Thirty-two male C57BL/6J mice (6–8 weeks old, 18.5–20.3 g) were obtained from the Laboratory Animal Center of the National Yang Ming University (Taipei, Taiwan), and randomly assigned to one of four groups (n=8 per group). Two animals were housed in each cage and maintained at 25°C in under a 12-h light/dark cycle. All experimental procedures were approved by the Animal Care and Use Committee of the Chang Gung Medical Foundation (Chiayi, Taiwan) and complied with the NIH Guide for Care and Use of Laboratory Animals (National Research Council, 1996) (32). Mice in the four groups were administered daily, via oral gavage for 28 consecutive days, with the following: Ethanol (200 mg/kg); RSV (200 mg/kg; Sigma-Aldrich, St. Louis, MA, USA); ethanol + RSV (100 mg/kg each); or distilled water (control group). RSV was diluted in sterile water. Food and tap water were available ad libitum and body weight was recorded weekly throughout the experiment.

Following treatment, mice were anesthetized with CO₂ and blood samples were extracted following a 12-h overnight fast. Plasma was isolated by centrifugation at 1,500 × g for 15 min and was stored at −35°C for subsequent biochemical analyses. Plasma concentrations of total cholesterol and triglycerol were determined spectrophotometrically using commercial kits (Merck Millipore, Darmstadt, Germany) according to manufacturer's instructions. The liver was rapidly removed, blotted dry, weighed, frozen in liquid nitrogen and stored at −80°C until use. To analyze antioxidative enzyme activity and expression, the liver tissue was homogenized in 10 volumes of 50 mM phosphate buffer (pH 7.4; Sigma-Aldrich) on ice using a Polytron homogenizer (model 099C-K54; Glas-Col LLC, Terre Haute, IN, USA). The homogenate was transferred into centrifuge tubes and centrifuged at 9,000 × g at 4°C for 20 min. The supernatant was separated for use in the subsequent measurement of antioxidative enzyme activity.

Plasma levels of MDA, an oxidative stress marker, were monitored by quantifying thiobarbituric acid (TBA)-reactive substances as previously described (33). Briefly, 1 g liver tissue was homogenized in 10 ml 1.15% KCl buffer (Sigma-Aldrich). The homogenate was mixed with 1% H₃PO₄ (Sigma-Aldrich) and 0.6% TBA (Sigma-Aldrich), and heated at 100°C for 45 min. The samples were cooled to room temperature and combined with n-butanol (Merck Millipore). Following vigorous vortexing, the butanolic phase was centrifuged at 4,000 × g for 10 min. 1,1,3,3-Tetraethoxypropane (Merck Millipore) was used as the standard. The histology of hepatic microvesicular steatosis was assessed using the Oil Red O (Sigma-Aldrich) staining method as previously described (34).

HepG2 is a well-differentiated human hepatocarcinoma cell line commonly used in hepatic studies. HepG2 cells were provided by Prof. An-Na Chiang and cultured in Dulbecco's Modified Eagle's Medium (HyClone; GE Healthcare, Logan, UT, USA) containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (GE Healthcare). After 24 h of growth at 37°C in 5% CO₂, cells were treated with ethanol or RSV (50, 100, 200 or 400 mM) for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich) was used to evaluate the cytotoxic effects of ethanol and RSV (35). The ROS levels in HepG2 cells were measured using the dye, 2′,7′-dichlorodihydrofluorescein diacetate (Molecular Probes; Thermo Fisher Scientific, Inc., Waltham, MA, USA), as described previously (36). This reduced dye was added to cells at a final concentration of 10 µM. The fluorescence of the oxidized dichlorofluorescein was recorded, with an excitation wavelength of 488 nm and an emission wavelength of 525 nm, using flow cytometry (model FC500; Beckman Coulter, Inc., Brea, CA, USA). The results were expressed as the relative fluorescence intensity. Measurements of ROS levels without ethanol or RSV treatment were used as the control.

