A total of 60 Sprague Dawley (SD) rats ((8–10 weeks; 30 males and 30 females) weighing 250–300 g were obtained from the Experimental Animal Center of Guilin Medical University (Guilin, China). The rats were kept in an environmentally controlled room with a temperature of 25±2°C, relative humidity of 55±10% and a 12-h light/dark cycle. The rats were allowed free access to food and water. The SD rats were randomly divided into six groups (n=10 in each). Rats in the control group and the CCl₄-treated model group only received an equivalent of distilled water containing 0.1% Tween 80 by oral gavage once a day for one week. Rats in the dimethyl diphenyl bicarboxylate (DDB)-treated group (positive control group) received DDB in distilled water containing 0.1% Tween 80 at a dose of 200 mg/kg body weight by oral gavage once a day for one week. Low, medium and high MFA-treated groups received MFA in distilled water containing 0.1% Tween 80 at a dose of 25, 50 or 100 mg/kg body weight by oral gavage once a day for a week. One hour after the last treatment, all rats in the CCl₄-treated model group, the DDB-treated group and the MFA-treated group received an intraperitoneal injection of CCl₄ (1 ml/kg body weight), while the control group received an equivalent volume of 0.9% physiological saline solution instead. At 24 h after CCl₄ treatment, all rats were sacrificed and a portion of liver tissues was immediately collected for analysis and placed in ice-cold 0.9% physiological saline solution to remove blood cells for ROS detection. The remaining liver tissues were immediately stored at −80°C for later use. The present study was performed in accordance with the Chinese legislation and the US National Institutes of Health guidelines for the use and care of experimental animals. All animal experiments were approved by the institutional ethical committee of Guilin Medical University (Guilin, China).

After blood collection, serum was separated by centrifugation at 3,200 × g for 20 min at room temperature. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum from rats were determined using commercially available diagnostic kits (Alanine aminotransferase assay kit; cat no. C009-2; Aspartate aminotransferase assay kit; cat no. C010-2; Nanjing Jiancheng Bio Co., Ltd., Nanjing, China) according to the manufacturer's instructions.

Liver tissue samples were homogenized in nine volumes of ice-cold 50 mM phosphate buffer (pH 7.4) and centrifuged at 3,200 × g for 20 min at 4°C. Supernatants were used to determine SOD, GSH-Px, MDA, CAT and total protein concentrations by using commercially available diagnostic kits (SOD assay kit; cat no. A001-3; GSH-PX assay kit; cat no. A005; cat no. MDA assay kit; cat no. A003-1; CAT assay kit; cat no. A007-1; total protein assay kit; cat no. A045-3; Nanjing Jiancheng Bio Co., Ltd.). The levels of MDA, GSH-Px, SOD and CAT were normalized to the content of total protein.

For histological examination, liver tissues were removed from a portion of the left lobe and fixed in 10% phosphate-buffered formalin. After being processed by routine histological procedures, the samples were cut into 5-µm slices. Sections were stained using hematoxylin for 5 min at 40°C and eosin solution for 1 min at room temperature, then the slides were observed for conventional morphological evaluation under a light microscope (BX41; Olympus, Tokyo, Japan) and images were captured at ×100 magnification. The degree of hepatic damage was evaluated. Histological changes were scored according to the following system: 0, no injury; 1, mild injury; 2, moderate injury; and 3, severe injury.

Total RNA was extracted from liver tissues using a tissue total RNA isolation kit (cat no. B518651; Shanghai Sangong Pharmaceutical Co., Ltd., Shanghai, China) according to the manufacturer's protocol. Total RNA was reversibly transcribed into complementary DNA (cDNA) using a cDNA synthesis kit (TIANScript MMLV; cat no. ER104; Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's protocol. An MJ PTC-200 PCR System (Bio-Rad, Hercules, CA, USA) and a qPCR kit (cat no. PC0902; 2× Taq PCR Master Mix; Aidlab Biotechnologies Co., Ltd., Beijing, China) were used based on the manufacturer's instructions for amplification of target genes. The primers used in the study are listed in Table I. The specific primers for target gene β-actin were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). As an internal standard control, the expression level of β-actin was simultaneously quantified. The PCR protocol was as follows: Initial denaturation for 3 min at 94°C; 30–40 cycles of denaturation for 30 sec at 94°C, annealing for 30 sec at 56–58°C, and extension for 1 min at 70°C; and a final extension for 5 min at 72°C. The PCR products were identified by electrophoresis using 1.5% agarose gel, and optical density of target gene bands was calculated in each sample using a Gel Doc XR+ automatic gel imaging analysis system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and with adjustment through β-actin correction to finally obtain the relative expression of target gene in each sample (25).

