Prolonged exposure of cholestatic rats to complete dark inhibits biliary hyperplasia and liver fibrosis

Abstract

Biliary hyperplasia and liver fibrosis are common features in cholestatic liver disease. Melatonin is synthesized by the pineal gland as well as the liver. Melatonin inhibits biliary hyperplasia of bile duct-ligated (BDL) rats. Since melatonin synthesis (by the enzyme serotonin N-acetyltransferase, AANAT) from the pineal gland increases after dark exposure, we hypothesized that biliary hyperplasia and liver fibrosis are diminished by continuous darkness via increased melatonin synthesis from the pineal gland. Normal or BDL rats (immediately after surgery) were housed with light-dark cycles or complete dark for 1 wk before evaluation of 1) the expression of AANAT in the pineal gland and melatonin levels in pineal gland tissue supernatants and serum; 2) biliary proliferation and intrahepatic bile duct mass, liver histology, and serum chemistry; 3) secretin-stimulated ductal secretion (functional index of biliary growth); 4) collagen deposition, liver fibrosis markers in liver sections, total liver, and cholangiocytes; and 5) expression of clock genes in cholangiocytes. In BDL rats exposed to dark there was 1) enhanced AANAT expression/melatonin secretion in pineal gland and melatonin serum levels; 2) improved liver morphology, serum chemistry and decreased biliary proliferation and secretin-stimulated choleresis; and 4) decreased fibrosis and expression of fibrosis markers in liver sections, total liver and cholangiocytes and reduced biliary expression of the clock genes PER1, BMAL1, CLOCK, and Cry1. Thus prolonged dark exposure may be a beneficial noninvasive therapeutic approach for the management of biliary disorders.

Keywords: biliary epithelium; cholestasis; clock genes; melatonin; secretin.

Fig. 1.

Serotonin N-acetyltransferase (AANAT) gene expression and melatonin secretion in pineal gland tissue in animals exposed to light-dark cycles or complete dark for 1 wk. AANAT mRNA expression and melatonin secretion increases in the pineal gland from normal and bile duct ligation (BDL) + dark rats compared with their corresponding rats. A: data are means ± SE of 4 experiments. *P < 0.05 compared with normal rats. #P < 0.05 BDL + dark compared with BDL. B: data (means ± SE, n = 6 experiments) are expressed as pg/μg protein. *P < 0.05 compared with normal rats. #P < 0.05 BDL + dark compared with BDL.

Fig. 2.

A and B: in BDL rats there was increased percentage of PCNA-positive cholangiocytes and intrahepatic bile duct mass (IBDM) compared with normal rats. Chronic exposure of BDL rats to complete dark decreases the percentage of PCNA-positive cholangiocytes and IBDM compared with BDL controls. Prolonged dark exposure had no effect on the proliferation of normal rats. Original magnification, ×40 (PCNA), ×10 (CK-19). Data are expressed as means ± SE. *P < 0.05 vs. normal rats. #P < 0.05 BDL + dark compared with BDL rats exposed to light-dark cycles. C: the architecture of normal rats exposed to 12:12-h light-dark cycles or prolonged dark was normal. BDL rats showed prominent biliary proliferation. BDL rats exposed to complete dark for 1 wk show lower cholangiocyte proliferation compared with BDL rats. Original magnification, ×10.

Fig. 3.

Chronic exposure of BDL rats to dark for 1 wk decreases PCNA and CK-19 expression in purified cholangiocytes. PCNA (A) and CK-19 (B) expression was evaluated by real-time PCR. Data are expressed as means ± SE of 4 experiments from cumulative preparations of cholangiocytes. *P < 0.05 BDL compared with normal rats. #P < 0.05 BDL + dark compared with BDL rats. C: PCNA protein expression was evaluated by immunoblots. Data are expressed as means ± SE of 4 immunoblots reactions from cumulative preparations of cholangiocytes. *P < 0.05 BDL compared with normal rats. #P < 0.05 BDL + dark compared with BDL rats. D: PCNA protein expression evaluated by FACS analysis (n = 3). P1 represents the pool cholangiocytes population gated from the range of forward-scattered light (FSC) and side-scattered light (SSC). *P < 0.05 BDL + dark compared with BDL rats.

Fig. 4.

Chronic exposure of BDL rats to dark for 1 wk decreases secretin-stimulated cAMP levels in purified cholangiocytes. Basal and secretin-stimulated cAMP levels were evaluated by ELISA. Secretin-induced cAMP levels were ablated in cholangiocytes isolated from BDL + dark rats compared with BDL rats. Data are expressed as means ± SE (n = 6). *P < 0.05 vs. the corresponding basal value of BDL rats.

Fig. 5.

Evaluation of fibrosis in liver sections. There was enhanced fibrosis (evaluated by Sirius red staining) and increased expression of α-smooth muscle actin (α-SMA) in BDL compared with normal rats. When BDL rats were exposed to complete dark for 1 wk, there was reduced fibrosis and expression of α-SMA compared with BDL rats exposed to light-dark cycles. Original magnification, ×40.

Fig. 6.

Prolonged exposure of BDL rats to dark decreases the expression of fibrotic markers. Following BDL, there was increased expression of α-SMA, COLA1, fibronectin-1, MMP-2, MMP-9, TIMP1, TIMP2, and TGF-β in total liver (A and C) and purified cholangiocytes (B and D) compared with normal rats. Prolonged exposure of BDL to dark decreases the expression of α-SMA, COLA1, fibronectin-1, MMP-2, MMP-9, TIMP1, TIMP2, and TGF-β in total liver samples (A and C) and purified cholangiocytes (B and D). Data are expressed as means ± SE of 4 experiments to GAPDH ratio from cumulative preparations of cholangiocytes. *P < 0.05 BDL compared with normal rats. #P < 0.05 BDL + dark compared with BDL rats.

Fig. 7.

There was increased expression of Per1 (A), BMAL1 (B), CLOCK (C), and Cry1 (D) in cholangiocytes from BDL rats compared with normal rats. Exposure of BDL rats to complete dark for 1 wk decreases the mRNA expression of Per1, BMAL1, CLOCK, and Cry1 compared with BDL rats exposed to 12:12-h light-dark cycles. Data were expressed as relative mRNA levels ± SE to GAPDH ratio. *P < 0.05 BDL compared with normal rats. #P < 0.05 BDL + dark compared with BDL rats.