Schisandra Lignan Extract Protects against Carbon Tetrachloride-Induced Liver Injury in Mice by Inhibiting Oxidative Stress and Regulating the NF-κB and JNK Signaling Pathways

Schisandra chinensis (S. chinensis) is a traditional Chinese herbal medicine widely used for the treatment of liver disease, whose main active components are lignans. However, the action mechanisms of the lignans in S. chinensis remain unclear. This study aimed to investigate the protective effect and related molecular mechanism of Schisandra lignan extract (SLE) against carbon tetrachloride- (CCl4-) induced acute liver injury in mice. Different doses of SLE at 50, 100, and 200 mg/kg were administered daily by gavage for 5 days before CCl4 treatment. The results showed that SLE significantly decreased the activities of serum ALT/AST and reduced liver pathologic changes induced by CCl4. Pretreatment with SLE not only decreased the content of MDA but increased SOD, GSH, and GSH-Px activities in the liver, suggesting that SLE attenuated CCl4-induced oxidative stress. The expression levels of inflammatory cytokines TNF-a, IL-1β, and IL-6 were decreased after oral administration of SLE, probably because lignans inhibited the NF-κB activity. Additionally, SLE also inhibited hepatocyte apoptosis by suppressing JNK activation and regulating Bcl-2/Bax signaling pathways. In conclusion, these results suggested that SLE prevented CCl4-induced liver injury through a combination of antioxidative stress, anti-inflammation, and antihepatocyte apoptosis and alleviated inflammation and apoptosis by regulating the NF-κB, JNK, and Bcl-2/Bax signaling pathways.


Introduction
Liver injury has been recognized as a serious health problem worldwide, which can be caused by viral infections, hepatotoxic drugs, and toxic chemicals [1,2]. There are few effective drugs available for the clinical treatment of acute and chronic liver injury at present. It is necessary to elucidate the possible molecular mechanism underlying liver injury for the sake of developing effective drugs [3]. Carbon tetrachloride (CCl 4 ) is a hepatotoxic chemical that has been widely used to induce experimental liver injury models [4][5][6]. Metabolic activation of CCl 4 to reactive free radicals by cytochrome P450 enzymes is the initiating event of CCl 4 -induced liver injury. These free radicals result in lipid peroxidation of hepatocellular membrane, ultimately leading to acute liver inflammation and hepatocyte death [4,7].
It is widely believed that liver injury is closely associated with oxidative stress, inflammation, and apoptosis in the process of CCl 4 -induced liver damage [7][8][9]. Several studies have demonstrated that persistent inflammatory response contributes to the progression of chronic liver diseases [10]. Nuclear factor kappa B (NF-B) is a key transcription factor that plays an important role in regulating cell growth, differentiation, apoptosis, and inflammation. Under pathological conditions, NF-B is activated to promote inflammatory response by inducing the expression of a series of proinflammatory cytokines, such as tumor necrosis factor (TNF-a), interleukin-1 (IL-1 ), and IL-6 [11]. Thus, downregulation of NF-B activity may serve to inhibit the production of proinflammatory cytokines and attenuate inflammatory response. Additionally, previous studies have confirmed that oxidative stress-mediated ROS can induce apoptosis by activating 2 Evidence-Based Complementary and Alternative Medicine c-Jun N-terminal kinase (JNK) signaling pathway [12]. It was reported that JNK can promote apoptosis by directly activating mitochondrial apoptotic proteins [13]. In recent studies, JNK activation was reported to play a critical role in the CCl 4 -induced liver injury [14,15], suggesting that JNK may be a potential target of liver injury. Theoretically, any compound that attenuates oxidative stress, inflammation, and apoptosis in the liver can protect or eliminate liver injury.
Schisandra chinensis (S. chinensis) is a traditional Chinese herbal medicine that has been used for the treatment of viral and chemical liver injury in Asia for thousands of years [16][17][18]. The lignans as the main active ingredients in S. chinensis have various pharmacological effects such as antioxidative, anti-inflammatory, antitumor, and hepatoprotective activities [18,19]. It was reported that S. chinensis exerted its effect against chemical-induced liver injury by regulating the antioxidation function and heat shock proteins [20,21]. However, further mechanisms underlying the protective effect of S. chinensis against CCl 4 -induced liver injury remain unclear, and whether this protective effect is associated with inhibition of inflammation and apoptosis needs to be further clarified. The present study aimed to evaluate the effect of Schisandra lignan extract (SLE) on oxidative stress, inflammation, and hepatocyte apoptosis after CCl 4 treatment and investigate inflammation and hepatocyte apoptosis related signaling pathways to explore its possible molecular mechanism.

