ASBT Vivo‐Morpholino Decreases Hepatic Mast Cell, Fibrosis and Biliary Senescence in Mdr2 ‐/‐ Mice

Abstract

Background Primary Sclerosing Cholangitis (PSC) is characterized by increased biliary senescence, liver fibrosis, and mast cell (MC) infiltration. Mdr2-/- mice, model of PSC, mimic some features of PSC. PSC features include (i) increased conjugated total bile acid (TBA) levels, (ii) dysregulation of BA synthesis and (iii) higher hepatic expression of the apical sodium bile acid transporter (ASBT), expressed only by cholangiocytes. Hydrophobic BAs enhance cholestasis, biliary proliferation and ASBT expression. MCs infiltrate the liver in PSC and reside near senescent cholangiocytes, increasing biliary damage and TBA levels. ASBT is regulated by farnesoid X receptor (FXR) and blocking MC-FXR decreases biliary damage, fibrosis, and biliary ASBT expression. No information exists to demonstrate if blocking hepatic ASBT inhibits MC activation and subsequent liver damage in Mdr2-/- mice. Methods 12 wk male Mdr2−/− mice were treated with control or ASBT Vivo-Morpholino (2 injections, 12.5 mg oligo/kg BW via tail vein for 1 wk). Liver damage was determined by H&E and serum enzyme levels. Ductular reaction (DR) was evaluated by immunohistochemistry (IHC) for CK-19, Ki-67 and immunofluorescence (IF) for p16, co-stained with CK-19. ASBT expression was measured by qPCR and IF in liver, isolated cholangiocytes and small intestine. MC infiltration was measured by IHC for Tryptase β-2 in liver and intestine. Hepatic fibrosis was determined by Fast Green-Sirius Red staining. TBA levels were measured in serum, liver and feces. BA receptors/transporters were co-stained with CK-19 in small intestine and liver. Results Mdr2−/− mice have increased DR, fibrosis, and serum/hepatic TBA levels, which decrease in Mdr2−/− ASBT Vivo-Morpholino mice. Inhibition of ASBT in Mdr2-/- mice reduced (i) biliary and intestinal ASBT expression and (ii) serum and hepatic CA and CDCA levels. Fecal TBA levels decreased, but CA and CDCA levels increased, following ASBT Vivo-Morpholino. ASBT Vivo-Morpholino treatment reduced hepatic and intestinal MC-infiltration/activation. Conclusion Elevated serum TBA levels seen in PSC lead to increased intestinal BA transport inducing MC infiltration and increased biliary ASBT. Inhibition of biliary ASBT blocks hepatic and intestinal MC recruitment and subsequent DR, hepatic fibrosis and TBA levels. Targeting MCs via ASBT inhibition is a novel strategy for the management of PSC.