Chronic nicotine exposure stimulates biliary growth and fibrosis in normal rats

Abstract

Background: Epidemiological studies have indicated smoking to be a risk factor for the progression of liver diseases. Nicotine is the chief addictive substance in cigarette smoke and has powerful biological properties throughout the body. Nicotine has been implicated in a number of disease processes, including increased cell proliferation and fibrosis in several organ systems.

Aims: The aim of this study was to evaluate the effects of chronic administration of nicotine on biliary proliferation and fibrosis in normal rats.

Methods: In vivo, rats were treated with nicotine by osmotic minipumps for two weeks. Proliferation, α7-nicotinic receptor and profibrotic expression were evaluated in liver tissue, cholangiocytes and a polarized cholangiocyte cell line (normal rat intrahepatic cholangiocyte). Nicotine-dependent activation of the Ca(2+)/IP3/ERK 1/2 intracellular signalling pathway was also evaluated in normal rat intrahepatic cholangiocyte.

Results: Cholangiocytes express α7-nicotinic receptor. Chronic administration of nicotine to normal rats stimulated biliary proliferation and profibrotic gene and protein expression such as alpha-smooth muscle actin and fibronectin 1. Activation of α7-nicotinic receptor stimulated Ca(2+)/ERK1/2-dependent cholangiocyte proliferation.

Conclusion: Chronic exposure to nicotine contributes to biliary fibrosis by activation of cholangiocyte proliferation and expression of profibrotic genes. Modulation of α7-nicotinic receptor signalling axis may be useful for the management of biliary proliferation and fibrosis during cholangiopathies.

Keywords: Biliary epithelia; Cholangiocytes; Fibrosis; Nicotinic receptors; Proliferation.

Figure 1

α7-nAChR gene and protein expression in liver sections and isolated cholangiocytes from control and nicotine-treated rats and NRIC. [A] Bile ducts in liver sections from control and nicotine-treated rats express α7-nAChR by immunohistochemistry. Original magnification, x40. Enlargement of areas containing bile ducts positive for α7-nAChR are shown. Positively stained bile ducts are indicated by yellow arrows, whereas hepatocytes are indicated by an orange arrow. [B] Cholangiocytes isolated from control and nicotine-treated rats express α7-nAChR by immunoblotting analysis. ß-actin was utilized as a loading control. [C] NRICs are positive for α7-nAChR (green) by immunofluorescence. Nuclei are stained with DAPI (blue). [D] Cholangiocytes isolated from normal, nicotine-treated rats and NRIC express the message for α7-nAChR by real-time PCR. Data are mean ± SEM of 6 experiments.

Figure 2

Evaluation of intrahepatic bile duct mass (IBDM) and biliary proliferation in liver sections and cholangiocytes isolated from control and nicotine-treated rats. [A] In liver sections from control and nicotine-treated rats, a significant increase in IBDM was observed in nicotine-treated rats compared with the normal control rats (for semiquantitative data see Table 1). Bile ducts are indicated with yellow arrows. Original magnification, x40. [B] A significant increase in PCNA protein expression is observed in cholangiocytes isolated from nicotine-treated compared to control rats by immunoblots. Data are mean ± SEM of 6 experiments. *p < 0.05 vs. control.

Figure 3

Evaluation of profibrotic gene expression in total liver samples and cholangiocytes isolated from normal and nicotine-treated rats. [upper panel] Chronic administration of nicotine stimulated a significant increase in the gene expression of α-SMA, collagen 1A and fibronectin in total liver samples from nicotine-treated rats compared to control. [lower panel] Chronic administration of nicotine stimulated a significant increase in the gene expression of α-SMA and collagen 1A in cholangiocytes isolated from nicotine-treated rats compared to normal control. No significant changes were observed for fibronectin gene expression. Data are mean ± SEM of 6 experiments. *p < 0.05 vs. normal control.

Figure 4

Evaluation of collagen deposition by Masson’s trichrome staining, fibronectin and α-SMA expression by immunohistochemistry in liver sections from control and nicotine-treated rats. Masson’s trichrome staining in control [A] and nicotine-treated rats [B]. Chronic administration of nicotine stimulated an increase in collagen deposition (blue staining) around the portal areas of nicotine-treated [B] compared with normal control rats [A] (for semiquantitative data see Table 1). Immunohistochemistry for fibronectin [C, D] and α-SMA [E, F] expression in control and nicotine-treated rats, respectively. An increase in both fibronectin and α-SMA expression is observed in in nicotine-treated [D, F] compared with normal control rats [C, D], respectively. Bile ducts are indicated with yellow arrows. Original magnification, x40.

Figure 5

Evaluation of nicotine-induced proliferation and ERK1/2 activation in NRIC. [A] Nicotine (0.01–20 μM) induced a dose-dependent increase in NRIC proliferation at 48 hr. [B] AR-R 17779 (0.01×20 μM; a specific α7-nAChR agonist) stimulated a dose-dependent increase in NRIC proliferation at 48 hr. [C] AR-R 17779 (1 μM)-induced NRIC proliferation is partially blocked by alpha-bungarotoxin (ABT; 0.5 μM; α7-nAChR selective antagonist), BAPTA/AM (5 μM) and PD98059 (10 nM). Data are mean ± SEM of six experiments. *p < 0.05 vs. basal values. [D] Nicotine-stimulated the phosphorylation and nuclear translocation of ERK1/2. p-ERK1/2 (red) and DAPI (blue). Phosphorylation and nuclear translocation of ERK1/2 was inhibited by preincubation with ABT. [E] NRIC were stimulated with 0.2% BSA or nicotine (10 μM for 0, 2, 5, 10, 20, 30, 60, min) and ERK1/2 phosphorylation was evaluated by a phospho-ERK Cell-based ELISA kit. Nicotine stimulated an increase in p-ERK/t-ERK at 10, 20 and 30 minutes of incubation. Data are mean ± SEM of six experiments. *p < 0.05 vs. basal.