Preparation of Schisandra Lignan Extract.
Firstly, the airdried S. chinensis raw material was powdered and extracted twice with ethanol (95%) for 2 h under reflux at 50 ∘ C. The filtered sample was extracted 3 times with ethyl acetate successively. The extracts were concentrated under reduced pressure at 37 ∘ C in a rotator evaporator. Then, the mixture was subjected to normal phase column chromatography and eluted with petroleum ether and ethyl acetate. The extracts were collected and evaporated under reduced pressure. Finally, the SLE was produced and maintained at 4 ∘ C for further study.

Animals and Experimental
Design. Male C57BL/6 mice weighing 20 ± 2 g (Shanghai SLAC Laboratory Animal Center, Shanghai, China) were housed under appropriate conditions (25 ± 2 ∘ C and 12-hour light/dark cycle) with free access to water and food. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Experimental Ethics Committee of the Second Military Medical University (Shanghai, China).
After one-week environmental adaptation, 60 mice were equally randomized into six groups: (1) control group, (2) model (CCl 4 ) group, (3) SLE (50 mg/kg) + CCl 4 group, (4) SLE (100 mg/kg) + CCl 4 group, (5) SLE (200 mg/kg) + CCl 4 group, and (6) Bicyclol (200 mg/kg) + CCl 4 group. SLE solution was prepared by suspending it in 0.5% (w/v) sodium carboxymethyl cellulose (CMC-Na). Mice were fed SLE solution (50, 100, and 200 mg/kg) daily for 5 days by intragastric administration; mice in the control and model groups were given 0.5% CMC-Na solution. Two hours after the last dose of SLE, mice in the model group and SLE + CCl 4 group were injected intraperitoneally with 0.3% CCl 4 (15 mL/kg, in olive oil). Subsequently, all mice were fasted overnight and sacrificed 24 h after CCl 4 injection. Serum samples and liver tissues were harvested, a portion of the liver tissue was immediately fixed in 10% formalin for pathological examination, and the remaining liver was stored at −80 ∘ C for further analysis.

ALT/AST Assessment.
Serum samples were separated from blood by centrifugation at 3000 ×g for 15 min at 4 ∘ C. Serum alanine transaminase (ALT) and aspartate transaminase (AST) were determined by using a clinical automatic analyzer (Hitachi, Japan) and commercial reagent kit (Roche Diagnostic, Mannheim, Germany) according to the manufacturer's protocol.

Determination of MDA, SOD, GSH, and GSH-Px Activity.
The liver tissue was homogenized in 9 volumes of 0.9% saline solution and centrifuged at 3000 ×g for 15 min at 4 ∘ C. The supernatant was used for determination of MDA, SOD, GSH-Px, and GSH according to the protocols of commercially available kits.
2.6. H&E Staining and TUNEL Assay. The liver tissue was fixed in 10% buffered formalin for 2 days, paraffin-embedded, sliced cut into 5 m thick sections, and stained with haematoxylin/eosin (H&E) according to a standard protocol. The liver sections were imaged under a microscope (Olympus, Japan). TUNEL staining was performed according to the manual protocols (Boster, MK1020). Nuclei were stained with DAPI for the assessment of nuclear morphology. All slices were imaged with a fluorescence microscope (magnification, 400x). Three slices in each group were randomly selected to count the positive cells.  Table 1. The expression levels were measured in terms of the cycle threshold (Ct) and then normalized to GAPDH expression using the 2 −ΔΔCt method [22].

Western Blotting Analysis.
Total proteins were extracted from the liver tissue using the strong RIPA lysate kit (Beyotime Biotechnology, P0013B) containing 1 mM PMSF (Beyotime Biotechnology, ST506), and the protein concentration was quantified with the BCA protein kit (Thermo Scientific, 23228). Then, Western blotting assays were performed as previously described. An equal amount of protein (60 g) was denatured by mixing with corresponding loading buffer at 100 ∘ C for 5 min. An equivalent volume of the supernatant was loaded onto SDS-polyacrylamide gels (10%) for electrophoretic separation and then transferred onto nitrocellulose membranes (Millipore, HATF00010). After being blocked with 5% skim milk (BD, 232100) for 2 h, the membranes were incubated with primary antibodies at 4 ∘ C overnight and then with the secondary antibodies for 2 h at room temperature. Protein expression was imaged by Odyssey Infrared Imaging System (LI-COR Biosciences, USA).

