Knockdown of the melatonin receptor, MT1, reduces biliary hyperplasia and liver fibrosis in cholestatic bile duct ligated (BDL) mice

Cholestatic liver diseases are characterized by increased biliary proliferation and intrahepatic bile duct mass (IBDM), liver damage and fibrosis. Melatonin, which is synthesized in the pineal gland as well as cholangiocytes, exerts its effects by interaction with MT1 and MT2 receptors. Knockdown of MT2 increases biliary proliferation and liver fibrosis in cholestatic mice. Also, melatonin inhibits biliary hyperplasia by interacting with MT1 receptors. However, since these studies were performed only in vitro, we evaluated the role of MT1 in regulating biliary proliferation and liver fibrosis in vivo in MT1 knockout (KO) mice, and wild-type (WT) mice in which the hepatic expression of MT1 was reduced by Vivo-Morpholinos for MT1. Methods: The studies were performed in normal (WT), MT1 Morpholino-treated or MT1 KO mice with or without BDL surgery. We evaluated the expression of MT1 in liver sections by immunohistochemistry (IHC) and immunoblots in cholangiocytes. Intrahepatic biliary ductal mass (IBDM) and biliary proliferation was evaluated by IHC for CK-19 and PCNA in liver sections, and qPCR for PCNA in cholangiocytes. Liver injury was determined by H&E staining in liver sections and serum levels of transaminases and bilirubin. Fibrosis was evaluated by Sirius red staining in liver sections and qPCR for TGFbeta1, alpha-SMA and fibronectin in cholangiocytes and total liver. In vitro, shRNA-MT1 transfected murine cholangiocytes (MCCs) and controls were used to measure proliferation and TGFbeta1, alpha-SMA and fibronectin (FN-1) mRNA expression by qPCR. Results: Normal cholangiocytes express low levels of MT1, which markedly increased after BDL. We demonstrated that in MT1 KO BDL and MT1 Morpholino-treated BDL mice, along with reduced MT1 expression, there was decreased biliary proliferation and IBDM. In MT1 KO BDL and MT1 Morpholino- treated BDL mice, there was decreased lobular damage, serum levels of transaminases and bilirubin, and liver fibrosis in liver sections and reduced expression of TGFbeta1, alpha-SMA and FN-1 compared to BDL WT mice. In shRNA-MT1 transfected MCCs, there was decreased proliferation and reduced expression of TGFb1, alpha-SMA and FN-1. The findings indicate that shifting the melatonin axis to signal via MT2 during the knockdown of MT1 expression decreases biliary proliferation and liver fibrosis. Conclusion: Our findings indicate that the balance of MT1/MT2 receptor expression for the melatonin signaling axis may play an important role in the regulation of biliary proliferation and hepatic fibrosis. Inhibition of MT1 may be a key approach for ameliorating biliary hyperplasia and liver fibrosis in cholestatic liver diseases.