Suppression of MT1 and Melatonin Treatment Improves Liver Phenotypes in Mdr2‐/‐ mice

Abstract

Background & AimPrimary Sclerosing Cholangitis (PSC) is a cholestatic liver disease characterized by hepatic fibrosis and portal inflammation. Melatonin is synthesized by arylalkylamine N‐acetyltransferase (AANAT) in the pineal gland, as well as extrapineal sites such as cholangiocytes. We previously found that: (i) the MT1 receptor is primarily expressed in cholangiocytes with low and absent expression in hepatic stellate cells and hepatocytes, respectively; (ii) melatonin reduces biliary proliferation via MT1 receptor signaling; and (iii) melatonin treatment for 1 wk decreases biliary proliferation and liver fibrosis in bile duct ligated rats by downregulation of MT1 and clock genes (PER1, CRY1, CLOCK and BMAL1). We aimed to evaluate the beneficial effects of long‐term melatonin treatment and MT1 signaling on biliary phenotypes, liver fibrosis and portal inflammation in Mdr2‐/‐ mice (a model of PSC).MethodsMale FVB/NJ and Mdr2‐/‐ mice had access ad libitum to drinking water with/without melatonin for 3 months. We evaluated: (i) ductular reaction (DR) by immunohistochemistry (IHC) for CK19, (ii) liver fibrosis by Sirius Red staining and (iii) portal inflammation by IHC for F4/80. MT1, AANAT, and clock genes immunoreactivity was evaluated by immunofluorescence (IF) co‐stained with CK19 and qPCR in isolated cholangiocytes. Male C3H‐Hej (WT for MT1‐/‐), FVB/NJ (WT for Mdr2‐/‐), MT1‐/‐, Mdr2‐/‐ mice and MT1‐/‐/Mdr2‐/‐ (DKO) mice were euthanized at 12 wk of age. We analyzed: (i) MT1 expression by IF and mRNA expression in total liver by qPCR to validate our model; (ii) liver damage by H&E, DR by IHC for CK19 and liver fibrosis by Sirius Red staining and (iii) portal inflammation by IHC for F4/80 and expression of the inflammatory markers by qPCR in total liver.ResultsLong‐term melatonin treatment reduces DR, liver fibrosis and portal inflammation in Mdr2‐/‐mice. Prolonged administration of melatonin in Mdr2‐/‐mice improves liver phenotype by decreasing the immunoreactivity of MT1, AANAT and clock genes in Mdr2‐/‐ mice, which suggests the chronobiotic action of melatonin on biliary circadian rhythm. DKO mice have no MT1 expression and display ameliorated liver phenotype compared to Mdr2‐/‐ mice.ConclusionWe demonstrated that chronic melatonin treatment improves liver histology and restores the biliary circadian rhythm by interaction with MT1. Suppression of MT1 receptor in Mdr2‐/‐mice ameliorates biliary/liver phenotypes through changes in clock genes and AANAT. Restoration of the circadian rhythm by modulation of melatonin/MT1 signaling may be key for the management of cholangiopathies.