All study protocols were approved by the Animal Ethics Committee of The Affiliated Hospital of Guizhou Medical University (Guiyang, China). A total of 40 specific-pathogen-free C57BL/6 male mice (weight, 20±2 g; age, 6–7 weeks) were acquired by the Experimental Animal Center of Guizhou Medical University and maintained on standard chow in an 25.1±1°C atmosphere with a 12-h dark/light cycle. Next, 40 mice were random allocated into four groups as follows: Control, CCl₄-induced cirrhosis model, 7.5 mg/kg ellagic acid and 15 mg/kg ellagic acid groups. In the control group, mice were given normal saline (100 µl; 11 weeks; via oral gavage, once daily for two days). CCl₄ (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was diluted into olive oil at a final concentration of 10%, and then this was used to induce a cirrhosis model by administration three times a week for 6 weeks, at a dose of 2 ml/kg each time (intraperitoneal injection). CCl₄-injected mice were divided into a further three groups (n=10 each) after 6 weeks of CCl₄ administration, including the cirrhosis model, 7.5 mg/kg ellagic acid and 15 mg/kg ellagic acid groups for 5 weeks.

Whole blood samples were collected from the eye socket using 2–3% chloral hydrate and centrifuged at 1,000 × g for 20 min at 4°C to collect serum. Serum levels of alanine aminotransferase (ALT; C009-2), aspartate aminotransferase (AST; C010-2) and albumin (ALB; A028-1; all Nanjing Jiancheng Biology Engineering Institute) were read at a wavelength of 560 nm using a spectrophotometer (GE Healthcare Life Sciences, Chicago, IL, USA).

Following treatment with ellagic acid, mice were sacrificed via decollation after anesthetization with 30 mg/kg pentobarbital sodium (Sinopharm Chemical Reagent Co., Ltd.). Following treatment with ellagic acid, liver tissue samples were acquired using 10% chloral hydrate and fixed with 4% formaldehyde. Next, the tissue samples were dehydrated in a graded ethanol series, cleared in xylene and embedded in paraffin. The tissues were then sectioned into 4-µm samples and stained hematoxylin and eosin (H&E). Images were captured using a microscope (TCS SP2; Leica Microsystems GmbH, Wetzlar, Germany).

Total RNA from liver tissue samples was isolated using TRI reagent (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer's protocol. Next, 2 µg total RNA was subjected to reverse transcription using a c-DNA synthesis kit (Revert Aid First Strand c-DNA synthesis kit; Fermentas; Thermo Fisher Scientific, Inc., Waltham, MA, USA). cDNA was then amplified by polymerase chain reaction (PCR) using specific primers of collagen I: Forward, 5′-CAGAGTGGAAGAGCGATTA-3′, and reverse, 5′-CAAGGACAGTGTAGGTGAA-3′. Amplification was performed in a DNA Thermal Cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.) at 95°C for 10 min, followed by 40 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 40 sec.

Liver tissue (50 mg) sections were collected and homogenized for 10 sec at 12,000 × g for 10 min at 4°C. Total protein in the samples was measured using a BCA assay reagent (Beyotime Institute of Biotechnology, Nanjing, China). Subsequently, 30 µg protein was used to measure ROS formation (S0033; Beyotime Institute of Biotechnology) Cells were incubated with DCFH-DA for 20 min at 37°C, and washed with PBS. ROS formation was measured at a wavelength of 488 nm and emission wavelength of 525 nm using a spectrophotometer (GE Healthcare Life Sciences, Chicago, IL, USA).

Whole blood samples were collected from the eye socket using 10% chloral hydrate and centrifuged at 1,000 × g for 20 min at 4°C to collect serum. After cooling to room temperature, 150 µl serum was used to detect the activities of glutathione peroxidase (GSH-Px; A005), glutathione (GSH; A006-2), superoxide dismutase (SOD; A001-3) and malondialdehyde (MDA; A003-1; all Nanjing Jiancheng Biology Engineering Institute) using ELISA kits and a spectrophotometer (GE Healthcare Life Sciences) according to the manufacturer's instructions.

Following treatment with ellagic acid, liver tissue samples were acquired and homogenized for 10 sec at 12,000 × g for 10 min at 4°C. Total protein was measured using a BCA assay reagent (Beyotime Institute of Biotechnology), and then 50 µg protein was subjected to 10–12% SDS-PAGE and electrotransferred onto a polyvinylidene difluoride membrane (BD Biosciences, San Jose, CA, USA). The membrane was blocked in 5% non-fat milk in phosphate-buffered saline (PBS; pH 7.4) for 2 h, and subsequently incubated overnight at 4°C with the following primary antibodies: Anti-inducible nitric oxide synthase (iNOS; 1:400; sc-649; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-vascular endothelial growth factor (VEGF; 1:500; sc-13083; Santa Cruz Biotechnology, Inc.), anti-VEGF receptor 2 (VEGFR2; 1:3,000; ab11939; Abcam, Cambridge, UK) and β-actin (1:500; sc-7210; Santa Cruz Biotechnology, Inc.). Next, the membranes were washed with PBS and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:5,000; 7074; Cell Signaling Technology, Inc., Danvers, MA, USA) at room temperature for 2 h. Protein expression in the samples was detected by Amersham ECL Prime western blotting detection reagent (GE Healthcare Life Sciences, Piscataway, NJ, USA) and analyzed using AlphaEase FC (FluorChem FC2) software (Cell Biosciences Inc., Santa Clara, CA, USA).

