In the present study, male C57BL/6 mice (6–8 weeks old; 15–20 g; n=30, 6 per group; Beijing HFK Bioscience Co., Ltd., Beijing, China) were raised in specific pathogen-free conditions (23±1°C; relative humidity, 39–43%; 12 h light/dark cycle; free access to food and water). Animal care and experimental protocols were conducted in accordance with guidelines provided by the Institutional Animal Care and Use Committee of Sichuan University (Chengdu, China). Ethics approval was obtained from the Institutional Ethics Committee of Sichuan University. Prior to subjecting the mice to 70% PH, mice were anesthetized with a subcutaneous injection of pentobarbital at a dose of 80 mg/kg. For the study group, Nam (250 mg/kg per day) was administered 6 h prior to PH via intraperitoneal injection and once daily subsequently for a total of 3 days. Control animals were treated with the same frequency and volume of PBS as Nam. All of the study mice were sacrificed at the indicated time points (24, 36, 48 and 72 h) and the liver samples were harvested and stored in liquid nitrogen. Liver recovery rates were calculated as the ratio of the regenerating liver weight to the mean liver weight prior to PH. The serum samples (1 ml) were additionally collected from the angular veins of the mice. Blood glucose (Glu), triglyceride (TG), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using enzymatic methods (ADVIA 165; Siemens Healthineers, Erlangen, Germany).

To obtain the total protein, the liver tissues were frozen in liquid nitrogen, washed twice with PBS, and homogenized in radioimmunoprecipitation assay lysis buffer (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The samples were incubated on ice for 30 min prior to centrifugation at 4°C for 15 min at a speed of 12,000 × g. Subsequent to centrifugation, the supernatant was divided into small fractions and stored at −80°C. The protein content of each supernatant was quantified using the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's protocol. Western blot analysis was performed as described previously (26). Cyclin D1 (1:1,000; rabbit monoclonal; cat. no. ab134175), PCNA (1:1,000; mouse monoclonal; cat. no. ab29), and SIRT1 (1:1,000; mouse monoclonal; cat. no. ab110304) primary antibodies were obtained from Abcam (Cambridge, UK); β-actin (1:1,000; mouse monoclonal; cat. no. sc-69879), as well as the secondary antibodies [horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit; both 1:5,000; cat. no. sc-2005 and sc-2004] were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Densitometric analysis was performed using ImageJ software (version 1.48; National Institutes of health, Bethesda, MD, USA).

For immunohistochemical analysis and morphological examination [hematoxylin and eosin (H&E) staining], formalin-fixed (10% buffered formalin at 4°C overnight), paraffin embedded liver tissues were sectioned to 5 µm. H&E staining was performed as described previously (27).

For immunohistochemical analysis, sections were blocked with 5% bovine serum albumin (Biowest, Nuaillé, France) diluted in PBS (pH 7.4) for 1 h at 37°C. Proliferation marker protein Ki-67 (Ki-67) staining was performed with an anti-Ki-67 antibody (1:4,000; rabbit polyclonal; cat. no. ab15580; Abcam) at 4°C overnight. Next, slides were incubated with the HRP-conjugated goat anti-rabbit antibody at 37°C for 1 h. Liver sections examined at each time-point were from at least three individual animals in the treatment and control groups. Images were captured with the AX10 imager A2 fluorescence microscope (magnification, ×40; Carl Zeiss AG, Oberkochen, Germany). Percentage Ki-67 expression was evaluated in five random fields from each immunostained HCC section. Quantification of Ki-67 staining was performed using Image-Pro Plus version 6.0 (National Institutes of Health, Bethesda, MD, USA).

The Kyoto Encyclopedia of Genes and Genomes database (https://www.kegg.jp) was used to explore the potential genes regulated by SIRT1.

To detect the deacetylation of peroxisome proliferator-activated receptor g coactivator 1-α (PGC-1α) and oxysterols receptor liver X receptor-α (LXRα) by SIRT1, lysine acetylation was analyzed by immunoprecipitation and western blotting, using an acetyl-lysine antibody. Primary antibodies against PGC-1α, (1:1,000; rabbit polyclonal; cat. no. ab54481) LXRα (1:1,000; mouse monoclonal; cat. no. ab41902) and acetyl-lysine (1:1,000; rabbit polyclonal; cat. no. ab21623) were purchased from Abcam. HRP-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were used, as described above, and the western blotting procedure was the same as aforementioned (26). An immunoprecipitation assay was performed as previously described (28,29) using the Pierce^™ Classic Immunoprecipitation kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA). A Student's t-test was performed to compare continuous values between the treatment and control groups. Results are presented as the mean ± standard deviation of three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of Nam on the process of liver regeneration, 70% PH was conducted on the treatment and control groups. As demonstrated in Fig. 1A, the Ki-67 immunohistochemistry staining was performed to validate hepatic cell proliferation following PH. It was observed that when compared with the control group, increased active proliferation was observed in the treatment group at 24–72 h post-operation. Furthermore, quantification of the Ki-67 staining revealed a significant restoration of hepatocyte proliferation in the treatment group 36–72 h following surgery (Fig. 1B; P<0.05). The treatment group additionally exhibited higher liver recovery rates compared with the control group (Fig. 1C). These results suggested that the proliferation of primary hepatocytes was enhanced in the presence of Nam.