Total protein was extracted from liver tissues with radio immunoprecipitation assay lysis buffer (cat no. P0013B, Beyotime Institutute of Biotechnology, Shanghai, China). The Mitochondrial protein was extracted from liver tissue using a Cytoplasmic and Mitochondrial Protein Extraction kit (cat no. C500051; Sangon Biotech Co., Ltd., Shanghai, China). Protein concentration was determined using a bicinchoninic acid assay kit (Beyotime Biotechnology, Inc.). A total of 50 µg/lane of sample proteins were separated by 12% SDS-PAGE (Bio-Rad Laboratories, Inc. USA). The separated proteins were then transferred to pure nitrocellulose blotting membranes. The membranes were then blocked for 1 h with 5% bovine serum albumin (cat no. B600036; Sangon Biotech Co., Ltd., Shanghai, China) in Tris-buffered saline containing 0.05% Tween 20 (TBST) at room temperature. The membranes were incubated with anti-NOX4 (1:500; cat no. D121050), anti-Bcl-2 (1:1,000; cat no. D151442), anti-caspase-3 (1:1,000; cat no. D220074), anti-cleaved caspase-3 (1:500; cat no. D260009), anti-Bax (1:1,000; cat no. D220073), anti-GAPDH (1:1,000; cat no. D110016; all from Sangon Biotech Co., Ltd., Shanghai, China), anti-p22^phox (1:500; cat no. BS60290; Bioworld Technology, Inc., Nanjing, China), anti-phospho-p38 MAPK (1:1,000; cat no. 4511T), anti-p38 MAPK (1:1,000; cat no. 14451), anti-phospho-JNK (1:1,000; cat no. 4668T), anti-JNK (1:1,000; cat no. 9252T; all from Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-IL-1β (1:1,000; cat no. sc-52012), anti-TNF-α (1:1,000; cat no. sc-33639; both from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) primary antibodies at 4°C overnight. The samples were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (1:50,000; cat no. ZB2301; horseradish peroxidase (HRP) Affinipure Goat anti-rabbit immunoglobulin G or 1:50,000; ZB2305 HRP Affinipure Goat anti-Mouse immunoglobulin G; Zhongshang Goldenbridge Bio, Beijing, China) at room temperature for one hour and protein bands were visualized by enhanced chemiluminescence (cat no. E002-100; 7seapharmatech Co. Ltd, Shanghai, China). The imaging system Chemi Doc XRS+ (Bio-Rad Laboratories, Inc., USA) was used for imaging and quantitative analysis of the blots. VCDA1 or GAPDH protein was used as an internal control.

The level of ROS was determined by detecting the fluorescence intensity of the oxidant-sensitive probe 2,7-dichlorodihydrofluorescein diacetate (Molecular Probes; Thermo Fisher Scientific, Inc., Waltham, MA, USA) as described in a previous study (26). The amount of formed dichlorofluorescein in the clear supernatant was determined using a microplate reader (Infinite M200 PRO; Tecan, Zurich, Switzerland) at an excitation wavelength of 502 nm and an emission wavelength of 523 nm.

Tissue lipid peroxidation was measured using a TBARS colorimetric assay. Liver homogenate was incubated with 8.1% (w/v) SDS for 10 min, followed by addition of 20% acetic acid (pH 3.5). The reaction mixture was incubated with 0.6% TBA (w/v) for 1 h in a boiling water bath. Pink color chromogen was extracted in butanol-pyridine solution (15:1) and spectrophotometrically quantified at 532 nm.