Statistical
Analysis. Data are presented as the mean ± SEM. Differences between groups were determined by a twotailed Student's -test in GraphPad Prism 5. < 0.05 was considered statistically significant. As shown in Figure 1, the total lignan content of SLE was over 50% (W/W), in which schisandrol A was 10.96%, schisandrol B was 2.76%, schisandrin A was 2.78%, and schisandrin B 7.22%.

SLE Protects against CCl 4 -Induced Acute Liver Injury.
Liver injury was evaluated by serum ALT/AST activities and histopathological analysis. As expected, severe liver injury was induced by CCl 4 treatment in the mice, as indicated by elevation of ALT/AST activities and histopathological analysis of H&E staining. The levels of ALT and AST were 39.40 ± 7.76 U/L and 66.20 ± 21.54 U/L, respectively, in the control group versus 202.70 ± 113.50 U/L and 249.60 ± 87.01 U/L in the model group (Figures 2(b) and 2(c)). In agreement with serum ALT/AST activities, liver damage was observed in the liver sections as evidenced by inflammatory cell infiltration, hepatocyte ballooning degeneration, and necrosis after CCl 4 treatment. However, pretreatment with SLE for 5 days not only decreased the upregulation of ALT/AST levels but ameliorated pathological lesions induced by CCl 4 (Figure 2(d)). Taken together, these data suggest that SLE has a protective effect against CCl 4 -induced acute liver injury in mice.

SLE Attenuates CCl 4 -Induced Oxidative Stress.
In this study, the activities of MDA, SOD, GSH, and GSH-Px were detected to investigate the effects of SLE on liver oxidative stress caused by CCl 4 . Malondialdehyde (MDA) is the product of cell membrane lipid peroxidation and often used as a biomarker of liver oxidative damage. After CCl 4 treatment, the content of MDA increased from 1.24 ± 0.27 nmol/mg protein to 7.25±1.82 nmol/mg protein in the liver, indicating that oxidative liver injury was induced by CCl 4 in the mice. Interestingly, SLE significantly decreased the MDA content 2-fold in response to 200 mg/kg SLE treatment (Figure 3(a)). Compared with the control group, the activity of SOD was   decreased ( < 0.05) in the model group, which was obviously reversed by 100 or 200 mg/kg SLE (Figure 3(b)). Additionally, the activities of GSH and GSH-Px declined after CCl 4 treatment in the model group (1.73 ± 1.24 mg/g protein; 443.50 ± 158.50 U/mg protein) compared to control (5.18 ± 0.57 mg/g protein; 896.20 ± 77.54 U/mg protein). Conversely, SLE pretreatment markedly increased GSH and GSH-Px activities in a dose-dependent manner compared with model group (Figures 3(c) and 3(d)). We Know that CCl 4 are primarily metabolized and activated by cytochrome P450 2E1 (CYP2E1) and thus SLE could attenuate CCl 4 -induced oxidative stress by inhibiting the expression of CYP2E1. As expected, the expression of CYP2E1 was decreased after CCl 4 treatment, but treatment with SLE significantly increased its expression (Figures 3(e) and 3(f)). These results indicate that SLE could suppress CCl 4 -induced oxidative stress maybe through regulating CYP2E1 activity.

SLE Suppresses CCl 4 -Induced Inflammatory Response and NF-B Activation.
Knowing that liver injury is associated with inflammatory response, the effect of SLE on the expression of inflammatory cytokines induced by CCl 4 was evaluated. Compared with the control group, the expressions of hepatic TNF-a, IL-1 , and IL-6 mRNA were significantly elevated after CCl 4 treatment ( < 0.01), whereas these inflammatory cytokines were reduced after 100 or 200 mg/kg SLE treatment (Figure 4(a)). Moreover, the level of serum TNF-a increased more than 10-fold after CCl 4 injection in the model group, but SLE treatment blocked this trend especially at the dose of 200 mg/kg in TNF-a level (Figure 4(b)).
Since the NF-B signaling pathway plays an important role in inflammation, we investigated whether NF-B signaling was inhibited when mice were treated with SLE. The Western blot results showed that CCl 4 treatment increased the p-NF-B expression in the liver compared with control mice. However, SLE treatment inhibited p-NF-B activity in CCl 4 -treated mice. All these results indicate that SLE could attenuate the release of proinflammatory cytokines and inhibit the activation of NF-B in the CCl 4 -induced liver injury (Figures 4(c) and 4(d)).

