expression level in ileum of mice fed HFD and 450 mg/Kg/day Pu-erh tea for 26 weeks. n = 3 individuals/group. c Gene expression of FXR and FGF15 in ileum of mice fed HFD and 225 mg/Kg/day theabrownin for 8 weeks. n = 8 individuals/group. d Serum FGF15 levels of mice fed HFD and 450 mg/Kg/day Pu-erh tea for 26 weeks. n = 8 individuals/group. e Serum FGF19 levels of human subjects that received standard diet and 50 mg/Kg/day Pu-erh tea for 4 weeks. n = 13 individuals/group. f Hepatic mRNA expression levels of BA synthetic enzymes of mice fed HFD and 450 mg/Kg/day Pu-erh tea for 26 weeks. n = 8 individuals/group. g Hepatic mRNA expression levels of BA synthetic enzymes of mice fed HFD and 225 mg/Kg/day theabrownin for 8 weeks. n = 8 individuals/group. h Hepatic protein expression levels of BA synthetic enzymes of mice fed HFD and 450 mg/Kg/day Pu-erh tea for 26 weeks. n = 3 individuals/group. i IHC staining of hepatic BA synthetic proteins of mice fed HFD and 450 mg/Kg/day Pu-erh tea for 26 weeks (scale bar, 50 μm). The mRNA expression and protein expression were normalized to GAPDH and β-Actin, respectively. Data were expressed as mean ± SEM. Differences between data in mice and human were assessed by Mann–Whitney U test and Wilcoxon rank-sum test, respectively, *p < 0.05, ^#p < 0.005

Fig. 7

FXR-FGF15 and FXR-SHP regulated BA synthesis in alternative pathway. a Dominant BAs in liver and ileum of HFD fed mice gavaged with 225 mg/Kg/day theabrownin and 50 mg/Kg/day TCA, TCDCA, TUDCA respectively for 8 weeks. n = 8 individuals/group. b mRNA expression of ileal FXR, FGF15 and hepatic FXR, SHP and BA synthetic genes in HFD fed mice gavaged with 225 mg/Kg/day theabrownin and 50 mg/Kg/day TCA, TCDCA, TUDCA respectively for 8 weeks. n = 8 individuals/group. c Immunofluorescence staining of ileal FXR, FGF15 and hepatic FXR, SHP of HFD fed mice gavaged with 225 mg/Kg/day theabrownin and 50 mg/Kg/day TCA, TCDCA, TUDCA respectively for 8 weeks (scale bar, 50 μm). d Fifty micromole per liter of TCDCA and TUDCA inhibited protein expression of nuclear FXR in the human FHs 74 Int and L02 cell lines, and 50 μM of CDCA and TCA activated the expression of nuclear FXR in human FHs 74 Int and L02 cell lines and promoted expression of CYP27A1 and CYP7B1 in alternative pathway of bile acids synthesis. n = 3 individuals/group. e Immunofluorescence staining of FXR, FGF19 in FHs 74 Int cells supplied with 50 μM CDCA, CDCA coupled with 50 μM of TCDCA, CDCA coupled with 50 μM of TUDCA, and 50 μM of TCA for 24 h (scale bar, 100 μm). f Immunofluorescence staining of FXR, SHP in L02 cell lines supplied with 50 μM of CDCA, CDCA coupled with TCDCA, CDCA coupled with TUDCA and TCA for 24 h (scale bar, 50 μm). g The mRNA expression of ileal FXR, FGF15 and hepatic BA synthetic genes in HFD fed mice gavaged with 225 mg/Kg/day theabrownin, theabrownin coupled with 100 mg/Kg/day fexaramine for 8 weeks. n = 8 individuals/group. h The mRNA expression of ileal FXR, FGF15 and hepatic BA synthetic genes in HFD fed mice gavaged with 225 mg/Kg/day theabrownin, theabrownin coupled with 50 μg/Kg/day recombinant FGF19 protein by intraperitoneal injection for 8 weeks. n = 8 individuals/group. The mRNA expression was normalized to GAPDH. Protein expression was normalized by β-Actin or Lamin B1. Data were expressed as mean ± SEM. Differences between data were assessed by the Mann–Whitney U test, *p < 0.05, ^#p < 0.005

Fig. 8

Proposed mechanism for theabrownin reduce cholesterol level. a Theabrownin promoted alternative bile acids synthetic pathway by simultaneous inhibition of intestinal FXR-FGF15 signaling with TCDCA or TUDCA and activation of hepatic FXR-SHP signaling with CDCA.Intestinal FXR-FGF15 signaling inhibits hepatic bile acid synthesis genes CYP7A1, CYP8B1, CYP27A1 and CYP7B1 with no selectivity, whereas hepatic FXR-SHP signaling selectively inhibits CYP8B1 in the classic pathway. Theabrownin in Pu-erh tea induced TCDCA and TUDCA in distal ileum to inhibit FGF15 production and thus, activated bile acid synthesis enzymes. Additionally, increased CDCA production in the liver promoted nuclear FXR expression as well as SHP to inhibit CYP8B1 expression. Ultimately, the combined regulation of decreased intestinal FXR-FGF15 and increased hepatic FXR-SHP on bile acid synthesis enzymes resulted in increased expression of CYP7B1 in alternative pathway and decreased expression of CYP8B1 in classic pathway leading to increased production of CDCA rather than CA. b Proposed mechanism for theabrownin induced attenuation of hepatic cholesterol levels via modulation of gut microbiome-mediated bile acid metabolism. Primary BAs, mainly CA and CDCA, were produced in the liver from cholesterol and conjugated to glycine or taurine to form conjugated BAs and then secreted to the intestinal. BSH enzymes are produced in intestinal microbes and function to hydrolyze conjugated BAs into unconjugated BAs. BSH microbes were suppressed by theabrownin in Pu-erh tea resulting in the accumulation of conjugated BAs in distal ileum. Conjugated BAs inhibited intestinal FXR-FGF15 signaling which subsequently alleviated the suppression of BA synthesis gene expression by this signaling pathway, resulting in increased BA production in alternative synthetic pathway and fecal BA excretion and ultimately, decreased cholesterol levels