SOD activity in the liver extracts of C57BL/6 mice or HepG2 cells was assayed using the hydroxylamine reduction method (37). The hypoxanthine/xanthine oxidase system (38) was used to measure the reduction of hydroxylamine by O₂^•−, which was monitored at 550 nm. One unit of SOD activity was recorded as the quantity of enzyme required to decrease the reduction of hydroxylamine by 50%. Mouse liver or HepG2 cell CAT activity in the extract was assayed using the method described by Aebi (39). Decomposition of H₂O₂ resulting from CAT activity was assayed by monitoring H₂O₂ and the reduction in absorbance at 240 nm. One unit of CAT activity was recorded as the quantity of enzyme catalyzing 1 µmol H₂O₂ per min at 25°C. GPx activity was quantified according to a coupled enzyme (GPx and glutathione reductase) procedure (40), which measures the decrease in absorbance at 340 nm as NADPH is converted to NADP. One unit of GPx activity was recorded as the quantity of enzyme oxidizing 1 µmol NADPH per min. The specific activity of SOD, CAT and GPx are expressed as U/mg protein. The protein content of the liver or cell extract was determined using the Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA, USA) (41).

Protein levels of antioxidative enzymes and PPARs were determined using western blot analysis in HepG2 cells supplemented with ethanol and/or RSV for 24 h. Total cell protein was extracted using lysis buffer containing 1% Triton X-100, 50 mM HEPES, 6 mM EDTA, and 150 mM NaCl supplemented with complete protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Cell lysates were centrifuged, and the supernatants were collected. The protein concentration was determined using the Bradford method (Bio-Rad Laboratories, Inc.) using bovine serum albumin as a standard, and equal quantities of protein (30 µg) were analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA). The immunoblots were blocked with 5% non-fat milk and then incubated at 4°C for 16–32 h with the following primary antibodies: Rabbit anti-human SOD polyclonal antibody (1:5,000; cat. no. ab13533; Abcam, Cambridge, UK); rabbit anti-human CAT polyclonal antibody (1:2,000; cat. no. ab16731; Abcam); goat anti-human GPx IgG (1:1,000; cat. no. sc-22145; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); rabbit anti-human PPARα IgG (1:1,000; cat. no. sc-9000; Santa Cruz Biotechnology, Inc.); rabbit anti-human PPARβ/δ IgG (1:1,000; cat. no. sc-7197; Santa Cruz Biotechnology, Inc.); goat anti-human PPARγ IgG (1:1,000; cat. no. sc-1981; Santa Cruz Biotechnology, Inc.). Following the washing stage, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (Sigma-Aldrich) for 1 h at 4°C, and the target protein bands were visualized using Western Lightning Plus-Enhanced Chemiluminescence reagents (PerkinElmer, Inc., Waltham, MA, USA). The blots were then stripped for further probing, using β-actin (rabbit anti-human β-actin polyclonal antibody; cat. no. ab189073; Abcam) as an internal control. Relative intensities of protein bands were quantified by densitometry using ImageQuant software version 5.0 (Molecular Dynamics, Sunnyvale, CA, USA).

Total RNA was extracted from HepG2 cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Total RNA (2 µg) was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). The resulting cDNA (1 µg) was used as a template for qPCR analysis using SYBR Green Master Mixture (QuantiTect SYBR Green PCR kit 200; cat. no. 204143; Qiagen, Inc., Valencia, CA, USA) in a Roche LightCycler system (Roche Diagnostics GmbH). The specific primers (50 µM in 0.08 µl) for SOD, CAT and GPx genes were used as previously described (42). The following primer sequences were used: Cu/Zn-SOD forward, 5′-CAGGTCCTCACTTCAATCC-3′ and reverse, 5′-CCAAACGACTTCCAGCAT-3′; CAT forward, 5′-CGAAGGCGAAGGTGTTTG-3′ and reverse, 5′-AGTGTGCGATCCATATCC-3′; GPx forward, 5′-CACAACGGTGCGGGACTA-3′ and reverse, 5′-CATTGCGACACACTGGAGAC-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse, 5′-GAAGATGGTGATGGGATTTC-3′. Comparative assessment of target gene mRNA expression was performed using GAPDH mRNA as an internal control. Reaction conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 20 sec, 60°C for 20 sec and 72°C for 20 sec. Relative quantification of antioxidative enzyme mRNA was calculated using the Cq method (ratio = 2 ^{− (Cq (antioxidant enzyme) - Cq (GAPDH)}) as described previously (43).

