Fig 2: 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 3: 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

Fig 4: PDAC cells expressed low levels of bile acids receptors. Relative mRNA expression of (A) LIFR (B) LIF compared to MKN45 (C) GPBAR1 compared to U937 and (D) FXR compared to HEPG2. Each value is normalized to GAPDH and is expressed relative to those of positive controls, which are arbitrarily set to 1. Results are the mean ± SEM of three samples for group. Immunofluorescence analysis of (E) Gpbar1 and (F) Fxr in MIA PaCa-2 cells (Magnification 100x on left and 200x on right).

Fig 5: Natural and synthetic bile acids antagonize LIFR. The figure shows two-dimensional structure of (A) LCA (B) CDCA (C) BAR502. Natural and synthetic bile acids inhibition activity of LIFR/LIF binding accessed by a cell-free AlphaScreen assay, particularly in (D) LCA (E) CDCA and (F) BAR502 IC50 are shown. (G) STAT3 transactivation on HepG2 cells. The table (H) summarizes EC50 on FXR and GPBAR1 and IC50 on LIFR of Natural and synthetic bile acids.