SLE Decreases CCl 4 -Induced Hepatocytes Apoptosis.
To validate the possible role of SLE on CCl 4 -induced apoptosis, TUNEL assay was performed to investigate cell apoptosis. The number of TUNEL-positive cells in the liver of CCl 4treated mice was significantly increased more than 8-fold compared with normal mice. However, the number of liver TUNEL-positive cells was decreased after 200 mg/kg SLE treatment as compared with the model group ( < 0.01) (Figures 5(a) and 5(b)). To further identify the effect of SLE on apoptosis, the expression of apoptotic protein cleaved caspase-3 was determined by Western bolting. As shown in Figure 5(c), the expression level of cleaved caspase-3 was significantly elevated in the CCl 4 -treated mice. However, SLE treatment inhibited this elevation in a dose-dependent manner. Next, in order to determine whether the antiapoptotic effect of SLE was associated with mitochondrial apoptosis, Bcl-2 and Bax were determined. The results showed that the expression level of Bcl-2 was decreased, whereas Bax was apparently increased in the model group, leading to Bcl-2/Bax ratio imbalance. Interestingly, SLE treatment decreased the expression level of Bax and increased the expression level of Bcl-2, thus recovering the Bcl-2/Bax ratio balance (Figures 5(c) and 5(d)).

SLE Suppresses JNK Signaling Pathway in CCl 4 -Induced
Liver Injury. Knowing that JNK activation is a critical event of mitochondrial dysfunction involved in CCl 4 -induced hepatotoxicity, the expression level of JNK and p-JNK was detected to determine whether SLE could inhibit JNK activation against hepatocyte death. The expression of total JNK in the liver was similar between each group, while the expression of phosphorylated JNK significantly increased in the model group as compared with the control group. In addition, compared with JNK1, JNK2 appeared to be markedly activated after CCl 4 challenge. Moreover, SLE treatment dramatically Evidence-Based Complementary and Alternative Medicine

