Figure 1.
Theonellasterol from Theonella swinhoei.
(A) Chemical structure of theonellasterol isolated from Theonella swinhoei. (B) Superimposition of the different docking poses of theonellasterol in the rat FXR (theonellasterol and 1OSV [20] yellow), and human FXRs (theonellasterol and 3BEJ [15] orange; theonellasterol and 1OSH [17] pink; theonellasterol and 3DCT [18] purple; theonellasterol and 3RUU [19] light blue). (C) Superimposition of theonellasterol (yellow) with 6-ECDCA (sky blue), and Z (pink)/ E (light pink) gugglusterone in the binding pocket of FXR (1OSV). Amino acids interacting with theonellasterol (yellow) are depicted in green, amino acids interacting with 6-ECDCA and theonellasterol are depicted in sky blue, amino acids interacting with Z/E gugglusterone and theonellasterol are depicted light pink, amino acids interacting with 6-ECDCA and Z/E gugglusterone in red, and amino acids interacting with all molecules are depicted in blue.
Figure 2.
Theonellasterol is an FXR antagonist.
Luciferase reporter assay performed in HepG2 transiently transfected with pSG5-PXR, pSG5-RXR, pCMV-βgal, pCYP3A4promoter-TKLuc vectors and stimulated 18 h with (A) 10 µM of CDCA or theonellasterol and (B) 10 µM of CDCA alone or in combination with theonellasterol 50 µM. Data are the mean ± S.E. of three experiments. *P<0.05 versus cells left untreated. #P<0.05 versus CDCA. (C) CHiP assay of NCoR binding to the OSTα promoter. CDCA displaces NCoR from OSTα and this effect is reversed by theonellasterol. RT-PCR analysis of proteins immune-precipitated with a control IgG are shown as control. Data are the mean ± S.E. of three experiments. *P<0.05 versus anti IgG immunoprecipitates. #P<0.05 CDCA versus cells left untreated; P<0.05 thenollasterol versus CDCA alone.
Figure 3.
Theonellasterol reverses the effect of CDCA on the expression of canonical FXR target genes.
Relative mRNA expression of (A) OSTα, (B) BSEP, (C) SHP and (D) MRP4 in HepG2 cells treated with 10 µM CDCA alone or with the combination of CDCA plus theonellasterol 50 µM. (E) CHiP assay performed in HepG2 cells not stimulated or primed with CDCA, 10 µM, alone or in combination with theonellasterol, 50 µM. Theonellasterol antagonizes the recruitment of FXR on the MRP4 promoter. Data are the mean ± S.E. of three experiments. *P<0.05 versus cells left untreated. #P<0.05 versus CDCA alone.
Figure 4.
Theonellasterol is a selective FXR antagonist.
(A) (C) and (D) HepG2 cells were co-transfected with the Gal4 luciferase reporter and a series of chimeras in which the Gal4 DNA binding domain is fused to the LBD of the indicated nuclear receptors. Cells were treated with the appropriate agonists or specific agonists in combination with theonellasterol. (B) HepG2 cells were co-transfected with pSG5-PXR, pSG5-RXR and with the reporter pCYP3A4promoter-TKLuc and then stimulated with rifaximin, a PXR agonist, alone or in combination with theonellasterol. Data are the mean ± S.E. of three experiments. *P<0.05 versus not treated cells. (E) Microarray analysis showing the relative mRNA expression of various nuclear receptors and nuclear receptors co-activators following stimulation of HepG2 cells with CDCA, 10 µM, alone or in combination with theonellasterol, 50 µM. Data are the mean ± S.E. of three experiments.
Figure 5.
FXR agonism and antagonism lead to development of different patterns of liver injury in response to BDL.
(A) Common bile duct dilation, 3 days after BDL, is worsened by 6-ECDCA and attenuated by theonellasterol. Data are mean ± SE of 6 mice per group. *P<0.05 versus sham operated. ** Theonellasterol versus BDL. (B) Representative macroscopic features of common bile duct (white arrows) in 3-day BDL mice administered theonellasterol and 6-ECDCA . (C) BDL increases serum levels of alkaline phosphatase, a marker of bile duct obstruction. Serum levels of alkaline phosphatase were not changed by administering BDL mice with theonellasterol, while were slightly increased by 6-ECDCA. Data are the mean ± S.E. of 6 mice per group. *P<0.05 versus sham operated. (D) Serum concentration of total bile acids. Data are the mean ± S.E. of 6 mice per group. *P<0.05 versus sham operated. (E) Quantitative analysis of tauro-conjugated bile acids in BDL animals. BDL increases serum concentrations of T-CA, T-MCA and T-CDCA while secondary bile acids were almost undetected. Challenging BDL mice with 6-ECDCA increases serum levels of T-6-ECDCA. Data are the mean ± S.E. of 4 mice per group. *P<0.05 versus sham operated.
Figure 6.
BDL causes liver cell injury and theonellasterol attenuates liver necrosis.
(A and B) Liver necrosis was robustly attenuated by theonellasterol as confirmed by assessment of ALT and histopathology analysis. Data are the mean ± S.E. of 6 mice per group. *P<0.05 versus sham. **P<0.05 versus BDL. #P<0.05 versus BDL plus theonellasterol. (C) Representative liver histology from an individual mice per group. Liver sections were stained with H&E, original magnification 10×.
Figure 7.
Theonellasterol alters the liver expression of genes involved in bile acid metabolism in BDL mice.
(A) Real-Time PCR analysis of OSTα, (B) BSEP, (C) SHP and (D) MRP4. Data are the mean ± S.E. of 4 mice per group. *P<0.05 versus sham. **P<0.05 versus BDL. #P<0.05 versus BDL plus theonellasterol.