Cell experiment:

For GPBAR1 mediated transactivation, HEK-293T cells are plated at 10000 cells/well in a 24 well-plate and transfected with 200 ng of pGL4.29, a reporter vector containing a cAMP response element (CRE) that drives the transcription of the luciferase reporter gene luc2P, with 100 ng of pCMVSPORT6-human GPBAR1, and with 100 ng of pGL4.70. At 24 h post-transfection, HepG2 and HEK293T cells are incubated with 10 μM BAR501 for 18 h and luciferase activities are assayed and normalized against the Renilla activities[1].

Animal experiment:

Mice: C57BL6 mice are administered i.p. 500 μL/Kg body weight of CCl4 in an equal volume of paraffin oil twice a week for 9 weeks. CCL4 mice are randomized to receive BAR501 (15 mg/Kg daily by gavage) or vehicle (distilled water). Serum bilirubin, albumin, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase are measured by routine biochemical clinical chemistry[1].

BAR501 is a selective agonist of the G protein-coupled bile acid-activated receptor (GP-BAR1; EC50 = 1 μM) that lacks activity at the farnesoid X receptor (FXR). In vivo, BAR501 reduces hepatic perfusion pressure and counteracts norepinephrine-induced vasoconstriction in na?ve rats. It protects against development of endothelial dysfunction by increasing liver cystathione-γ-liase (CSE) expression and activity, while reducing endothelin 1 (ET-1) gene expression in a rat model of cirrhosis. In vitro, BAR501 increases Akt-dependent phosphorylation of CSE and endothelial nitric oxide synthase (eNOS) and inhibits ET-1 transcription in human liver sinusoidal cells. BAR501 also shifts macrophages from the inflammatory (M1) phenotype to the anti-inflammatory (M2) phenotype and reduces trinitrobenzenesulfonic acid-induced colitis in mice in a dose-dependent manner.