Dried Schisandra chinensis fruits (500 g), provided by Zhixin Pharmaceutical Company (Guangdong, China), were authenticated by the pharmacist at The First Hospital of China Medical University (Shenyang, China) and macerated in 70% ethanol for 30 min at room temperature. The fruits were then refluxed three times (for 1 h each) with 70% ethanol. The combined extract was filtered and condensed by rotary evaporation (Rotary evaporator, Shyarong Biochemical Instruments Inc., Shanghai, China) under reduced pressure. The condensed extract was then freeze-dried to obtain a powder, which was placed in a desiccator at room temperature until use.

Wild-type C57BL/6 male mice (aged 7–9 weeks, weighing 20–25 g) were obtained from Charles River Laboratories, Inc. (Wilmington, MA, USA). All animals were maintained on food and water ad libitum and housed in microisolation cages. All experiments with animals were approved by and performed according to the guidelines of the China Medical University Ethics Committee (Shenyang, China). The mice were fasted overnight prior to administration of acetaminophen (300 mg/kg, intraperitoneal) or phosphate-buffered saline (PBS) control. A number of the mice with acute liver failure were then treated with Schisandra chinensis 3 h after the acetaminophen treatment (50 mg/kg, intraperitoneal).

Blood samples were collected by cardiac puncture 12 h after the end of treatment. The samples were analyzed for serum alanine transaminase (ALT) and aspartate transaminase (AST) (Beijing Gersion Bio-Technology Co., Ltd., Beijing, China). Briefly, the values of the serum ALT and AST activities were derived according to the ‘absorptivity micromolar extinction coefficient’ of NADH at 340 nm and were expressed in terms of unit per liter. Pyruvate is reduced to lactate by lactate dehydrogenase with the simultaneous oxidation of NADH to NAD, which was monitored by measuring the rate of decrease in absorbance at 340 nm.

Liver tissue samples were collected following the blood collection. The samples were fixed with 10% formaldehyde in PBS for 24 h, dehydrated in a graded ethanol series, embedded in paraffin and sliced at a thickness of 5 μm. The paraffin sections were stained with hematoxylin and eosin for histopathological analysis.

Samples of liver (50 mg) were minced in ice-cold 5% metaphosphoric acid (1:10), homogenized and then centrifuged at 3,000 × g for 10 min at 4°C. The supernatants were filtered through a 0.2-μm syringe filter, and the reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG) were quantified using the respective colorimetric assay kits (Beyotime Institute of Biotechnology, Beijing, China).

To prepare the microsomes, liver samples (1 g) were homogenized at 4°C in two volumes (w/v) 10 mM Tris-base (pH 7.4) containing 1.5% KCl using a Teflon-glass homogenizer (DuPont, Wheaton, NJ, USA). The homogenates were centrifuged at 1,000 × g (10 min, 4°C), and then the supernatants were collected and centrifuged at 12,000 × g (20 min, 4°C) to remove cellular debris, followed by centrifugation at 100,000 × g (1.5 h, 4°C). The microsomes were resuspended in homogenization buffer containing 0.5 mM phenylmethanesulfonylfluoride and centrifuged at 100,000 × g (90 min, 4°C). The pellets were resuspended in 0.25 M sucrose containing 10 mM Tris-base (pH 7.4) and stored at −80°C. The levels of Cyp2e1, Cyp1a2 and Cyp3a activity were measured according to the methods of Gardner et al (17). To assess Cyp isoform specificity, enzyme activity levels were measured following the addition of either 1 μM of the Cyp1a2 inhibitor, rutaecarpine, or of the Cyp3a inhibitor, ketoconazole.

According to the methods of Qian et al (18), hepatocytes were isolated from overnight-fasted wild-type C57BL/6 male mice by collagenase digestion and plated on type 1 collagen-coated 24-well microtiter plates, 6-cm culture dishes or glass bottom Petri dishes in Waymouth’s medium MB-752/1 (HiMedia Laboratories, Mumbai, India) supplemented with 2 mM L-glutamine, 10% fetal calf serum, 100 nM insulin, 100 nM dexamethasone, 100 U/ml penicillin and 100 μg/ml streptomycin. Cell viability was identified as >90% by trypan blue exclusion, according to the manufacturer’s instructions (Beyotime Institute of Biotechnology). After 4 h, the hepatocytes were placed in hormonally defined medium consisting of RPMI-1640 supplemented with 240 nM insulin, 2 mM L-glutamine, 1 μg/ml transferrin, 0.3 nM selenium, 1.5 μM free fatty acids, 100 U/ml penicillin and 100 μg/ml streptomycin.

The cells were plated in 96-well plates (1,500 cells/well) and allowed to attach overnight. Subsequently, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Sigma-Aldrich, Carlsbad, CA, USA; final concentration, 0.5 mg/ml) was added to the wells and the cells were incubated for 4 h. Absorbance was measured at 550–560 nm by using a microplate reader (Bio-Rad, Hercules, CA, USA).

Following the manufacturer’s instructions (Apoptosis Detection kit; KeyGen Biotech Co., Ltd., Nanjing, China), the cells were washed and resuspended in binding buffer prior to incubation in FITC-labeled Annexin V and PI for 10 min. The suspensions were immediately analyzed by a FACSCalibur machine (BD Biosciences, Baltimore, MD, USA).

Cells were collected and centrifuged at 1,500 × g for 5 min, and the pellet was resuspended in 100 μl PBS at a density of 1×10⁶ cells/ml. Cold ethanol (900 μl, 70%) was added to the mixture for 1 h on ice. The cells were collected by centrifugation at 1,500 × g for 5 min. The pellet was then resuspended in 100 μl PBS containing RNase A (0.2 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and maintained at room temperature for 30 min. The cells were recovered by centrifugation and the pellets were resuspended in 350 μl PBS containing PI (50 μg/ml; KeyGen Biotech Co., Ltd.) and analyzed by flow cytometry using the FACSCalibur machine.

