Abstract

Background:

Primary sclerosing cholangitis (PSC), a chronic cholangiopathy, results in cholestasis, inflammation, and stricturing of intrahepatic and extrahepatic bile ducts. Recently, we have shown that the transcription factor STAT3 mediated, in part, the protective role of the FXR agonist, GW4064, in parenteral nutrition (PN)-associated cholestasis (Ghosh et al., Hep comm 2023). The objective of this study is to determine the interaction of STAT3 and FXR agonists (IV GW4064 and obeticholic acid [OCA]) in a mouse model of acute cholangiopathy and in vitro.

Methods:

Acute DDC dietary mouse model (diethyl1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate diet feeding x1 day) and cell culture were used.

Results:

DDC mice had increased AST, ALT, bilirubin, bile acids, ductular reaction and macrophage infiltration and reduced hepatocyte mRNA expression of Abcb11, Abcg8, Nr0b2 (SHP), Nr5a2 and Abcc2 mRNA relative to chow fed mice; all of these were normalized by either FXR agonist treatment. Macrophage mRNA expression of pro-inflammatory (M1) and anti-inflammatory (M2) genes showed that FXR agonists led to upregulation of M2 (Clec10a1, Msr1, Klf4) and down regulation of M1 genes (Cd68, Il-1b, Itgam, Cox1) in DDC mice vs. controls. A significant increase in hepatic STAT3 phosphorylation and acetylation and binding of FXR to Stat3 promoter, as well as decreased apoptosis markers by flow cytometry (FAS, caspase 8 and caspase 3), were present in FXR agonist/DDC mice compared to DDC mice. DDC mice showed elevated hepatic arachidonic acid (AA) levels (mass-spec) associated with apoptosis, both of which were ameliorated by FXR agonist treatment. AA incubation with Huh7 cells caused increased apoptosis (by Annexin V flow cytometry). In THP1 cells that were exposed to AA cocultured with Huh7 cells, treatment with GW4064 or OCA increased anti-inflammatory Il-10 and PPARA mRNA in THP1 and ABCB11/BSEP mRNA in Huh7. In Huh7, AA suppressed the protective upregulation by OCA or GW4064 of BSEP and SHP, and when STAT3 was knocked down by siRNA ,then the expression was further suppressed. Finally, in DDC mouse liver, increased binding of STAT3 to Cpt1b promoter with its increased expression (likely enhanced fatty acid oxidation) was induced by GW4064.

Conclusion:

In the DDC acute cholangiopathy model, FXR agonists are protective through STAT3 signaling, M1 to M2 macrophage polarization, and by inducing fatty acid oxidation, suggesting STAT3 as a potential therapeutic target in PSC.