A total of 120 ICR mice (n=24/group, n=6/time interval; male; 25–30 g; 8–15 weeks old) obtained from the Animal Experimental Center, Nanjing Drum Tower Hospital, Medical School of Nanjing University (Nanjing, China) were used for simple 70% PH experiments. Mice were housed under conditions of 60% humidity, a temperature of 18–22°C, a 12 h light/dark cycle and free access to food and water. Mice were fasted overnight prior to PH. Anesthesia was induced with chloral hydrate (10%; 350 mg/kg) by intraperitoneal (i.p.) injection. Following laparotomy, the median and left liver lobes were surgically removed resulting in 70% PH. Mice were sacrificed via carbon dioxide inhalation asphyxia to collect blood and liver specimens prior to PH (days −2 and 0) and following PH (days 1, 2, 3 and 5), and the recovery of liver mass was estimated by the liver-to-body weight ratios. The experimental design was examined and approved by the Experimental Animal Ethics Committee of the Nanjing Drum Tower Hospital, Medical School of Nanjing University (Nanjing, China).

All animals were divided randomly into 5 groups (n=24 per group): i) Sham; ii) Control; iii) Compound C (Selleck Chemicals, Houston, TX, USA), which is the AMPK inhibitor dorsomorphin; iv) FO [comprising 40% docosahexaenoic acid (DHA) and 40% eicosapentaenoic acid (EPA); Wuhan Shengtianyu Biotechnology, Wuhan, China]; and v) Compound C + FO. Mice in the Sham group had laparotomy and exposure of the liver, but no PH. Mice in the Control group had 70% PH without any treatment. Mice in the Compound C group had 70% PH and treatment with Compound C (8 mg/kg) by i.p. injection daily, beginning half an hour prior to the operation until the day prior to the indicated time. Mice in the FO group had 70% PH and treatment with FO (12 ml/kg) by oral gavage daily, beginning at 2 days prior to the operation until the day prior to the indicated time. Mice in the Compound C + FO group had 70% PH and treatment with FO (12 ml/kg) by oral gavage daily from 2 days prior the operation until the day prior to the indicated time and Compound C (8 mg/kg) by i.p. injection daily from half an hour prior to the operation until the day prior to the indicated time.

A total of 50 mg liver tissues were lysed in cold radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 1:100 volume of phenylmethylsulfonyl fluoride. Following lysis at 4°C for 1 h, cell lysates were centrifuged at 14,000 × g for 5 min at 4°C. The total protein concentration in the supernatant was determined by Bicinchoninic Acid assay (Beyotime Institute of Biotechnology). Proteins were separated by 10% SDS-PAGE (30 µg/lane) and transferred to a nitrocellulose membrane at 100 V for 60 min. Following blocking in TBS + 0.1% Tween-20 (TBST) buffer containing 5% bovine serum albumin (BSA; Absin Bioscience Inc., Shanghai, China) at 37°C for 1 h, the membranes were incubated in TBST containing 2% BSA and antibodies against Occludin (cat. no. ab64482; 1:1,000; Abcam, Cambridge, UK), Claudin-3 (cat. no. sc-17662; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), tight junction protein ZO-1 (ZO-1; cat. no. sc-10804; 1:1,000; Santa Cruz Biotechnology, Inc.), phosphorylated (p)-AMPK (cat. no. sc-33524; 1:1,000; Santa Cruz Biotechnology, Inc.), AMPK (cat. no. sc-33524; 1:1,000; Santa Cruz Biotechnology, Inc.) and BSEP (cat. no. sc-74500; 1:1,000; Santa Cruz Biotechnology, Inc.) at 37°C for 1 h. An antibody against β-actin (cat. no. 4970; 1:5,000; Cell Signaling Technology, Inc., Danvers, MA, USA) was used as an internal control. The membranes were washed three times with TBST and incubated with the following secondary antibodies at 37°C for 1 h: Goat anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (cat. no. BA1055; 1:5,000; Boster Biological Technology, Pleasanton, CA, USA), HRP-goat anti-mouse antibody (cat. no. BA1051; 1:5,000; Boster Biological Technology) and HRP-rabbit anti-goat antibody (cat. no. BA1060; 1:5,000; Boster Biological Technology). Following three washes with TBST, the target proteins were detected using an Enhanced Chemiluminescence kit (WBULS0500; EMD Millipore, Billerica, MA, USA). Densitometry analysis was performed using the Intel iPP 6.0 software (Intel Corporation, Santa Clara, CA, USA).

Paraffin-embedded liver tissue sections (~4 µm) were fixed in 4% paraformaldehyde at room temperature for 24 h, deparaffinized and dehydrated through graded ethanol. The sections were washed 3 times with PBS for 5 min each, and then treated with 3% hydrogen peroxide for 10 min at room temperature to block the endogenous peroxidase activity. The sections were subsequently washed 3 times with PBS for 5 min each. Following blocking with 5% BSA for 30 min at 37°C, sections were incubated with primary antibodies against Occludin, ZO-1, BESP or Ki-67 (1:200 dilution in PBS) overnight and then with a HRP-conjugated anti-rabbit immunoglobulin G secondary antibody (cat. no. BA1055; 1:100; Boster Biological Technology) for 1 h at room temperature. Finally, the sections were incubated with 3,3-diaminobenzidine reagent for 10 min, counterstained with hematoxylin, dehydrated and mounted for inverted phase contrast microscopy (CK30 OLYMPUS; Olympus Corporation, Tokyo, Japan) analysis.