Based on the oxidation of intracellular anti-oxidants with iron (III) in acidic medium, the TAC in the liver was assayed with a commercially available assay kit (cat no. A015-1; Nanjing Jiancheng Bio Co., Nanjing, China). The TAC of the samples was measured according to the manufacturer's protocol. One unit of TAC was defined as the capability of increasing the optical density value at 520 nm by 0.01 per mg protein per min at 37°C.

All statistical analyses were performed using SPSS software (version 17.0; International Business Machines, Corp., Armonk, NY, USA). One-way analysis of variance was used to determine significant differences between groups. The Student-Newman-Keuls test was used for comparisons between groups. Values are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

To determine whether MFA attenuates liver damage in CCl₄-treated rats, the activities of ALT and AST in serum were measured. Compared with those in the normal control, the activities of ALT and AST in serum from the model group were significantly increased (P<0.01). Of note, administration of MFA at all doses significantly inhibited the elevation of ALT levels, and MFA at 50 and 100 mg/kg significantly inhibited the elevation of AST levels in CCl₄-treated rats in a dose-dependent manner (P<0.05; Table II). These results suggested that MFA provides protection against CCl₄-induced liver injury.

To quantify oxidative liver injury, the hepatic levels of SOD, GSH-Px, MDA and CAT were assayed. The hepatic levels of MDA were assessed as an indicator of lipid peroxidation in oxidative liver damage, and CCl₄ treatment obviously increased the hepatic MDA levels compared with those in the control group (P<0.01), which was significantly inhibited by pre-administration of MFA (P<0.05; Table III). Furthermore, the results demonstrated that the activities of SOD, GSH-Px and CAT in the model group were significantly decreased compared with those in the control group, but pre-treatment with DDB or MFA (50 or 100 mg/kg) significantly increased the activities of SOD, GSH-Px and CAT compared with those in the model group (P<0.01). In addition, CCl₄ treatment significantly increased hepatic MDA levels compared with those in the control group (P<0.01), but pre-treatment with DDB or MFA significantly decreased MDA levels compared with those in the model group (P<0.05). Of note, the low dose of MFA had no significant effect on SOD or GSH-Px (Table III). These results indicated that MFA suppressed CCl₄-induced oxidative liver injury.

To detect histological changes in the liver, H&E staining was performed. Visual observation revealed that livers from the normal control group were reddish brown, soft and elastic, but livers from the CCl₄ model group exhibited significantly increased liver volume, blood stasis, edge thickening and liver surface with petechial hemorrhage. In the MFA (25 mg/kg) group, the liver was slightly enlarged, and spot bleeding was significantly reduced. By contrast, the appearance of the liver in the medium and high MFA groups was close to normal. H&E staining of liver sections from the normal control group demonstrated an intact hepatic lobular structure, normal hepatic cells with well-preserved cytoplasm, prominent nuclei, hepatocytes that were radially arranged around the central vein, well-defined sinusoidal line, uniform size, no degeneration, no necrosis, and hepatic cords that were arranged in neat rows. In addition, no inflammatory cell infiltration was observed (Fig. 1A). In the model group treated with CCl₄, typical pathological characteristics were observed, including destroyed hepatic lobule structure, liver sinus and central venous dilatation, hyperemia, a disorder in the arranged of hepatic cords, ballooning degeneration, broad infiltration of inflammatory cells, centrilobular fatty changes, apoptosis and widespread hepatocellular necrosis, particularly significant bridging necrosis, and inflammatory cell infiltration in hepatic lobules and portal area (Fig. 1B). By contrast, CCl₄-intoxicated rats pre-treated with DDB had nearly normal liver tissues with no significant changes in hepatocytes (Fig. 1C). In the low MFA group, liver sections exhibited moderate hypertrophy of hepatocytes with a relatively intact central vein, spotty necrosis, a rare large area of necrosis, shrinking sinusoidal line and reduced number of inflammatory cells (Fig. 1D). Of note, hepatic lesions were markedly ameliorated in the medium and high MFA groups, with slight inflammatory cell infiltration (Fig. 1E and F). The inflammation score of CCl₄-treated rats was significantly higher than that of normal control rats, while pre-treatment with MFA reduced the inflammation score (Fig. 1G). These results indicated that CCl₄ treatment caused obvious histological changes in the liver, while pre-treatment with MFA prevented CCl₄-induced damage.

