7A-C).8 INT-747 and INT-767 increased the size and amount of bile infarcts, as well as LW/BW ratio, in CBDL mice (Supporting Fig. 12A,B), whereas only INT-767 significantly decreased SW/BW ratio (Supporting Fig. 12C) and showed a trend to reduction of serum ALT (Supporting Fig. 13A). Although histological examination

of H&E-stained livers revealed bile infarcts in all the groups, only INT-747 increased infiltration of inflammatory cells within the portal fields (Supporting Fig. 13B). In line with serum ALT levels, INT-767-fed CBDL mice had reduced expression of proinflammatory genes Tnf-α and Il-1β and less CD-11b- and F4/80-positive cells around bile infarcts (Supporting Fig. 14A,B). However, keratin 19 (K19) and Vcam-1 gene expression remained unchanged in CBDL mice after INT-747, INT-777, and INT-767 feeding (Supporting Fig. 15). In this study, we have addressed the DNA Methyltransferas inhibitor therapeutic mechanisms of BA receptor signaling through the nuclear BA receptor, FXR, and the G-protein-coupled membrane BA receptor, TGR5, in the Mdr2−/− mouse cholangiopathy model. We report herein that, in this model, the novel FXR/TGR5 agonist, INT-767, reduces bile toxicity by decreasing biliary BA output and inducing HCO-rich

AZD6244 cell line choleresis in an FXR-dependent manner. BAs are important signaling molecules with hormonal actions through dedicated nuclear and G-protein-coupled receptors, such as FXR and TGR5, respectively.8 TGR5 and FXR polymorphisms19, 20 further support the importance

of BA signaling in human cholestastic diseases, such as PSC. Liver injury in Mdr2−/− mice is considered to evolve because of detergent properties of nonmicellar-bound free biliary BAs,29 leaving many open questions for the potential role of BA signaling in modulating biliary pathophysiology. Only the dual FXR/TGR5 agonist, INT-767, Doxacurium chloride was hepatoprotective in the Mdr2−/− model, as reflected by reduced serum ALT, decreased hepatic inflammation, improved reactive cholangiocyte phenotype, and reduced fibrosis. We could neither observe significant direct anti-inflammatory effects of INT-767 in RAW264.7 macrophages (with very low endogenous Fxr and Tgr5 expression), BEC cholangiocytes, or HepG2 hepatocytes (both with high levels of Fxr and very low Tgr5; data not shown) nor direct antifibrotic effects in primary MFBs (with very low endogenous Fxr and Tgr5 expression) as major fibrogenic cells in the Mdr2−/− model. Absent expression of FXR and TGR59, 11 in hepatic stellate cells further indicates that FXR and TGR5 signaling may have no direct antifibrotic effects. These findings led us hypothesize that INT-767 might improve liver injury by directly impacting on bile formation and composition. Indeed, via Fxr activation, INT-767 inhibited BA synthesis (by ileal Fgf15 and hepatic Shp induction), thus resulting in decreased biliary BA output while significantly increasing bile flow and-unexpectedly-HCO output.