Male Sprague-Dawley rats (weight, 250–300 g) were obtained from the Experimental Animal Center of Jiamusi University (Jiamusi, China) and housed in a temperature- and light-controlled room at 19-22°C with a 12 h light/dark cycle. The animals had free access to food and water. The animal study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Jiamusi University.

Rats were fasted for 12 h and assigned to one of the following three groups: Control group (rats were fed with normal diet), bile acid group (rats were fed with normal diet plus 0.2% bile acid) and glucose group (rats were fed with normal diet plus 10% glucose). There were 36 rats in each group. At day 7, rats were subjected to 50 or 70% hepatectomy and the weight of removed liver tissues was recorded as mR. At 24, 48 or 72 h following hepatectomy, the rats were sacrificed by cervical dislocation following anesthesia induction, and liver tissues and blood were collected immediately. Total liver mass was calculated based on the ratio of removed liver weight and volume, and recorded as mT. That is, for rats in which 50% of the was liver removed, the weight of the removed portion was divided by 0.5 to obtain the total volume, and for those in which 70% was removed, it was divided by 0.7. The liver weight of sacrificed rats was recorded as mS and the hepatic regeneration rate was calculated as [mS-(mT-mR)]/mT × 100.

Fresh liver samples were immediately fixed in neutral formalin and embedded in paraffin. Paraffin-embedded liver samples were cut into 5-μm sections deparaffinized in xylene, rehydrated in serial dilutions of ethanol, placed in antigen retrieval solution and microwaved at low power for 10 min. The activity of endogenous peroxidase was blocked by incubation with 3% H₂O₂ (Sinopharm Chemical Reagent, Shanghai, China). A mouse monoclonal antibody against proliferating cell nuclear antigen (PCNA; 1:50; sc-252820; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was added and incubated at 4°C overnight. Sections were then incubated with a horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (1:200; A0216; Beyotime Institute of Biotechnology, Haimen, China) for 30 min at 37°C. Positive signals were visualized by 3,3′-Diaminobenzidine (Solarbio, Beijing, China) and sections were then counterstained with hematoxylin. Images of the sections were captured using a microscope (x400; BX51, Olympus, Tokyo, Japan). A brown or yellow color was regarded as a positive reaction.

Hepatocyte isolation was performed as previously described (13). Briefly, following administration of anesthesia with 10% chloral hydrate (Sinopharm Chemical Reagent), a cannula was introduced into the portal vein and D-Hank’s solution (Solarbio) was perfused in order to remove the blood. Collagenase (0.05%; Invitrogen, Carlsbad, CA, USA) was then perfused to hydrolyse the collagen molecules. The liver was removed from the capsule, cut into sections and passed through a 200 mesh sieve. The viability of isolated hepatocytes was >95%, as assessed by trypan blue (Beyotime Institute of Biotechnology) exclusion. Primary hepatocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) at 37°C in a humidified 5% CO₂ atmosphere. The medium was replaced every 2 days, and cells were passaged when they reached 80–90% confluence. The experiments were initiated following the third passage, with cells being divided into nine groups: The control group (hepatocytes were cultured in normal medium); the bile acid group (hepatocytes were cultured in normal medium with 40 μmol/l bile acid; Sigma-Aldrich, St. Louis, MO, USA); the glucose group (hepatocytes were cultured in normal medium with 25 mmol/l glucose); the FXR agonist group (hepatocytes were cultured in normal medium with 10 μmol/l FXR agonist, GW4064; Sigma-Aldrich); the FXR antagonist group (hepatocytes were cultured in normal medium with 100 μmol/l FXR antagonist, Guggulsterones; Sigma-Aldrich); the bile acid and FXR agonist group (hepatocytes were cultured in normal medium with 40 μmol/l bile acid and 10 μmol/l FXR agonist, GW4064); the bile acid and FXR antagonist group (hepatocytes were cultured in normal medium with 40 μmol/l bile acid and 100 μmol/l FXR antagonist, Guggulsterones); the glucose and FXR agonist group (hepatocytes were cultured in normal medium with 25 mmol/l glucose and 10 μmol/l FXR agonist, GW4064); and the glucose and FXR antagonist group (hepatocytes were cultured in normal medium with 25 mmol/l glucose and 100 μmol/l FXR antagonist, Guggulsterones). Cells were incubated at 37°C with 5% CO₂ and saturated humidity conditions in a culture box (Heal Force, Shanghai. China) and collected at 72 h.