To evaluate oxidative stress in the liver, the levels of ROS and TBARS as well as the TAC were measured. The results demonstrated that CCl₄ treatment markedly improved hepatic ROS and TBARS levels, while decreasing the TAC compared with those in the control group (P<0.01; Fig. 2A-C). Of note, pre-treatment with MFA significantly reduced CCl₄-induced ROS and TBARS expression and significantly increased the TAC (P<0.01; Fig. 2A-C). These results indicated that MFA inhibited oxidative stress induced by CCl₄ in rat livers.

To assess whether MFA affects the generation of ROS by inhibiting NOX4 and p22^phox in acute liver injury induced by CCl₄, the present study determined the expression of NOX4 and p22^phox in liver tissues. qPCR revealed that the levels of NOX4 and p22^phox mRNA in the livers of CCl₄-treated rats were significantly increased compared with those in the control group (P<0.01). By contrast, pre-treatment with DDB or MFA (100 mg/kg) decreased the expression of NOX4 and p22^phox mRNA compared with that in rats treated with CCl₄ only (P<0.01; Fig. 3A and B). Western blot analysis demonstrated significantly increased expression of NOX4 and p22^phox protein in the liver of CCl₄-treated rats compared with that in the control group (P<0.01). Of note, the protein expression of NOX4 and p22^phox was significantly reduced by pre-treatment with DDB or MFA (100 mg/kg) compared with that in rats treated with CCl₄ only (P<0.01; Fig. 3C and D). These results suggested that pre-treatment with MFA inhibited the mRNA and protein expression of NOX4 and p22^phox in the livers of rats treated with CCl₄.

To determine the expression of TNF-α and IL-1β in liver tissues, qPCR and western blot analysis were employed. The results demonstrated that CCl₄ treatment significantly increased the hepatic TNF-α and IL-1β mRNA and protein expression compared with that in the control group (P<0.01; Fig. 4A-D). Of note, pre-administration of DDB or MFA significantly suppressed the CCl₄-induced mRNA and protein expression of hepatic TNF-α and IL-1β (P<0.01; Fig. 4A-D). These results indicated that pre-treatment with MFA prevented CCl₄-induced pro-inflammatory responses by inhibiting the expression of TNF-α and IL-1β.

To investigate the effects of MFA on apoptosis induced by CCl₄, western blot analysis was used to determine the ratio of Bax/Bcl-2 and the ratio of cleaved caspase3/caspase3. The results demonstrated that CCl₄ treatment markedly increased the expression of Bax compared with that in the control group, while reducing the expression of Bcl-2, leading to a significantly increased Bax/Bcl-2 ratio. However, pre-treatment with MFA significantly decreased the CCl₄-induced expression of the pro-apoptotic protein Bax and prominently decreased the Bax/Bcl-2 ratio as compared with that in the CCl₄ treatment group (P<0.01; Fig. 5A and B). In addition, cleaved caspase3 levels in the livers of CCl₄-treated rats were significantly elevated as compared with those in the controls (P<0.01). However, pre-treatment with MFA significantly inhibited this CCl₄-induced elevation (P<0.01; Fig. 5C and D). These results suggested that MFA inhibits CCl₄-induced apoptosis in the livers of rats.

To investigate whether JNK and P38 MAPK signaling was involved in the mechanism of action of MFA, the present study investigated the effects of MFA on JNK and P38 MAPK in livers using western blot analysis. The results revealed that the levels of p-JNK and p-P38 MAPK were significantly increased in the livers of CCl₄-treated rats compared with those in the controls (P<0.01; Fig. 6A and B). However, this upregulation of p-JNK and p-p38 MAPK was significantly suppressed by pre-treatment with MFA or DDP (P<0.01; Fig. 6A and B). These results indicated that JNK and P38 MAPK activation is involved in the anti-apoptotic effects of MFA.