Discussion
S. chinensis is a traditional Chinese herbal medicine that has been widely used as a tonic and medicinal ingredient in China. In this study, we investigate the protective effect and possible molecular mechanism of SLE against CCl 4induced acute liver injury in mice. The results of this study showed that SLE could attenuate liver injury via inhibiting oxidative stress, inflammation, and apoptosis process in mice (summarized in Figure 6(c)). In the model group, the activities of ALT/AST were markedly increased after CCl 4 treatment. Histopathological analysis of H&E staining also reflected the severity of CCl 4 -induced liver injury. When SLE was administered before CCl 4 injection, mice were protected against CCl 4 hepatotoxicity as evidenced by lower serum ALT/AST levels and improved liver morphology and histology. These results confirm the hepatoprotective effect of SLE as reported previously. Oxidative stress is the crucial step in the development of CCl 4 -induced liver injury [23]. CCl 4 is known to be metabolized to trichloromethyl free radicals (•CCl 3 ) by cytochrome P450 enzymes [24,25] and these free radicals may attack intracellular nucleic acid, protein, and lipid, ultimately leading to hepatocyte oxidative damage and death [4]. MDA is the end product of lipid peroxidation and often used as a biomarker of liver oxidative damage. It was found in this study that SLE pretreatment significantly decreased the level of liver MDA induced by CCl 4 in mice, indicating that SLE could prevent CCl 4 -induced lipid peroxidation. SOD is an important antioxidant enzyme by transforming superoxide radical to H 2 O 2 , while GSH and GSH-Px could degrade H 2 O 2 to protect cells against oxidative injury. Previous reports showed that S. chinensis extract and its lignans stimulated GSH related enzymes and enhanced hepatic antioxidant and free radical scavenging activities [26,27], which was consistent with our observations. In the current study, our data showed that the activities of SOD, GSH, and GSH-Px were significantly decreased in the injured livers, indicating that the liver redox state was damaged. However, the levels of SOD, GSH, and GSH-Px were increased in the mice pretreated with SLE in a dose-dependent manner after CCl 4 injection. These results demonstrate that SLE effectively attenuated CCl 4 -induced oxidative stress in the mice via enhancing the antioxidant capability. Furthermore, several reports showed that SLE could inhibit oxidative stress in CCl 4 -induced liver injury by inhibiting CYP450 enzymes [28,29]. Since CYP2E1 is the major isozyme involved in the metabolism of CCl 4 , the expression of CYP2E1 was investigated. A similar result was obtained in this study. Expression of CYP2E1 protein was decreased after CCl 4 treatment, but SLE blocked this decline in CYP2E1 expression. In addition, S. chinensis may activate the Nrf2-ARE pathway to induce antioxidant effects to prevent liver toxicity induced by acetaminophen (APAP) [30]. These studies suggested that SLE could suppress oxidative stress induced by hepatotoxic chemicals. CCl 4 -induced liver injury was reported to be associated with inflammatory response [31,32]. CCl 4 and excessive free radicals probably activate kupffer cells, which can mediate the hepatic inflammation process by producing proinflammatory cytokines, such as TNF-a, IL-1 , and IL-6. TNF-a is a main proinflammatory cytokine and plays an important role in cell death, immune cell activation, and inflammation through different signaling pathways. In our study, the mRNA level and serum content of TNF-a were significantly increased after CCl 4 treatment, which was attenuated by SLE treatment in a dose-dependent manner. Our results also demonstrated that the mRNA levels of other inflammatory cytokines, IL-1 and IL-6, were remarkably decreased by SLE treatment in mice with CCl 4 -induced liver injury. To further study the antiinflammatory effect of SLE, NF-B signaling was investigated. NF-B is famous for a transcription factor that regulates the expression of various proinflammatory cytokines in kupffer cells. Also, NF-B signaling pathway is considered a major role in the pathophysiology of liver injury. In CCl 4 -induced liver injury, NF-B was activated by CCl 4 -induced oxidative stress and inflammatory cytokines. Previous studies have found that schisandrin exerts its anti-inflammatory effect by modulating NF-B signaling pathway in vitro [33][34][35]. In the current study, CCl 4 treatment activated NF-B and increased p-NF-B expression, whereas SLE treatment significantly decreased the expression level of p-NF-B. Taken together, our findings demonstrate that SLE could attenuate the release of proinflammatory cytokines and inhibit the activation of NF-B in the CCl 4 -induced liver injury.
Additional studies have focused on elucidating how SLE protects against hepatocyte death. Apoptosis and necrosis are two patterns of cell death. It is widely believed that necrosis is the main way of cell death in CCl 4 -induced hepatocyte death. However, several studies have reported that severe apoptosis of hepatocytes was involved in CCl 4 -induced acute liver injury [8,36,37]. Reactive oxygen species produced by oxidative stress can induce intrinsic mitochondrial apoptosis and ultimately lead to caspase-dependent apoptotic signaling [38]. The Bcl-2 family is the dominant regulators in the process of mitochondrial apoptosis. In this study, we found that CCl 4 increased the number of TUNEL-positive cells compared with the normal mice, but SLE pretreatment decreased this trend. Furthermore, the expression levels of Bcl-2, Bax, and cleaved caspase-3 were investigated by Western blot. As expect, CCl 4 treatment increased the Bax expression and downregulated the Bcl-2 expression, which could be reversed by SLE. Meanwhile, the cleaved caspase-3 expression was decreased by SLE. These results support that SLE protects the liver against hepatocyte apoptosis during CCl 4 -induced liver injury.
To further investigate the effect of SLE on hepatocyte apoptosis, we examined the JNK signaling pathways, knowing that JNK signaling plays a critical role in CCl 4 -induced mitochondrial dysfunction and hepatocyte death [15]. In this study, we found that the phosphorylation level of JNK was increased by CCl 4 compared with the control group. However, SLE treatment significantly decreased the phosphorylation level of JNK. How does SLE suppress JNK signaling in the CCl 4 -treated mice? These results showed that SLE suppressed JNK signaling pathway by suppressing the phosphorylation of JNK. In addition, it has been demonstrated that oxidative stress can activate JNK to cause apoptosis by mitochondrial intrinsic apoptotic pathways [12,39]. Sustained JNK activation contributes to Bcl-2 family-mediated apoptosis and mitochondrial permeability transition (MPT) induced necrosis [40]. In this study, CCl 4 treatment increased oxidative stress, thereby activating JNK and downstream signaling pathways. Correspondingly, SLE suppressed JNK activation in the CCl 4 -treated mice, which may be associated with decreased oxidative stress. Taken together, our data indicated that SLE ameliorates hepatocyte apoptosis maybe through regulating JNK signaling pathway.