1E). After LPS administration, the numbers of F4/80-positive cells in WT and TGR5−/− mouse livers were increased with 47% and 57%, respectively. Results indicated that LPS increased Kupffer cell infiltration more significantly in TGR5−/− mouse liver. It has been well reported that LPS induces hepatocyte apoptosis in vitro and in vivo.21-24 Liver injury induced by LPS was further confirmed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Compared with WT controls, TUNEL-positive percentage was enhanced in the livers of TGR5−/− mice after injection of LPS (Fig. 1F).

Together, both in vitro and in vivo results suggest that macrophages, primary Kupffer cells, and livers from TGR5−/− mice are more sensitive to LPS-induced inflammation. We then tested whether ligand-activated TGR5 could inhibit these proinflammatory NF-κB target selleck screening library genes induced by LPS.

23(S)-mCDCA is a new synthetic, highly selective TGR5 agonist.25 We synthesized 23(S)-mCDCA and confirmed its TGR5-specific activity by observing the expected dose-dependent increase in cyclic adenosine monophosphate (cAMP) in WT macrophages, but not TGR5−/− macrophages, and by activation of a CRE-reporter construct only in TGR5-expressing cells (data not shown). Measurements of ALT, AST, and alkaline phosphatase (ALP) and histological staining indicated that administering 23(S)-mCDCA to mice did not induce toxic effects (data not shown). 23(S)-mCDCA treatment repressed LPS-induced IP-10 and IL-6 PLX 4720 expression in WT macrophages, but not TGR5−/− macrophages (Fig. 2A). A similar inhibition of mRNA levels of iNOS, MCP-1, cyclooxygenase-2 (COX-2), and IL-6 by the TGR5 agonist was observed in response to stimulation with LPS in WT Kupffer cells, but not TGR5−/− Kupffer cells (Fig. 2A). Kawamata et al.14 and Keitel et al.13 observed a similar down-regulating TNF-α, IL-6, IL-1α, and IL-1β in rabbit macrophages Carnitine palmitoyltransferase II and rat Kupffer cells upon treatment with bile acids. Furthermore, we examined the effects of the TGR5 agonist on the NF-κB pathway in vivo. Livers from WT mice that were pretreated with the TGR5 agonist, 23(S)-mCDCA, showed significantly

less LPS-induced expression of IP-10, MCP-1, iNOS, and IFN-γ mRNA than did nonpretreated livers. This inhibition was abolished, or considerably reduced, in TGR5−/− mouse groups (Fig. 2B). Collectively, these results demonstrate that TGR5 plays a protective role against LPS-induced inflammation in vitro and in vivo. We next tested whether TGR5 agonist inhibited NF-κB activity at the levels of gene transcription. We cotransfected HepG2 cells with a NF-κB reporter plasmid and the control plasmid, phRL-TK, and assessed the effects of the TGR5 ligand on the regulation of NF-κB reporter activity. Treatment with known NF-κB pathway activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or LPS resulted in 5.1- and 1.8-fold higher NF-κB reporter activity, respectively (Fig.