Food and Chemical Toxicology 64 (2014) 41–48
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Hepatoprotective properties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathway Jie-Qiong Ma a,⇑, Jie Ding a, Li Zhang a, Chan-Min Liu b a b
School of Chemistry and Pharmaceutical, Sichuan University of Science and Engineering, 643000 Zigong City, Sichuan Province, PR China School of Life Science, Jiangsu Normal University, No. 101, Shanghai Road, Tongshan New Area, Xuzhou City 221116, Jiangsu Province, PR China
a r t i c l e
i n f o
Article history: Received 19 September 2013 Accepted 13 November 2013 Available online 25 November 2013 Keywords: Sesamin CCl4 Oxidative stress Apoptosis JNK Liver
a b s t r a c t Sesamin (Ses), one of the major lignan derived from sesame seeds, has been reported to have many benefits and medicinal properties. However, its protective effects against carbon tetrachloride (CCl4) induced injury in liver have not been clarified. The aim of the present study was to investigate the hepatoprotective effects of sesamin on oxidative stress and apoptosis in mice exposed to CCl4. Our data showed that sesamin significantly prevented CCl4-induced hepatotoxicity in a dose-dependent manner, indicated by both diagnostic indicators of liver damage (serum aminotransferase activities) and histopathological analysis. Moreover, CCl4-induced profound elevation of reactive oxygen species (ROS) production and oxidative stress, as evidenced by increasing of lipid peroxidation level and depleting of the total antioxidant capacity (TAC) in liver, were suppressed by treatment with sesamin. Furthermore, TUNEL assay showed that CCl4-induced apoptosis in mouse liver was significantly inhibited by sesamin. In exploring the underlying mechanisms of sesamin action, we found that activities of caspase-3 were markedly inhibited by the treatment of sesamin in the liver of CCl4 treated mice. Sesamin increased expression levels of phosphorylated Jun N-terminal kinases (JNK) in liver, which in turn inactivated pro-apoptotic signaling events restoring the balance between mitochondrial pro- and anti-apoptotic Bcl-2 proteins and decreasing the release of mitochondrial cytochrome c in liver of CCl4 treated mice. JNK was also involved in the mitochondrial extrinsic apoptotic pathways of sesamin effects against CCl4 induced liver injury by regulating the expression levels of phosphorylated c-Jun proteins, necrosis factor-alpha (TNF-a) and Bak. In conclusion, these results suggested that the inhibition of CCl4-induced apoptosis by sesamin is due at least in part to its anti-oxidant activity and its ability to modulate the JNK signaling pathway. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Oxidative stress is regarded as a mediator of acute and chronic liver injury. Carbon tetrachloride (CCl4) is a well known hepatotoxin that is widely used to induce acute toxic liver injury in a large range of laboratory animals (Kodai et al., 2007; Campo et al., 2008; Leong et al., 2011). A number of studies have shown that CCl4 is metabolized by the P450 enzyme system to yield reactive metabolic products trichloromethyl free radicals, which can initiate the process of lipid peroxidation and ultimately results in the over-
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCl4, carbon tetrachloride; JNK, the c-Jun N-terminal kinases; ROS, reactive oxygen species; Ses, sesamin; TAC, the total antioxidant capacity; TBARS, thiobarbituric acid reactive substances; TNF-a, necrosis factor-alpha; TUNEL, deoxyribonucleotidyl transferase (TdT)-mediated dUTP-fluorescein isothiocyanate (FITC) nick-end labeling.. ⇑ Corresponding author. Tel.: +86 013700950121; fax: +86 516 83500171. E-mail address: [email protected] (J.-Q. Ma). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.11.017
production of reactive oxygen species (ROS) and hepatocyte injuries (Kodai et al., 2007; Tien et al., 2011). Jun N-terminal kinases (JNKs), which belong to the superfamily of MAP-kinases, play a critical role in death receptor-initiated extrinsic as well as mitochondrial intrinsic apoptotic pathways (Dhanasekaran et al., 2008). JNKs activate apoptotic signaling by the upregulation pro-apoptotic genes via the transactivation of specific transcription factors or by directly modulating the activities of mitochondrial pro- and anti-apoptotic proteins through distinct phosphorylation events. Many researches showed that ROS can induce apoptosis by activating JNK signaling pathway (Dhanasekaran et al., 2008; Sinha et al., 2013). Many studies also revealed that CCl4 induced hepatic apoptosis by mitochondrial intrinsic and extrinsic apoptotic pathways (Huang et al., 2008; Leong et al., 2011; Borkham-Kamphorst et al., 2013; Monga et al., 2013). Sesame seeds (Sesamun indicum L.) are widely used as dietary supplements. Sesame seeds and their oil have been used in human diets for thousands of years and are believed to provide health ben-
