The serum activities of ALT and AST were significantly elevated in the CCl₄-treated group (p < 0.001) where ESC (100 and 500 mg/kg BW) significantly decreased the activities of serum ALT and AST (p < 0.001). The effect of supplement of silymarin was similar to that observed for the ESC-treated group (p < 0.001) (Figure 1).

Serum levels of ALT and AST are the most commonly used biochemical markers of liver injury [23]. Hepatic damage such as liver fibrosis, cirrhosis and liver atrophy can initiate the actions of regeneration, and thus increase the weight of liver [24]. In this study, it is shown that chronic administration of CCl₄ led to a marked increase in ALT and AST levels. In addition, when liver injury was evaluated by a histological approach [25], infiltration of mononuclear cells around the Glisson’s sheath after 8 weeks of CCl₄ treatment was detected. Furthermore, CCl₄ was also found to cause a variety of histological changes in the liver, including centrizonal necrosis, portal inflammation, and Kupffer cell hyperplasia. These histological changes as well as the increase in hepatic enzymes were significantly attenuated by ESC (100 and 500 mg/kg/BW) (Table 1).

As shown in Figure 2, normal hepatic architecture with a central vein and radiating hepatic cords were seen. In the CCl₄-treated group, sections showed degeneration and necrosis, fibrosis, hepatocyte infiltration containing neutrophils and mononuclear cells. In addition, central-to-central bridging necrosis was also noted. Moreover, treatment with of ESC (100 and 500 mg/kg BW) or silymarin significantly decreased the degree of inflammation, necrosis and fibrosis in CCl₄-treated groups rats.

We next explored the effect of ESC on catalase, SOD and GPx activities in CCl₄-induced hepatic fibrosis rats. It was found that the activities of SOD, GPx and catalase were markedly increased in rats treated with ESC (100 and 500 mg/kg BW). Hepatic GPx activities were significantly increased in rats treated with ESC (100 and 500 mg/kg BW) and silymarin (200 mg/kg BW) (P < 0.001) (Table 1).

Living tissue is a major defense mechanism involving anti-oxidative enzymes that convert active oxygen molecules into non-toxic compounds [26,27]. Anti-oxidative enzymes, such as SOD, GPx and catalase, are easily inactivated by lipid peroxides or reactive oxygen species during CCl₄ exposure [28]. Glutathione (GSH) is a known radical scavenger, and GSH-related enzymes, such as GPx and GR, play detoxifying and anti-oxidative roles through conjugation with glutathione or free radical reduction [29]. There are three classes of enzymes known to provide protection against reactive oxygen species: SOD, catalase, and GPx [30]. We have demonstrated that ESC (100 and 500 mg/kg/BW) significantly reversed the CCl₄-induced decrease in activities of anti-oxidative enzymes, such as Catalase, SOD and GPx. These results suggested that ESC reduced CCl₄-induced hepatic fibrosis in rats, probably by exerting a protective effect against hepatocellular fibrosis with its free-radical scavenging ability.

To further investigate the downstream signal components of cytochrome c release in the mitochondrial-dependent signaling pathway, pro-caspase-9 levels, activated caspase-9, pro-caspase-3 levels and activated caspase-3 were measured by Western blotting. Compared with the control group, it was found that the activated caspase-3, activated caspase-9, and cytochrome c levels were significantly increased in the CCl₄-treated group. In addition, the activated-caspase-9 levels and activated-caspase-3 levels were significantly decreased in ESC-treated groups (100 and 500 mg/kg) (Figure 3).

Carbon tetrachloride is a common hepatotoxin used in liver injury research. Early studies showed that the damage induced by CCl₄ in liver is partly involved in the apoptosis pathway in vivo. At least 2 different apoptosis pathways—the Mitochondrial pathway and the Death-receptor pathway—lead to caspase activation [31]. Although past reports have disclosed caspase 3 activation and other histopathological changes in CCl₄-induced apoptotic hepatocytes [32,33], little is known about the precise molecular mechanisms of apoptosis induction. Cytochrome c release will form a complex with pro-caspase-9 and its cofactor Apaf-1 (apoptotic protease-activating factor-1). Therefore, it is responsible for activating caspase-9, which further activates caspase-3 and executes the apoptotic program.

To further understand the effect of ESC on the mitochondrial-dependent apoptotic pathway, we examined the levels of three pro-apoptotic proteins (Bak, t-Bid, and Bax) in the liver tissue of the CCl₄-treated group. Bak, t-Bid and Bax levels in the CCl4-treated group were significantly higher than those in the control group. Also, Bak, t-Bid and Bax levels were significantly decreased in the ESC-treated groups (100 and 500 mg/kg) (Figure 4).

We examined the levels of three anti-apoptotic Bcl-2 proteins, namely Bcl-2, p-Bad and Bcl-xL, in liver tissue of apoptosis rats. Bcl-2 and Bcl-x L levels were significantly lower in the CCl₄-treated group than those in the control group. Bcl-xL and Bcl-2, but not p-Bad, were significantly increased in ESC-treated groups (100 and 500 mg/kg) (Figure 5).

Mitochondria are known to be a vulnerable target of various toxins and oxidative stress. The mitochondrial apoptotic pathway is regulated by the Bcl-2 family of proteins, which consist of both anti-apoptotic (such as Bcl-2) and pro-apoptotic (such as Bax) proteins [34]. Activation of the pro-apoptotic Bcl-2 family of proteins such as Bax is known to play an important role in the oxidative stress-induced apoptotic pathway [35]. It has been shown that Bax can translocate from the cytosol to mitochondria and exhibit conformational change under the apoptotic process.

