TCPOBOP was purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany), PB was purchased from Spectrum (Scottsdale, NJ, USA) and dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich; Merck Millipore.

A total of 48 male mice (8–10 weeks old; 20–25 g) on a C57BL/6 J background were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China) and housed in a specific pathogen-free animal facility under constant temperature (20–22°C), controlled humidity (45–55%) and a standard 12-h light/dark cycle. All experimental procedures were approved by the Institutional Animal Committee of Wenzhou Medical University (Wenzhou, China) and performed according to the Guide for the Care and Use of Laboratory Animals (Wenzhou Medical University, Wenzhou, China).

The regular Lieber-DeCarli ethanol liquid diet (ETOH group) or an isocaloric control diet (pair-fed group) were purchased from Trophic Animal Feed High-tech Co., Ltd. (Nantong, China), and were fed to the male mice ad libitum, as previously described (20). The percentages of caloric intake from ethanol (maltose dextrin), proteins, carbohydrates and fats were 35.5, 18, 11.5 and 35%, respectively. In the first week, mice were gradually introduced to the ethanol liquid diet containing 0.75% ethanol (w/v) for 2 days, 1.50% ethanol (w/v) for 2 days and 3.75% ethanol (w/v) for 3 days to acclimatize the mice to the liquid diet, and were then introduced to a 5% ethanol (w/v) diet for 4 weeks. No differences in body weight or the quantity of food consumed were found among the treated groups. In the following 3 days, the clinically used CAR ligand, PB (100 mg/kg/d; dissolved in DMSO) and the specific rodent CAR ligand, TCPOBOP (3 mg/kg/d; dissolved in corn oil), which has a higher potency, were administered intraperitoneally (i.p) to the mice in the ETOH group (11). As a vehicle, mice were injected i.p with 100 µl of DMSO (ETOH/DS group) or corn oil (ETOH/CO group) for comparison with the ETOH/PB and ETOH/TCP groups, respectively. A total of four animals were investigated in each treatment group. A total of 3 days following injection, the animals were anesthetized with 5% chloral hydrate (40 mg/kg, i.p) and sacrificed using the cervical dislocation method following blood collection. Blood was collected from the orbital sinus of the animals by traumatic avulsion of the globe from the orbit using a pair of tissue forceps. Sections of liver tissue were fixed in 4% paraformaldehyde solution and paraffin-embedded for light microscopy. In addition, liver tissues were cut into sections measuring <5 mm in thickness, embedded in OCT compound and frozen in liquid nitrogen. The remaining liver samples and the serum samples were stored at −80°C for further analysis.

The paraformaldehyde-fixed, paraffin-embedded liver samples were cut into 5-µm-thick sections and the OCT compound-embedded liver samples were cut into 8-µm-thick frozen sections, respectively. A light microscope was used to visualize hematoxylin and eosin staining, as well as Oil Red O (Sigma-Aldrich; Merck Millipore) staining to quantify steatosis and inflammation in each group.

Serum was prepared from the blood by centrifugation at 1,200 g at 4°C for 10 min. The levels of total serum bilirubin (TB), conjugated bilirubin (CB), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were determined in the pair-fed, ETOH, ETOH/PB, ETOH/TCP, ETOH/CO and ETOH/DS mice using a total/direct bilirubin kit (Sigma-Aldrich; Merck Millipore) and an ALT/AST assay kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the respective manufacturers' protocols.