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efits. Sesamin (Ses) is one of the major lignan in sesame seeds and oil. Several studies have shown that sesamin exerts antioxidative (Nakai et al., 2003; Liu et al., 2013) anti-inflammatory (Lee et al., 2009, 2011; Bournival et al., 2012), anti-apoptosis (Bournival et al., 2012; Liu et al., 2013), antihypertensive (Kong et al., 2009; Wu et al., 2012), cholesterol-lowering (Peñalvo et al., 2006), anticarcinogen (Hirose et al., 1992; Lee et al., 2011), hepatoprotective (Chang et al., 2009; Liu et al., 2013), renoprotective (Kong et al., 2009; Wu et al., 2012) and neuroprotective effects (Lee et al., 2011). Reports from our laboratory and others had shown that sesamin can protect liver from injury induced by hepatotoxins (Hemalatha et al., 2004; Chang et al., 2009; Liu et al., 2013). Despite those pharmacological benefits, the molecular mechanisms by which sesamin elicits hepatoprotective effects are still unclear. In the present study, we, for the first time, aimed to determine whether sesamin can protect mouse liver from CCl4-induced oxidative stress and apoptosis via JNK pathway.
2.5. Deoxyribonucleotidyl transferase (TdT)-mediated dUTP-fluorescein isothiocy -anate (FITC) nick-end labeling (TUNEL) assay For the TUNEL staining, the standard protocol for frozen sections was followed (BD ApoAlertTM DNA Fragmentation assay kit, BD Biosciences Clontech, Palo Alto, CA, USA). Apoptosis was assayed by TUNNEL staining using our previous method (Liu et al., 2013). 2.6. Assay of ROS level ROS was measured as described previously, which is based on the oxidation of 20 70 -dichlorodihydrofluorescein diacetate to 20 70 -dichloro-fluorescein (Shinomol and Muralidhara, 2007; Liu et al., 2012). 2.7. Assay of thiobarbituric acid reactive substances (TBARS) levels in liver Tissue lipid peroxidation was measured by our previous method (Liu et al., 2012, 2013). Liver homogenate was incubated with 8.1% SDS (w/v) for 10 min followed by addition of 20% acetic acid (pH 3.5). Reaction mixture was incubated with 0.6% TBA (w/v) for 1 h in boiling water bath. Pink color chromogen was extracted in butanol-pyridine solution (15:1) and read at 532 nm.
2. Materials and methods
2.8. Measurement of the total antioxidant capacity (TAC)
2.1. Chemicals and reagents
The total antioxidant capacity (TAC) in liver was assayed with a commercially available assay kit (Jiancheng Biochemical, Inc., Nanjing, China). This method is based on the oxidation of intracellular antioxidants with iron (III) in acidic medium. The liberated iron (II) reacts with 1,10-phenanthroline to form a colored complex, which is measured at 520 nm. The TAC of the samples was measured according to the manufacturer’s protocol. One unit of TAC was defined as the capability of increasing 0.01 optical densities (OD) value per mg protein per min at 37 °C.
Sesamin (>95% purity) and Carbon tetrachloride (CCl4) were obtained from Sigma Chemical Co. (St. Louis, MO, USA); anti-Bcl-2 antibody, anti-cleaved caspase-3, anti-Bax antibody, anti-Bak antibody, anti-JNK1/2 antibody, anti-phospho-JNK1/2 antibody, anti-phospho-c-Jun antibody, anti-c-Jun antibody, anti-cytochrome c antibody and goat anti-rabbit IgG-HRP from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (Beverly, MA, USA); Reagents and kits used in the assays of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); BCA assay kit from Pierce Biotechnology, Inc. (Rockford, IL, USA); TUNEL apoptosis detection kit from GenScript Corporation (Piscataway, NJ, USA). All other reagents unless indicated were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
2.9. Western blot analyses Nuclear and cytoplasmic extracts for western blotting were obtained by using a nuclear/cytoplasmic isolation kit (Beyotime Institute of Biotechnology, Bijing, China). Protein levels were determined using the BCA assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Western blot analysis was performed as previously described by us (Liu et al., 2012, 2013).