Cell cycle progression serves a vital role during liver regeneration (30). To examine the cell cycle of proliferating hepatocytes following PH, the expression of G₁/S-specific cyclin D1 and proliferating cell nuclear antigen (PCNA), two key regulators of the cell cycle, was analyzed by western blotting (Fig. 2A). It was identified that the expression levels of cyclin D1 and PCNA reached a peak at 72 h in the livers of mice from the treatment and control groups. Additionally, the livers of mice injected with Nam exhibited significantly increased expression of cyclin D1 at 24, 36, 48 and 72 h and PCNA at 0 and 24 h when compared with the control (Fig. 2B and C; P<0.05). These results suggested that Nam may accelerate liver regeneration following PH.

A previous study reported that severe liver damage may occur in short time periods following PH (31). To assess whether Nam may reduce liver damage following PH, H&E staining was conducted on liver tissue sections. The results of the H&E staining revealed that the liver tissues of the control group exhibited evident swelling, fatty degeneration and nuclear condensation of hepatocytes 48 h following PH, along with severe sinusoidal narrowing (Fig. 3A). By contrast, sections from the treatment group demonstrated patent sinusoids, and better preservation of the cytoplasm and nuclear morphology, suggesting that treatment with Nam may repair liver damage following PH.

ALT and AST are markers of liver injury following PH. According to a previous study, these two markers increase markedly following PH (31). To further determine whether Nam has the potential to reduce liver injury following PH, the serum content of ALT and AST was analyzed (Fig. 3B and C). It was observed that the serum levels of ALT and AST were significantly decreased following PH in the treatment group compared with the control group (Fig. 3B and C; P<0.001), suggesting that Nam may protect liver function following trauma caused by PH.

Our previous study demonstrated that the addition of Nam promoted the expression of SIRT1, a key regulator of liver regeneration, suggesting that Nam may promote liver regeneration by upregulating the expression of SIRT1 (25). To verify the mechanisms of Nam in promoting liver regeneration, western blotting analysis was conducted to determine the expression of SIRT1 in the treatment and control groups. As demonstrated in Fig. 4A and B, although the expression of SIRT1 following PH increased in the two groups in a time-dependent manner, the addition of Nam further facilitated the significantly increased expressions of SIRT1 24–72 h following PH (Fig. 4A and B; P<0.05).

Glu and TG metabolisms have been reported to be a prerequisite for liver regeneration (23,32). Previous studies have demonstrated that the levels of Glu and TG are modulated by SIRT1 during the process of liver regeneration (24,33,34). To further elucidate the mechanisms of Nam in promoting liver regeneration, the levels of TG and Glu from serum samples were analyzed. Compared with the control group, the levels of Glu and TG were restored faster in the treatment group (Fig. 4C and D), which was consistent with the result that the expression of SIRT1 was upregulated by Nam. These results suggest that Nam may elevate the levels of Glu and TG by promoting the expression of SIRT1, thereby accelerating the process of liver regeneration.

To further investigate the mechanism underlying SIRT1-mediated metabolic processes following treatment with Nam, bioinformatics analysis was conducted (data not shown), which identified two substrates of SIRT1, PGC-1α and LXRα, which may be deacetylated by SIRT1 and have been previously implicated in the process of glucogenesis and lipid synthesis, respectively (35,36). As demonstrated in Fig. 5A and C, compared with the control group, mice injected with Nam demonstrated a significant decrease in the lysine acetylation level of PGC-1α (P<0.01), suggesting that Nam may regulate Glu levels through the SIRT1-PGC-1α signaling pathways. Likewise, it was additionally observed that there was a significant decrease in the lysine acetylation level of LXRα in the treated group (Fig. 5B and D; P<0.01), suggesting that Nam may regulate lipid levels through the SIRT1-LXRα signaling pathways.