Following treatment with ellagic acid, liver tissue samples were acquired and homogenized via centrifugation at 12,000 × g for 10 min at 4°C. Total protein was measured using BCA assay reagent (Beyotime Institute of Biotechnology), and 5 µg protein was incubated with Ac-DEVD-pNA-caspase-3 activity kit (C1115; Beyotime Institute of Biotechnology) for 30 min at room temperature. The absorbance values were assessed at 405 nm using a spectrophotometer (GE Healthcare Life Sciences) according to the manufacturer's protocol.

Data are expressed as the mean ± standard error of the mean. Statistically significant differences between the groups were determined using the one-way analysis of variance test, followed by the post-hoc Tukey's test. P<0.05 was considered to indicate statistically significant differences.

At the beginning of the experiment, the protective effects of ellagic acid against CCl₄-induced cirrhosis were determined. As shown in Fig. 2 (P=0.0018, 0.0073 and 0.0089), the serum levels of ALT, AST and ALB in the cirrhosis model mice were effectively increased when compared with those in the control group. Following treatment with ellagic acid for 5 weeks, the serum levels of ALT, AST and ALB were effectively reduced in the cirrhosis mice (Fig. 2; P=0.0056, 0.0093 and 0.0072). These data suggest that ellagic acid reduced ALT, AST and ALB serum levels and thus may serve as an adjuvant for the treatment of cirrhosis.

H&E staining of liver tissue samples was used to analyze the effect of ellagic acid in the cirrhosis mice. As shown in Fig. 3, CCl₄-induced cirrhosis was markedly observed in mice of the model group, compared with the control mice. However, ellagic acid treatment was found to effectively inhibit the cirrhosis degree compared with the model mice (Fig. 3). These data suggest that ellagic acid remitted hepatic histopathological changes and thus may be a novel therapeutic option for the treatment of cirrhosis.

The collagen I gene expression in CCl₄-induced cirrhosis model mice was detected by PCR analysis and found to be significantly increased compared with the control group (Fig. 4; P=0.0021). However, treatment with ellagic acid effectively inhibited the collagen I gene expression in cirrhosis mice (Fig. 4; P=0.0056). These data suggest that ellagic acid reduced collagen I gene expression and may thus be used to treat cirrhosis.

iNOS protein expression in tissue samples is shown in Fig. 5. CCl₄ significantly induced iNOS protein expression in cirrhosis mice, compared with that of the control group (P=0.0023). However, administration of ellagic acid significantly suppressed the protein expression of iNOS in cirrhosis mice (Fig. 5; P=0.0081 and 0.0066). These data suggested that the protective effects of ellagic acid may prevent against CCl₄-induced cirrhosis through the suppression of iNOS expression.

CCl₄ administration to induce cirrhosis in mice was observed to inhibit the activities of GSH-PX (P=0.0022), GSH (P=0.0034) and SOD (P=0.0037), whereas it increased the MDA (P=0.0014) level in cirrhosis mice, when compared with the control group levels (Fig. 6). By contrast, treatment with ellagic acid for 5 weeks significantly reversed the GSH-PX (P=0.0082 and 0.0037), GSH (P=0.0065 and 0.0024), SOD (P=0.0056 and 0.0029) and MDA (P=0.0052 and 0.0041) activities in cirrhosis mice (Fig. 6). These data suggest that ellagic acid may have anti-oxidative effects and thus be used in the treatment of cirrhosis.

ROS formation was determined in order to evaluate the protective effect of ellagic acid against ROS formation in CCl₄-induced cirrhosis mice. The ROS formation was significantly increased in the untreated cirrhosis mice compared with that of the control group (Fig. 7; P=0.0023). However, administration of 7.5 or 15 mg/kg ellagic acid significantly inhibited ROS formation in the cirrhosis mice (Fig. 7; P=0.0062 and 0.0046). These data indicated that ellagic acid suppressed ROS formation to inhibit oxidative stress in mice with CCl₄-induced cirrhosis.

In addition, the present study examined the mechanism of ellagic acid against VEGF expression in CCl₄-induced cirrhosis mice by western blot analysis. A significant decrease in VEGF protein expression was identified in the cirrhosis model mice, compared with the control group (Fig. 8; P=0.0023). However, treatment with ellagic acid significantly induced the VEGF protein expression in the cirrhosis mice (Fig. 8; P=0.0076 and 0.0037).

The protective effect of ellagic acid against VEGFR2 expression in CCl₄-induced cirrhosis mice was further investigated using western blot analysis. As shown in Fig. 9, the protein expression of VEGFR2 in cirrhosis model mice was significantly higher compared with that of the control group (P=0.0027). Treatment with 7.5 and 15 mg/kg ellagic acid significantly decreased the protein expression of VEGFR2 in the cirrhosis mice (Fig. 9; P=0.0059 and 0.0043). These data indicate that ellgaic acid may function via VEGF to exert its protective effects against cirrhosis.

The anti-apoptosis effect of ellagic acid treatment against cirrhosis in mice was examined by recording the caspase-3 activity using an ELISA kit. Compared with the control group, the caspase-3 activity was significantly enhanced in cirrhosis model mice (Fig. 10; P=0.0013). Treatment with ellagic acid, however, significantly reduced caspase-3 activity in the cirrhosis mice (Fig. 10; P=0.0041 and 0.0026).