Opposite effects of knocking out MT1 and MT2 melatonin receptor on senescence and fibrosis of cholangiocytes and hepatic stellate cells during cholestatic liver injury
Authors: Wu Nan , Kyritsi Konstantina , Venter Julie , Meng Fanyin , Invernizzi Pietro , Bernuzzi Francesca Francesca , Sato Keisaku , Gaudio Eugenio , Zhou Tianhao , Francis Heather L. , Onori Paolo , Franchitto Antonio , Alpini Gianfranco , Glaser Shannon S. 
Published: June 2021

Senescent cholangiocytes (a key feature in primary sclerosing cholangitis, PSC and primary biliary cholangitis, PBC) contribute to liver fibrosis through activation of hepatic stellate cells (HSCs) by decreased HSCs senescence. Melatonin decreases biliary hyperplasia and liver fibrosis in bile duct ligated (BDL) rats. No data exists on the role of MT1 and MT2 melatonin receptors on biliary homeostasis, cellular senescence and liver fibrosis. Therefore, we hypothesized that there is a differential role of melatonin MT1 and MT2 on biliary proliferation, cellular senescence and liver fibrosis during BDL.MethodsStudies were performed with Wild‐type (WT), MT1−/− or MT2−/− mice with/without BDL for 1 wk. MT1 and MT2 levels were evaluated in liver sections by immunofluorescence (IF). Intrahepatic biliary mass (IBDM) and proliferation were evaluated by immunohistochemistry (IHC) for CK‐19 in liver sections. SA‐β‐gal staining and qPCR for p16 and p21 were used to evaluate biliary senescence. Liver fibrosis was measured by Sirius red staining and qPCR for fibrosis genes (TGF‐β1, α‐SMA, fibronectin and collagen alpha 1) in total liver, cholangiocytes and Laser Capture Microdissection (LCM)‐isolated HSCs. In vitro, human HSCs (hHSCs) were treated with biliary supernatants from all groups of mice before measuring the expression of fibrosis genes by qPCR.ResultsMT1 and MT2 in bile ducts was increased in BDL compared to WT mice. Biliary senescence, proliferation and liver fibrosis were increased in BDL compared to WT mice. Biliary senescence, proliferation and liver fibrosis were decreased in MT1−/− mice during BDL, but there was aggravated biliary senescence, proliferation and liver fibrosis in MT2−/− mice during BDL. In LCM‐HSCs from BDL mice, fibrotic genes expression was decreased in MT1−/− but increased in MT2−/− mice. However, expression of senescent genes was increased in MT1−/− but decreased in MT2−/− mice in LCM‐HSCs during BDL. In hHSCs treated with biliary supernatants from BDL WT mice, there was an expected increased expression of fibrotic genes compared to controls. Fibrotic gene expression was reduced in hHSCs treated with biliary supernatants from BDL MT1−/− mice and enhanced by biliary supernatants from BDL MT2−/− compared to hHSCs treated with supernatants from BDL WT mice. In contrast with fibrosis, senescence changes in the opposite trend simultaneously in hHSCs.ConclusionBiliary proliferation, cellular senescence and liver fibrosis are differentially regulated in MT1 and MT2 knockout mice. Specific targeting the MT1 receptor may be provide a key approach for the treatment of liver fibrosis during chronic liver diseases.Support or Funding InformationNIH NIDDK R01s; Veteran's Affairs Merit Review Awards; VA CD‐2 Awards; BSW Health Operational Funds; Baylor Scott and White Nicholas Hightower Chair; Department of Internal Medicine; Ladies Auxiliary of Veterans of Foreign Affairs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.