For isolation of total liver and kidney membranes, tissue was homogenized in 1 mM NaHCO₃ (pH 7.4), containing complete protease inhibitor (Beyotime Institute of Biotechnology, Haimen, China). Homogenates were gauze filtered, and total membranes were isolated by centrifugation at 100,000 × g for 1 h at 4°C. as previously described (12,21). Nuclear protein was extracted using the CelLytic™ NuCLEAR™ Extraction kit (Sigma-Aldrich; Merck Millipore). In addition, protein concentrations were determined using a BCA protein Assay kit (Beyotime Institute of Biotechnology). A total of 20 µg protein was loaded to each well of 10% polyacrylamide gels for electrophoresis. Proteins were blotted onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore). PVDF membranes were blocked using TBS containing 0.1% Tween 20 and 4% skim milk powder. Blots were incubated with polyclonal rabbit anti-mouse lamin-B (dilution, 1:5,000; cat. no. ab-65986; Abcam, Cambridge, UK), OATP1A1 (dilution, 1:1,000; cat. no. sc-47265; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), OATP1A4 (dilution, 1:1,000; cat. no. sc-33610; Santa Cruz Biotechnology, Inc.), OATP1B2 (dilution, 1:1,000; cat. no. sc-134460, Santa Cruz Biotechnology. Inc.), MRP2 (dilution, 1:2,000; cat. no. sc-20766, Santa Cruz Biotechnology, Inc.), CAR (dilution, 1:1,000; cat. no. ab-186869; Abcam), GAPDH (dilution, 1:1,000; cat. no. AG019; Beyotime Institute of Biotechnology) primary antibodies or monoclonal goat anti-mouse UGT1A1 primary antibody (dilution, 1:1,000; cat. no. sc-27419; Santa Cruz Biotechnology, Inc.) at 4°C overnight, respectively. Following washing with 1X TBS containing 0.1% Tween 20 for 3 times, there was incubation conducted using horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (dilution, 1:10,000; cat. no. A0208; Beyotime Institute of Biotechnology) and enhanced chemiluminescence substrate reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The time for secondary antibody incubation was <1 h at 37°C. The quantification of protein expression was determined via image processing and analysis using Image Lab 3.0 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

RNA was extracted from the frozen liver and kidney samples using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and complementary DNA (cDNA) synthesis for qPCR analysis was performed using a commercial kit (Toyobo Co., Ltd., Osaka, Japan) with subsequent melting curve analysis, according to the manufacturer's protocol. The PCR reaction mixture (forward primer, 0.4 µl; reverse primer, 0.4 µl; cDNA, 1 µl; diethylpyrocarbonate, 3.2 µl) was prepared using SYBR Green Real-time PCR Master Mix-Plus (Toyobo Co., Ltd.). The thermocycling parameters were as follows: pre-denaturation at 94°C for 1 min, denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec, extension at 72°C for 15 sec for 40 cycles of amplification. PCR amplification was performed using specific primers (Table I). Data were analyzed using Bio-Rad CFX manager (Bio-Rad Laboratories, Inc.). The levels of gene expression were calculated as a ratio to the expression of the housekeeping gene, β-actin.

The NCTC1469 normal mouse liver cell line was purchased from Zhongqiao (Shanghai, China). The cells were maintained at 37°C in a humidified atmosphere of 5% CO₂ in RPMI 1640 supplemented with penicillin/streptomycin (100 IU/ml and 100 µg/ml, respectively) and 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). Prior to the experiment, cell viability was measured via Trypan blue exclusion. The hepatocytes were plated on glass coverslips at a density of 2.0×10⁵ cells/ml in 6 wells and incubated in a CO₂ incubator for 3 h at 37°C. The cells were pretreated with or without 2 mM phenobarbital (PB) for 6 h followed by treatment with or without 100 µM alcohol for 12 h at 37°C. The cells were fixed with 4% formaldehyde/PBS for 15 min. 10% Goat serum (Gibco; Thermo Fisher Scientific, Inc.) in TBS containing 0.1% Triton X-100 was used to block nonspecific binding, as previously described (22). Membranes were incubated with CAR primary antibody at a dilution of 1:100 overnight and with corresponding secondary antibodies conjugated to Delight 594 (Bioworld Technology, Inc., Minneapolis, MN, USA) at a dilution of 1:200 for another 2 h at room temperature. The coverslips were sealed with antifade mounting medium (Beyotime Institute of Biotechnology) following staining with DAPI (Beyotime, Haimen; China) for 10 min, and were visualized using a fluorescence microscope (Olympus, Tokyo, Japan).