HepG2 cells were transiently transfected with 0.2 µg tk-PPREx-Luc reporter plasmid and 0.2 µg PPARα (pGShPPARα), PPARβ (pCMX-hPPARβ/δ) or PPARγ (pCMX-hPPARγ) expression vectors, which were constructed by Prof. An-Na Chiang's laboratory. For each transfection, the internal control vector, pCMV-β-gal, was also co-transfected. The luciferase assay was measured using a luminometer (EG&G Berthold; Berthold Technologies USA LLC, Oak Ridge, TN, USA) and normalized against the activity of β-galactosidase (44).

Data are presented as the mean ± standard error. Each experiment was repeated at least three times. Statistical analysis between two groups was performed using the Student's t-test. One-way analysis of variance combined with Tukey's multiple-comparison test was used to evaluate the statistical significance of differences among the four groups. Statistical analysis was performed using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Compared with the control mice, no significant differences were detected in the body weight and concentrations of plasma cholesterol levels among the groups (Table I). However, plasma triglyceride and MDA levels were 75.3 and 56.1% higher, respectively, in the ethanol-treated mice in comparison with the control mice (Table I). After 28 days of treatment, mice in the RSV group presented a 79.5% increase in SOD activity; however, no change was observed in the ethanol and ethanol + RSV groups. Moreover, ethanol and RSV exerted no significant effect on CAT and GPx activity compared with the control group in the livers of C57BL/6 mice. Furthermore, lipid accumulation was markedly increased in the liver of mice treated with ethanol, moderately increased in mice treated with ethanol + RSV and remained unchanged in mice treated with RSV (Fig. 1).

No significant alteration of cell viability was detected by the MTT assay in the HepG2 cells exposed to ethanol, RSV or ethanol + RSV (Fig. 2A). By contrast, ethanol treatment appeared to enhance ROS production in a dose-dependent manner (Fig. 2B). However, no significant change in ROS production was observed in the ethanol + RSV group. This indicates that RSV is able to scavenge ROS from ethanol-induced ROS generation.

To assess whether antioxidative enzymes are involved in RSV-mediated protection against oxidative stress, the activity of SOD, CAT and GPx was determined in HepG2 cells. As presented in Fig. 3, SOD activity significantly increased in cells exposed to RSV in comparison with the control (P<0.05), whereas the activity of CAT and GPx was not affected by ethanol or RSV treatment. The expression levels of SOD, CAT, and GPx protein (Fig. 4A–C) and mRNA (Fig. 4D–F) in HepG2 cells were determined by western blot and qPCR analysis. In comparison with the control cells, the expression levels of SOD protein and mRNA were 1.9- and 1.95-fold higher in the RSV group, respectively. There was no change in the expression of CAT and GPx in the three experimental groups in comparison with the control group.

To determine the mechanism underlying the induction of SOD gene expression by RSV, the expression and transactivation activity of PPARs in HepG2 cells was determined. PPARα, PPARβ and PPARγ protein expression was not affected in cells treated with ethanol and/or RSV (Fig. 5A and B). However, the transfection of RSV-treated HepG2 cells with the tk-PPREγ-Luc reporter construct and pCMX-hPPARγ resulted in a 2.1-fold increase in promoter activity in comparison with the untreated control cells (Fig. 5C). The treatment of HepG2 cells with 200 mM ethanol alone or 100 mM ethanol + 100 mM RSV did not exert any significant effect on PPAR transactivation.