2.2. Animals and treatment 2.10. Statistic analysis Male ICR mice (20–25 g) were obtained from the Branch of National Breeder Center of Rodents (Shanghai) and kept in an environmentally controlled room (23 ± 2 °C, 55 ± 10% humidity) with a 12-h light/dark cycle and allowed free access to food and water. After acclimation for 1 week, the animals were randomly divided into four groups (n = 10). Group I (normal control) was given distilled water (10 ml/ kg body weight). Group II (model) was given distilled water. Groups III and IV were administrated UA (60 and 120 mg/kg body weight, suspended in 0.5% carboxymethylcellulose sodium, daily). The choice of sesamin dose is based on previous findings, which showed that sesamin has protective effects on tissue damage (Chang et al., 2009; Liu et al., 2013). After the oral administration for 7 days, the animals were treated as described previously (Zhang et al., 2013). Two hours after the final administration, mice in Groups II–VI were injected intraperitoneally with 0.3% (v/ v) CCl4 (10 ml/kg, dissolved in olive oil), while the mice in Groups I received appropriate vehicle (olive oil, i.p.). Twenty-four hours after the CCl4 challenge followed by fasting, the animals were anesthetized for obtaining the blood and sacrificed to collect the livers. The right lobe of the liver was fixed in 10% formalin to prepare paraffin sections. The present research reported in this paper was conducted in accordance with the Chinese legislation and NIH publication on the use and care of laboratory animals and were approved by the respective university committees for animal experiments.
2.3. Measurement of serum aminotransferase activities The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were estimated spectrophotometrically using commercial diagnostic kits (Jiancheng Institute of Biotechnology, Nanjing, China) (Liu et al., 2013).
2.4. Histological evaluations The histological changes of liver were evaluated as described previously (Liu et al., 2013). Cryosections were collected on 3-aminopropyl-trimethoxysilanecoated slides (Sigma–Aldrich). The extent of hepatic damage was evaluated on H&E slides. The histological changes were scored according to the following criteria: 0, absent; 1, mild; 2, moderate; and 3, severe (Kim et al., 2011).
All statistical analyses were performed using the SPSS software, version 11.5. A one-way analysis of variance (ANOVA; P < 0.05) was used to determine significant differences between groups and the individual comparisons were obtained by Turkey’s HSD Post Hoc test. Statistical significance was set at P < 0.05.
3. Results 3.1. Sesamin protects against CCl4-induced hepatic dysfunction Serum aminotransferase (such as AST and ALT) is commonly used as an indicator of liver disease (Kodai et al., 2007; Campo et al., 2008; Liu et al., 2013). In order to determine whether sesamin can attenuate the liver damage in the CCl4-treated mouse, we measured the activities of serum ALT and AST (Fig. 1). In CCl4-treated mice, the activities of serum ALT and AST significantly increased by 332% and 608% as compared with these of the controls, respectively (P < 0.05). Interestingly, the treatment with sesamin on the other hand showed dose-dependent inhibition in this elevation (P < 0.05) (Fig. 1). 3.2. Sesamin alleviated CCl4-induced histology changes in liver Liver histological study was used to determine the protective effect of sesamin on CCl4-induced injury. As shown in Fig. 2, the results of histopathological evaluation showed that sesamin exhibited protective effect against CCl4-induced liver injury. CCl4 treatment caused visible histology changes including structure damage including large areas of extensive hepatocellular necrosis, leukocyte infiltration in mouse liver. Moreover, the score for liver injury was significantly higher in the CCl4-treated mice as com-
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pared with vehicle controls. Whereas, sesamin treatment significantly alleviated the CCl4-induced damage in mouse liver (Fig. 2). 3.3. Sesamin inhibited CCl4-induced apoptosis in liver We used the TUNEL assay to investigate the effect of sesamin on the CCl4-induced apoptosis (Fig. 3). The number of TUNEL-positive cells in the liver of CCl4-treated mice was significantly increased (P < 0.05). Whereas, sesamin markedly decreased the liver TUNEL-positive cells of mice treated with CCl4 in a dose-dependent manner (Fig. 3). 3.4. Sesamin inhibited CCl4-induced oxidative stress in liver
Fig. 1. Effect of sesamin on hepatic functional markers in CCl4 treated mice. (A) ALT activity; (B) AST activity. All values are expressed as mean ± S.E.M. (n = 7). ## P < 0.05, compared with the control group; **P < 0.05, vs. nickel-treated group.