Data are shown as the mean ± standard deviation. Statistical analysis was performed using two-tailed Student's t-test or one-way analysis of variance followed by post hoc tests (LSD-t or Dunnett's T3) for multigroup comparisons. SPSS 12.0 program (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

To investigate the role of CAR in bilirubin clearance in ALD, wild-type C57BL6 mice were fed with a Lieber-DeCarli liquid diet or a pair-fed control diet for 4 weeks, followed by i.p. injections with different CAR ligands for 3 days. The levels of ALT, AST and ALP in the EtOH group were higher, compared with those in the pair-fed controls (105±30.66, vs. 26.75±2.99 U/l; 187.8±55.34, vs. 111.3±19.52 U/l and 155±12.83, vs. 65.25±37.35 U/l, respectively; P<0.05) and were also elevated in the EtOH/TCP group, compared with those in its vehicle (EtOH/CO) group (510.3±151.2, vs. 103.8±25.63 U/l; 287.8±91.09, vs. 174.5±42.15 U/l and 284.5±46.65, vs. 148.8±13.74 U/l, respectively; P<0.05), as shown in Table II. The levels of AST and ALP in the EtOH/DS group (EtOH/PB vehicle) were lower, compared with those in the EtOH group (115±25.59, vs. 187.8±55.34 U/l and 106±18.94, vs. 155±12.83 U/l, respectively; P<0.05). Compared with the pair-fed controls, the levels of hepatic total bilirubin and direct bilirubin were significantly elevated following alcohol ingestion (0.33±0.10, vs. 0.75±0.17 µmol/l and 0.25±0.10, vs. 0.45±0.13 µmol/l, respectively; P<0.05), and were markedly decreased following administration of the high affinity CAR agonist, TCPOBOP (0.55±0.13, vs. 0.15±0.06 µmol/l and 0.43±0.10, vs. 0.13±0.05 µmol/l, respectively; P<0.05). The level of total bilirubin was reduced following indirect ligand PB injection whereas only a marginal alteration was observed in the level of direct bilirubin (Table II). Furthermore, the levels of total and direct bilirubin in the EtOH/DS group (EtOH/PB vehicle) were downregulated, compared with those in the EtOH group (0.45±0.06, vs. 0.75±0.17 µmol/l and 0.2±0.08, vs. 0.45±0.13 µmol/l, respectively; P<0.05). Taken together, chronic alcohol consumption led to ALD, and increased the levels of total and direct bilirubin. In addition, the CAR agonists, TCPOBOP and PB, decreased total and direct bilirubin levels.

H&E staining and oil red O staining revealed that chronic alcohol ingestion caused liver macrovesicular and microvesicular steatosis, cellular edema and ballooning degeneration, and nuclear pycnosis. These liver injuries were ameliorated by the CAR ligands, TCPOBOP and PB (Fig. 1A and B). In addition, TCPOBOP treatment resulted in hepatocyte enlargement, based on an elevated liver/body weight ratio (P<0.001; Fig. 1C). Chronic alcohol intake also significantly dysregulated the liver/body weight ratio, as shown in Fig. 1C (P<0.01).

The mRNA and protein levels of the primary mouse hepatic bilirubin uptake transporters, OATP1A4 and OATP1B2, remained unaltered following 4 weeks of chronic alcohol consumption (Fig. 2A and C). However, the mRNA level of hepatic OATP1A1 in the EtOH group was markedly reduced by ~60-fold (Fig. 2A). The protein levels of OATP1A1 were decreased by 60%, compared with those of the pair-fed group (Fig. 2C), in accordance with the downregulated mRNA levels (Fig. 2A). Furthermore, alcohol intake induced the mRNA and protein expression of bilirubin-glucuronidating UGT1A1 4.9- and 1.5-fold (Figs. 2B and C), respectively, as previously observed in a rat model of ALD (5). The mRNA levels of the excretion pump, MRP2, were significantly decreased 2.2-fold (Fig. 2B), whereas the protein level of MRP2 was deregulated (P<0.05; Fig. 2C), compared with that of the pair-fed group.

