Melatonin Therapy Reduces Biliary Proliferation and Hepatic Fibrosis in the Mdr2(-/-) Mouse Model of Primary Sclerosing Cholangitis (PSC) By Decreased Expression of miR-200b and Angiogenic Factors

Primary sclerosing cholangitis (PSC) is characterized by progressive cholestasis, fibrosis and damage to bile ducts. The circadian hormone melatonin plays a key role in the pathogenesis of biliary hyperplasia in the bile duct ligation model of extrahepatic cholestasis. We have previously shown that VEGFA is a key regulator of autocrine/paracrine biliary proliferation in models of cholestatic liver damage. In addition, miRNAs such as miR-200b play key roles in the regulation of proliferation and fibrosis in models of biliary atresia. We have previously shown that melatonin regulates miR-200b expression levels. However, molecular studies aimed to evaluate the mechanisms by which melatonin therapy regulates biliary proliferation and liver fibrosis in a mouse model of PSC remains unknown. The aim of our study was to evaluate the effects of chronic melatonin therapy on hepatic fibrosis and miR-200b and angiogenic factors. Methods: Studies were performed in male FVB/NJ and Mdr2-/- mice (12 weeks of age). The mice had access to drinking water containing melatonin (20 mg/l corresponding to a melatonin intake of 2 mg/g BW per day) or control drinking water for 1 week. Biliary proliferation (CK-19 immunohistochemistry), fibrosis (Sirius Red) and expression of pro-angiogenesis factors (VEGFA, VEGFC, and its receptors, VEGFR2 and VEGFR3, angiopoietin 1/2 and its receptors, Tie1 and Tie2) and markers of fibrosis (Fibronectin, Fn-1, and collagen alpha 1, col1a1) were evaluated in liver sections by immunohistochemistry (IHC). Serum levels of alkaline phosphatase (ALP) (marker of biliary damage) were evaluated. Expression of markers of fibrosis and angiogenesis were evaluated in isolated cholangiocytes and total liver samples by qPCR. miR-200b expression levels were evaluated in isolated cholangiocytes from control and Mdr2-/- mice treated with melatonin. Results: As expected, there was a significant increase in biliary mass, hepatic fibrosis and serum ALP levels in the Mdr2-/- control drinking water group compared to the FVB/NJ controls. In the Mdr2-/- mice receiving melatonin therapy, there was a significant reduction in biliary mass, hepatic fibrosis and serum ALP levels compared to the Mdr2-/- control drinking water group. Melatonin therapy also decreased the expression levels of pro-fibrogenic and pro-angiogenic markers by IHC and qPCR in the compared Mdr2-/- control drinking water group. miR200b expression levels were elevated in Mdr2-/- mouse cholangiocytes, whereas melatonin therapy dramatically decreased miR-200b expression levels. Conclusions: Melatonin therapy reduces both biliary mass and hepatic fibrosis in the PSC mouse model, which was associated with decreased expression of factors regulating angiogenesis and miR-200b. Melatonin therapy may be useful for reducing biliary damage and hepatic fibrosis during cholestatic liver injury