In this study, we have also demonstrated that ESC increased the levels of Bcl-2 and Bcl-x L and decreased the levels of Bak, Bax, t-Bid, cytochrome c, activated caspase-9, and activated caspase-3 proteins in rats exposed to CCl₄. Our data show that Bax protein content markedly increased in rats receiving CCl₄ alone, suggesting that oxidative stress caused by CCl₄ administration has activated Bax, Bak and t-Bid. We also found that Bcl-2 protein content markedly increased in rats receiving CCl₄ alone. Administration of ESC effectively decreased the levels of Bak, Bax, and t-Bid.

In summary, mitochondria-initiated apoptosis triggered by ROS plays an important role in CCl₄-induced hepatotoxicity in rats. ESC significantly reduced CCl₄-induced hepatic fibrosis in rats, probably by exerting a protective effect against hepatocellular fibrosis with its free-radical scavenging ability and inhibiting the Mitochondrial-Dependent Apoptotic Pathway in CCl₄-induced liver apoptosis in rats (Figure 6).

Male Sprague-Dawley rats were obtained from the BioLASCO Taiwan Co., Ltd., and fed with a standard laboratory diet and tap water ad libitum. The experimental animals were housed in an air-conditioned room of 22 ± 1 °C and 12 h of light. The rats were allowed free access to powdered feed, and mains water that was supplied through an automatic watering system. When they reached 250–300 g, the rats were used for experiments. Rats were divided randomly into groups according to the body weight in a proper range one day before administration of the test substance. All experimental procedures were performed according to the NIH Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Committee on Animal Research, China Medical University.

Apoptosis was induced in five groups of 10 rats by an oral administration of 0.5 mL/rat CCl₄, diluted 1:5 (v/v) in olive oil, twice a week for 8 weeks. The animals received only CCl₄, CCl₄ with ESC (10, 100 and 500 mg/kg BW, po, daily) or silymarin (Sigma-Aldrich, Steinheim, Germany; 200 mg/kg BW, daily). The ESC and silymarin were given when the CCl₄-induced chronic injury model started, and the total drug treatment duration was 8 weeks.

After the blood was drawn from rats at the eighth week, the animals were sacrificed at the same time and the livers were quickly taken out. They were then weighed after being clearly washed with cold normal saline and the moisture sucked up. The largest lobe of liver was divided into two parts for each liver sample, one part was submerged in 10% neutral formalin for the preparation of pathological section, and the other part was stored as a reserve at −80 °C.

The blood was centrifuged at 3024 g (BACKMAN, Krefeld, Germany) at 4 °C for 15 min to separate serum. The levels of serum Alanine Aminotransferase (sALT) and serum Aspartate Aminotransferase (sAST) were assayed using clinical test kits (Roche, Berlin, Germany) spectrophotometrically (Cobas Mira Plus, Frankfurt, Germany).

Livers were homogenized in nine volumes of isotonic phosphate buffer (0.01 M, pH 7.0). The prepared liver homogenate was centrifuged at 700 g for 5 min at 4 °C. Catalase was assayed by measuring the destruction of H₂O₂ at 240 nm according to the method of Aebi [36]. One unit (U) of this enzyme activity is defined as the amount of the enzyme giving K⁻¹; where K is the rate constant of the enzyme. Activity is expressed as U per mg of protein (U/mg protein). Superoxide dismutase (SOD) activity was determined according to the method of Misra and Fridovich [37] at room temperature. One hundred microliters of tissue extract was added to 880 μL (pH 10.2, 0.1 mM EDTA) carbonate buffer. Twenty microliters of 30 mM epinephrine (in 0.05% acetic acid) was added to the mixture and measured at 480 nm for 4 min on a Jassco V-550 Spectrophotometer. The enzyme activity was expressed as the amount of enzyme that inhibits the oxidation of epinephrine by 50%, which is equal to 1 unit.

Glutathione peroxidase (GPx) activity was determined according to the method of Flohe and Gunzler [38] at 37 °C. A reaction mixture was composed of 500 μL phosphate buffer, 100 μL 0.01 M GSH (reduced form), 100 μL 1.5 mM NADPH and 100 μL GRx (0.24 units). One hundred microliters of the tissue extract was added to the reaction mixture and incubated at 37 °C for 10 min. Then 50 μL of 12 mM t-butyl hydroperoxide was added to 450 μL tissue reaction mixture and measured at 340 nm for 180 s. The molar extinction coefficient of 6.22 × 10⁻³ M⁻¹ cm⁻¹ was used to determine the enzyme activity. One unit of activity is equal to the mM of NADPH oxidized/min per mg protein.

For histopathological examination, the formalin fixed liver was embedded in paraffin, cut into 4–5 mm thick sections, stained with hematoxylin-eosin, and observed under a photomicroscope.

Liver tissue was homogenated in PBS buffer at a ratio of 100 mg tissue/0.5 mL PBS for 5 min. The homogenates were placed on ice for 10 min and then centrifuged at 12,000 g for 30 min. Samples containing equal proteins (40 g) were loaded and analyzed by Western blot analysis. Briefly, proteins were separated by 12% SDS-PAGE and transferred onto PVDF membranes (Millipore, Belford, NJ, USA). Membranes were blocked with blocking buffer (5% non-fat dry milk, 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) for at least 1 h at room temperature and then incubated with primary antibodies in the above solution on an orbit shaker at 4 °C overnight. Following primary antibody incubations, membranes were incubated with horseradish peroxidase-linked secondary antibodies (anti-rabbit, anti-mouse, or anti-goat IgG).

Data were expressed as mean ± SEM. Statistical evaluation was carried out by one-way analysis of variance (One-way ANOVA) followed by Scheffe’s multiple range tests. P values of less than 0.05 were considered significantly.