OATP1A1, OATP1A4 and MRP2 also have been detected in the apical membrane of renal proximal tubular cells (10,17,23,24). Under pathological conditions, including cholestasis, bilirubin glucuronides are secreted into the plasma and then eliminated by the kidney (10). Adaptive renal transporter induction has been confirmed in mouse cholestatic models of common bile duct ligation (23). To investigate the extrahepatic pathway of bilirubin clearance in the ALD mouse model, the present study examined the renal expression of these transporters. The mRNA and protein expression levels of OATP1A1 in the kidney were decreased by 25- and 1.6-fold, respectively, compared with those in the pair-fed group (Figs. 2D and E). The expression levels of renal OATP1A4 and MRP2 remained unaffected (Figs. 2D and E). Previous reports on renal bilirubin transport in an ALD mouse model are limited.

The present study also examined whether the expression of CAR was affected by chronic alcohol ingestion. The alcohol-fed mice showed limited variation in mRNA and protein levels of CAR in livers of the alcohol-fed mice, whereas the protein level of CAR in nuclear extracts was negatively regulated by 60%, compared with that of the pair-fed group (Fig. 2B and F), suggesting that alcohol inhibited CAR translocation to the nucleus.

To determine the role of the TCPOBOP and PB CAR ligands in the regulation of bilirubin clearance in ALD mice, TCPOBOP, PB and their vehicles were administered (i.p.). A marked increase was observed in the mRNA expression levels of liver Oatp1a1, Oatp1a4 and Ugt1a1 in the EtOH/PB group, compared with that of the vehicle EtOH/DS group (2.5-, 1.3- and 4.6-fold, respectively) whereas no significant change in the levels of Oatp1b2 or Mrp2 were observed (Fig. 3A and B). An increase in the mRNA expression of Mrp2 of >2-fold was observed in the EtOH/TCP group, compared with its vehicle EtOH/CO group, whereas the mRNA levels of Oatp1a1, Oatp1a4, Oatp1b2 and Ugt1a1 were not altered significantly (Fig. 3A and B). Accordingly, the protein levels of UGT1A1 in the liver were induced following treatment with TCPOBOP and PB, compared with the levels in their vehicles (1.5- and 2-fold, respectively, P<0.05; Fig. 3C). MRP2 was also increased significantly in the EtOH/TCP group (1.4-fold; P<0.05) and marginally induced in the EtOH/PB group (1.3-fold; P>0.05; Fig. 3C). No significant enhancement in the relative protein levels of OATP1A1, OATP1A4 or OATP1B2 were found in the EtOH/TCP or EtOH/PB groups (Fig. 3C).

To further elucidate the effect of CAR ligands on the extrahepatic bilirubin clearance pathway in ALD mice, the present study detected the renal expression of OATP1A1, OATP1A4 and MRP2 transporters. The mRNA levels of Oatp1a1 and Oatp1a4 remained unchanged following treatment with the two agonists, whereas the mRNA level of Mrp2 was induced 2-fold by TCP (Fig. 3D), with no clear difference in protein levels (Fig. 3E).

The present study also determined whether the expression of CAR was affected by CAR ligands in the ALD mice. The total liver mRNA and protein expression levels of CAR were not induced by TCPOBOP or PB treatment (Fig. 3B and F), as described previously (17). However, it was found that the protein expression of CAR in the nuclear extract was increased 1.6-fold following PB administration (Fig. 3F), as previously described (18). However, no significant alteration in the protein level of CAR was observed in the nuclear extract following TCP administration, compared with that of its vehicle.

To further determine whether alcohol suppressed CAR translocation to the nucleus, the present study measured the expression of CAR using immunofluorescence analysis in normal mouse hepatocytes. As shown in Fig. 4, fluorescence was observed in the alcohol-treated cells, although few fluorescence dots were identified in the nuclei of the alcohol-treated cells. These results indicated that alcohol impaired the PB-induced translocation of CAR from the cytoplasm to the nucleus.