Serum expression levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), albumin (ALB) and C-reactive protein (CRP) were determined by the Laboratory of Biochemistry, Nanjing Drum Tower Hospital, Medical School of Nanjing University (Nanjing, China).

GraphPad Prism software version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA) and PASW statistics version 18.0 (SPSS, Inc., Chicago, IL, USA) were used for statistical analyses. Results are expressed as the mean ± standard deviation. One-way analysis of variance followed by the Least Significant Difference post hoc test was used to detect the statistically significant variations between groups. Each experiment was performed in triplicate. P<0.05 was considered to indicate a statistically significant difference.

Serum levels of ALT, AST, TBIL, ALB and CRP were evaluated at 1, 2, 3 and 5 days following PH to evaluate postoperative hepatic function (Table I). Following PH, the serum levels of ALT and AST in the Control mice were significantly increased compared with Sham (P<0.05; Table I), followed by a gradual trend to be restored to normal over time. When PH was accompanied by perioperative FO supplementation, the restoration of serum ALT and TBIL levels was significantly faster than those in the Control group (P<0.05 from day 2 and day 3 post-PH, respectively; Table I). To further examine the mechanism involved in the effects of perioperative FO supplementation on liver function, AMPK was inhibited by i.p. injection of Compound C. The levels of ALT and AST were significantly higher in the Compound C group compared with the Control group on day 2 post-PH, with or without perioperative FO supplementation (P<0.05; Table I). These results indicated that liver function improvement following PH in mice with perioperative oral FO supplementation may be mediated through AMPK signaling.

To investigate the mechanisms of FO supplementation on improving liver function through AMPK activation, hepatocyte proliferation was examined following PH by immunohistochemical staining for the Ki-67 proliferation marker. The proportion of Ki-67 positive hepatocytes in the livers of the FO group was significantly increased compared with the Control group on days 1, 2 and 3 following PH (P<0.05; Fig. 1). On day 5 following PH, there was no difference between the FO group and the Control group (P>0.05; Fig. 1). Measurement of the liver-to-body weight ratio reflected the results of hepatocyte proliferation. Perioperative oral supplementation with FO significantly increased postoperative liver-to-body weight ratio in mice undergoing PH compared with Control (P<0.05; Fig. 2). To further examine the mechanism involved in the effects of perioperative FO supplementation on hepatocyte proliferation, AMPK was inhibited by use of Compound C by i.p. injection. The proportion of Ki-67 positive hepatocytes was significantly decreased in the livers of the Compound C-treated group compared with the Control group from day 1 to 3 following PH, which was not resolved by perioperative oral supplementation with FO (P<0.05 vs. Control; Fig. 1). The discrepant results of proliferation at day 5 may be explained in that the AMPK inhibitor may have delayed the process of hepatocyte proliferation and FO treatment was unable to reverse it, which was consistent with the liver-to-body weight ratio. The liver-to-body weight ratio was also decreased in the Compound C group compared with the Control group from day 1 to 5 following PH, and treatment with perioperative FO supplementation did not improve it (P<0.05; Fig. 2). These data suggested that perioperative oral supplementation with FO promoted hepatocyte proliferation in mice following PH and this effect was mediated through AMPK signaling.

To further investigate the effects of perioperative FO supplementation on liver regeneration following PH, the expression levels hepatocyte polarization markers were examined. Polarization was evaluated by western blot analysis and immunohistochemistry for the expression of epithelial differentiation markers Occludin, Claudin-3 and ZO-1, and the hepatocyte marker BSEP. Phosphorylation levels of AMPK were also evaluated as a marker of AMPK signaling activation. The ratio of p-AMPK/total AMPK and the protein expression levels of Occludin, Claudin-3, ZO-1 and BSEP were gradually increased between day 1 and day 5 following PH in the Control group (Figs. 3 and 4). Perioperative oral supplementation with FO further enhanced this change (Figs. 3 and 4). To evaluate the effects of AMPK signaling on tight junction and BSEP protein expression, AMPK was inhibited by i.p. injection with Compound C (Fig. 5). The level of p-AMPK expression was reduced in the Compound C group compared with the Control group (P<0.05; Fig. 5A and B), as was expression of Occludin, Claudin-3, ZO-1 and BSEP; however, the level of p-AMPK expression was still reduced in the Compound C+FO group compared with the Control group between days 2 and 5 (P<0.05; Fig. 5). In conclusion, the results demonstrated that perioperative FO oral supplementation promoted the expression of hepatocyte polarization markers in mice following PH and this effect was probably mediated through AMPK activation.