The levels of TBA in liver and serum were measured with the TBA assay kit (Nanjing Jiancheng Bioengineering Institute, China) according to manufacturer’s instructions. The levels of TG, TC, HDL, LDL and FFA in hepatocytes were assessed using an ELISA kit (Rat TG ELISA kit, Ximei, Shanghai, China; Rat TC ELISA kit, Rat HDL ELISA kit, Hyperheal, Shanghai, China; Rat LDL ELISA kit, J&L Biological, Shanghai, China; Rat FFA ELISA kit, Lianshuo Biological, Shanghai, China, respectively). Optical density was measured using a microplate reader (BioTek Instruments, Winooski, VT, USA).

Total liver protein was extracted with a radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, China) and total cellular protein was extracted with NP-40 buffer (Beyotime Institute of Biotechnology). Equal quantities of total protein (40 μg) were loaded and separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA) and blocked in 5% fat-free milk for 1 h. Membranes were incubated with primary antibody at 4°C overnight. The following primary antibodies were used: A rabbit polyclonal antibody to FXR (1:100; sc-13063; Santa Cruz Biotechnology, Inc.), a rabbit polyclonal antibody to Caveolin 1 [1:1,000 (animal) or 1:2,000 (cell); ab2910; Abcam, Cambridge, UK], a rabbit polyclonal antibody to ASBT (1:1,000; bs-4189R; Bioss, Beijing, China), a rabbit polyclonal antibody to BSEP (1:1,000; ab99088), a rabbit monoclonal antibody to NTCP (1:1,000; ab133670) (Abcam) a goat polyclonal antibody to SHP (1:100; sc-15283) and a rabbit polyclonal antibody to CYP7A1 (1:100; sc-25536) (Santa Cruz Biotechnology, Inc.). Membranes were then washed and incubated with goat anti-rabbit (A0208) or donkey anti-goat (A0108) HRP-conjugated secondary antibodies (1:5,000, Beyotime Institute of Biotechnology) at 37°C for 45 min. Immunoreactive bands were visualized by enhanced chemiluminescence solution (Qihai Biotech, Shanghai, China) according to the manufacturer’s instructions.

Data are presented as the mean ± standard deviation. Two-tailed Student’s t test was used to assess statistical significance. Data were analyzed using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

The restituted liver mass in the bile acid group was markedly increased, with a peak at 72 h, and was higher than that in the control group. By contrast, the hepatic regeneration rate of the rats fed with glucose was reduced compared with the control group (Fig. 1). The calculated regeneration rate of rats that survived 70% liver resection was less than that in the 50% resection group (survival, n=6 per group). PCNA is a protein marker of DNA synthesis that is involved in the initiation of cell proliferation. The expression of PCNA is correlated with the S-phase of the cell cycle. At 72 h following hepatectomy, the expression of PCNA was highest in the bile acid group (Fig. 2), which was in accordance with the results of the hepatic regeneration rate.

FXR is known to be an important receptor for bile acid and its activation affects lipid and glucose metabolism (14). Caveolin-1 is involved in the regulation of intracellular homeostasis, such as lipid metabolism, cell activation and cell proliferation (15). Based on the results of the initial experiments, which demonstrated the facilitation of hepatic regeneration by bile acid, the effect of bile acid on the expression of FXR and Caveolin-1 was investigated. As shown in Fig. 3, the protein expression of FXR and Caveolin-1 was elevated in response to administration of bile acid. By contrast, treatment with glucose decreased the expression of FXR and Caveolin-1.

Serum and liver bile acid levels were similar to those measured in a previous study (16). Oral administration of 0.2% bile acid for 7 days significantly increased the concentration of serum and liver bile acid, whereas the concentration decreased in the glucose-fed group (Fig. 4).

In order to investigate the effects of bile acid and FXR on lipid metabolism in hepatocytes, TG, TC, HDL, LDL and FFA levels in liver cells were measured (Table I). Levels of TG, LDL and FFA were reduced in the bile acid and FXR agonist groups, whilst they were increased in the glucose and FXR antagonist groups. By contrast, levels of TC and HDL were increased in the bile acid and FXR agonist groups, and decreased in the glucose and FXR antagonist groups. These differences were statistically significant (P<0.05).

In order to verify the mechanism by which bile acid and FXR affect lipid metabolism, the expression of FXR signaling- and lipid metabolism-associated proteins was determined. As shown in Fig. 5, the expression of bile salt export pump (BSEP), Caveolin-1 and small heterodimer partner (SHP) were elevated in response to bile acid and FXR agonist, whereas that of apical sodium-dependent bile acid transporter (ASBT), Na⁺/taurocholate cotransporting polypeptide and cholesterol 7α-hydroxylase (NTCP) and CYP7A1 were downregulated. The effects of glucose and the FXR antagonist on FXR signaling and lipid metabolism proteins were the opposite of those of bile acid and the FXR agonist.