Many studies suggested that the levels of ROS, TBARS and TAC might be indicators of oxidative stress (Li et al., 2004, 2012, 2013).The results showed that sesamin could decrease CCl4-induced ROS and TBARS levels (Fig. 4). CCl4 treatment markedly increased hepatic ROS and TBARS levels by 121% and 72% as compared with those of the controls, respectively (P < 0.05). However, the treatment with sesamin on the other hand showed dosedependent inhibition in this elevation (P < 0.05) (Fig. 4). As shown in Fig. 4C, the TAC level in CCl4-treated mice was markedly decreased by 37% as compared with that of the control (P < 0.05). However, treatment with semamin in CCl4-treated mice significantly increased the hepatic TAC level in a dose-dependent manner (P < 0.05).
Fig. 2. Morphological and histological evaluation of liver in mice. (A) The control group; (B) Mice treated with CCl4; (C) Mice treated with CCl4 and fed with sesamin (60 mg / kg); (D) Mice treated with CCl4 and fed with sesamin (120 mg /kg). The black arrow indicates infiltrating leukocytes. The white arrow indicates hepatic cell necrosis. Original magnification, 20 10.
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3.5. Sesamin-mediated protective action involves JNK and c-Jun activation Activation of JNK is known to promote apoptosis (Dhanasekaran et al., 2008; Sinha et al., 2013). To investigate whether JNK signaling was involved in the action of sesamin, we determined the effects of sesamin on JNK pathway in mouse liver. The results showed the level of phospho-JNK was markedly increased in the livers of CCl4-treated mice as compared with the vehicle controls (Fig. 5). However, the up-regulation of phospho-JNK was markedly suppressed in the sesamin and CCl4 co-treated mice (P < 0.05). Activation of c-Jun was involved in mitochondrial extrinsic apoptotic pathways. As shown in Fig. 5, the level of phospho-c-Jun was markedly increased in the livers of CCl4-treated mice as compared with the vehicle controls (P < 0.05). However, the treatment with sesamin on the other hand showed dose-dependent inhibition in this elevation (P < 0.05). 3.6. Sesamin modulates CCl4-induced expression of pro-apoptotic proteins JNK has been shown to regulate the expression of pro-apoptotic and anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bax (Dhanasekaran et al., 2008; Sinha et al., 2013). Therefore, we examined the effects of sesamin on JNK regulated intrinsic proapoptotic proteins. As shown in Fig. 6, CCl4 treatment increased the expression of Bax, Bak, TNF-a and cytosol cytochrome c and
caused a reduction in the expression of the anti-apoptotic protein Bcl-2 (P < 0.05). However, sesamin treatment abolished the CCl4evoked pro-apoptotic signaling events in the liver of mice (P < 0.05) (Fig. 6). 3.7. Sesamin reduced CCl4-induced caspase-3 activation In response to both the extrinsic and intrinsic apoptotic stimuli, JNK plays an essential role through its ability to interact and modulate the activities of caspase proteins (Dhanasekaran et al., 2008; Sinha et al., 2013). Caspase-3 is one of the key executioners of apoptosis, capable of cleaving or degrading many key proteins such as nuclear lamins, fodrin, and the nuclear enzyme poly (ADPribose) polymerase (PARP) (Sinha et al., 2013; Liu et al., 2013). In order to determine whether sesamin can attenuate apoptosis in the liver of CCl4-treated mice, the activity of caspase-3 was also examined. As shown in Fig. 6F, cleaved caspase-3 levels were significantly elevated as compared with that of the controls in the CCl4-treated mouse liver (P < 0.05). Interestingly, the treatment with sesamin on the other hand inhibited this elevation in a dose-dependent manner (P < 0.05). 4. Discussion The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemicals necessary for
Fig. 3. In situ detection of fragmented DNA [deoxyribonucleotidyl transferase-mediated dUTP-FITC nick-end labeling (TUNEL) assay] in the liver of mice. The merged images (down) show that apoptotic cells appear yellow and non-apoptotic cells appear red. Scale bars = 100 mm. The histogram showed the relative proportion of TUNEL-positive cells in the liver of mice. All values are expressed as mean ± S.E.M. **P < 0.05, compared with the control group; ##P < 0.05, vs. nickel-treated group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Effect of sesamin on the oxidative stress in liver of CCl4-treated mice. (A) Level of ROS; (B) Level of TBARS; (C) TAC. Each value is expressed as mean ± S.E.M. (n = 7). ##P < 0.05, compared with the control group; **P < 0.05, vs. nickel-treated group.
