Prolonged darkness reduces liver fibrosis in a mouse model of primary sclerosing cholangitis by miR-200b down-regulation

Melatonin therapy or prolonged exposure to complete darkness reduces biliary hyperplasia and liver fibrosis in bile-duct–ligated (BDL) rats; however, no information exists in primary sclerosing cholangitis (PSC). Thus, we aimed to determine the therapeutic effects of prolonged dark therapy or melatonin administration on hepatic fibrosis in the multidrug resistance gene 2–knockout (Mdr2^-/-) mouse model of PSC. Melatonin levels, biliary mass, liver fibrosis, angiogenesis and miR-200b expression were evaluated in wild-type and Mdr2^-/- mice exposed to darkness or melatonin treatment or in male patients with PSC and healthy controls. Mdr2^-/- mice were also treated with miR-200b inhibitor or control before evaluating biliary mass, liver fibrosis, and angiogenesis. After overexpression of arylalkylamine N-acetyltransferase (AANAT; the enzyme regulating melatonin synthesis) or inhibition of miR-200b in cholangiocytes and hepatic stellate cells in vitro, we evaluated angiogenesis and fibrosis gene expression. After exposure to darkness or administration of melatonin, Mdr2^-/- mice show elevated serum melatonin levels and inhibition of biliary mass, along with reduction of liver fibrosis and angiogenesis. MicroRNA PCR analysis demonstrated that miR-200b expression increased in Mdr2^-/- mice and patients with PSC compared with controls and decreased in Mdr2^-/- mice subjected to dark exposure or melatonin treatment. Inhibition of miR-200b in Mdr2^-/- ablates biliary proliferation, liver fibrosis, and angiogenesis. In vitro, overexpression of AANAT or inhibition of miR-200b in cholangiocytes and hepatic stellate cells decreased the expression of miR-200b, angiogenesis, and fibrosis genes. Dark therapy or targeting melatonin/miR-200b axis may be important in the management of biliary damage and liver fibrosis in cholangiopathies including PSC.