MMP was analyzed using the fluorescent dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) according to the manufacturer’s instructions (KeyGen Biotech Co. Ltd.). Briefly, cells were plated on a six-well culture plate. Following treatment for 24 h, the cells were washed twice with PBS, harvested and incubated with 20 nM JC-1 for 30 min in the dark. MMP was then analyzed using the FACSCalibur machine.

Cells (5×10⁵) were cultured in 12-well tissue culture plates overnight, and then cotreated with drugs and 2′,7′-dichlorofluorescin diacetate (DCF-DA), an ROS-sensitive dye. Following treatment, the cells were harvested and suspended in PBS. The relative fluorescence intensities of the cells were quantified using the FACSCalibur machine.

The 26S proteasome function was assayed as described previously (19). The assay was based on the detection of the fluorophore 7-amino-4-methylcoumarin (AMC) following cleavage from the labeled substrate Suc-LLVY-AMC (Boston Biochem Inc., Cambridge, MA, USA). This fluorogenic proteasome substrate was added to the cell lysate at a final concentration of 80 μM in 1% dimethylsulfoxide. Adenosine triphosphate-dependent cleavage activity was monitored continuously by detection of free AMC using a microplate reader (Bio-Rad) at 380/460 nm at 37°C.

Cell extracts were resolved on SDS-PAGE and then transferred to nitrocellulose membranes. These membranes were developed and visualized with electrochemiluminescence (Pierce, Waltham, MA, USA). The primary antibodies used are listed in Table I.

All values are presented as the mean ± the standard error of the mean. Student’s paired t-test was used to identify statistically significant differences. Kaplan-Meier survival plots were generated and comparisons were made with log-rank statistics. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using GraphPad Prism 4 software (GraphPad Software Inc., San Diego, CA, USA).

The effects of a diet containing Schisandra chinensis on mice with acetaminophen-induced liver injury were analyzed. Hepatocellular cytoplasmic degeneration, bridging necrosis and severe congestion were observed in mice treated with acetaminophen (Fig. 1A). Compared with those of the untreated mice, the mice administered acetaminophen exhibited a rapid induction of hepatotoxicity with significant elevations in the levels of serum ALT and AST (Fig. 1B, P<0.05). Treatment with Schisandra chinensis appeared to inhibit acetaminophen-induced hepatotoxicity, as reduced levels of serum transaminases and marginal structural alterations in the liver were observed (Fig. 1A and B). Acetaminophen treatment alone resulted in a rapid reduction in the levels of GSH and an increase in the levels of GSSG. However, the levels of GSH in the acetaminophen and Schisandra chinensis combined treatment group showed a restored trend compared with those of the untreated group (Fig. 1C, P<0.05). No significant differences were identified in the levels of microsomal Cyp2e1 and Cyp3a activity among the mice in the different groups. However, the levels of Cyp1a2 were significantly suppressed by Schisandra chinensis administration compared with those in the acetaminophen-treated mice (Table II, P<0.05). Furthermore, a significantly different survival rate between the untreated group and the acetaminophen-treated group, as well as between the acetaminophen-treated group and the acetaminophen- and Schisandra chinensis-treated group in the Cox model was identified (Fig. 1D, P<0.05).

The proliferation of hepatocytes was inhibited by acetaminophen as revealed using the MTT assay (Fig. 2A, P<0.05). PI staining of cells revealed that acetaminophen-treated cells were arrested in the G₁ phase (Fig. 2B). Annexin V-FITC and PI double staining was performed to detect apoptotic cells. In the cells with acetaminophen treatment, the apoptotic ratio was 11–12-fold higher than that of the untreated cells (Fig. 2C, P<0.05). As shown in Fig. 2D, the red/green ratio, used to measure the MMP, in the normal cells (1.2% green, 98.8% red) was reversed following acetaminophen treatment (62.5% green, 37.5% red). The fluorescent dye DCF-DA was used to measure the ROS content in cells following acetaminophen treatment. As shown in Fig. 2E, acetaminophen treatment directly induced an increase in the fluorescence intensity of the cells (42.7%) when compared with that of the normal cells (13.4%, P<0.05). Furthermore, acetaminophen was found to inhibit proteasome activity in hepatocytes (Fig. 2F, P<0.05). Consistent with the results obtained in vivo, Schisandra chinensis protected hepatocytes against acetaminophen-induced apoptosis, ROS release and injury to mitochondria and proteasomes (Fig. 2).

The aforementioned results revealed the changes of mitochondria in hepatocytes. Due to these results, the changes were further analyzed; expression of Bax, Bcl-xL, Bcl-2 and p-Bcl-2 was detected using western blot analysis. As shown in Fig. 3 (lanes 1 and 2), a reduction in the Bcl-2 and Bcl-xl expression levels and an increase in Bax and P-Bcl-2 expression levels were identified in hepatocytes with acetaminophen treatment compared with those in the normal cells. The levels of these proteins were markedly reversed following Schisandra chinensis treatment (Fig. 3, lanes 2 and 3). The levels of phosphor-c-Jun N-terminal kinase (p-JNK) were upregulated by acetaminophen treatment but the levels of total JNK did not change compared with those in the normal cells (Fig. 3, lanes 1 and 2). However, p-JNK was inhibited following Schisandra chinensis treatment (Fig. 3, lane 3). β-actin served as an internal control for all western blotting.