digestion. However, the liver is the most common target organ for chemically induced injuries (Liu et al., 2013). CCl4 is considered as a direct hepatotoxin which produces centrilobular necrosis and steatosis in liver (Kodai et al., 2007; Tien et al., 2011; Kim et al., 2011). The present investigation reveals that the activities of AST and ALT in the serum of CCl4 treated mice were markedly increased (Fig. 1), which in response to CCl4 have been attributed to hepatic structural damage because these enzymes are normally localized to the cytoplasm and are released into the circulation after cellular damage has occurred (Ozturk et al., 2009; Liu et al., 2013). Moreover, the histological changes of liver, such as structure damage, hepatocellular necrosis and leukocyte infiltration, had been observed in CCl4-treated animals (Fig. 2). Our previous study had demonstrated that sesamin has hepatoprotective effects (Liu et al., 2013). In this study, treatment with sesamin effectively protected mice against CCl4-induced liver damage by reducing elevated serum ALT & AST activities (Fig. 1) and alleviating hepatic histological changes (Fig. 2). These results suggest that sesamin could protect mice against CCl4-induced hepatic dysfunction and histopathologic damage. Many studies suggest that one possible molecular mechanism involved in CCl4 hepatotoxicity is the disruption of delicate oxidant/antioxidant balance, which can lead to liver injury via oxidative damage (Kodai et al., 2007; Chang et al., 2009; Leong et al., 2011). Accumulating evidence has also shown that CCl4 is metabolized to produce highly toxic trichloromethyl free radical (CCl3) and/or trichloro methyl peroxyl (OOCCl3) free radicals by cytochrome P450 enzyme and causes damage (Kodai et al., 2007; Tien et al., 2011; Sinha et al., 2013). Both trichloromethyl and its peroxy radical are capable of binding to proteins or lipids, or of abstracting
Fig. 5. The protective effect of sesamin against CCl4-induced apoptosis depends on the JNK pathway. (A) Relative density analysis of the phospho-JNK protein bands; (B) Relative density analysis of the phospho-c-Jun protein bands; b-actin was probed as an internal control in relative density analysis of the JNK and c-Jun protein bands. The relative density is expressed as the ratio (phospho-JNK/totalJNK, phospho-c-Jun/total-c-Jun). The vehicle control is set as 1.0. Values are averages from seven independent experiments. Each value is expressed as mean ± S.E.M. ##P < 0.05, compared with the control group; **P < 0.05, vs. CCl4treated group.
a hydrogen atom from an unsaturated lipid, initiating lipid peroxidation and liver damage (Weber et al., 2003; Chang et al., 2009; Sinha et al., 2013). These free radicals combine with polyunsaturated fatty acids of hepatic and testicular cell membranes, cause elevation of thiobarbituric acid reactive substances (TBARS) concentration with subsequent necrosis and increase lysosomal enzymes activities (Yen et al., 2009; Shim et al., 2010; Zhang et al., 2013). In the present research, levels of ROS and TBARS were remarkably increased in CCl4-treated mouse liver as compared with these of the control, indicating that CCl4 exposure induced oxidative stress (Fig. 4). Sesamin has been reported to have many benefits and medicinal properties (Nakai et al., 2003; Bournival et al., 2012). In this study, sesamin decreased the ROS and TBARS productions in the CCl4-treated mouse liver (Fig. 4), which may be due to the powerful antioxidant and free radical scavenging activities (Hemalatha et al., 2004; Shinomol and Muralidhara, 2007). Our finding suggests that sesamin could at least partly attenuate oxidative stress by decreasing levels of ROS and lipid peroxide in CCl4-treated mouse liver. JNKs have been shown to be have involved in stimulating apoptotic signaling. Oxidative stress can activate JNK to cause apoptosis by receptor-initiated extrinsic and mitochondrial intrinsic apoptotic pathways (Dhanasekaran et al., 2008; Sinha et al., 2013). The
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Fig. 6. Immunoblotting analysis of apoptosis-provoking proteins in response to CCl4 and sesamin. (A) Relative density analysis of the Bax protein bands; (B) Relative density analysis of the Bcl-2 protein bands; (C) Relative density analysis of the cytosol cytochrome c bands; (D) Relative density analysis of the Bak protein bands; (E) Relative density analysis of the TNF-a protein bands; (F) Relative density analysis of the cleaved caspase-3 protein bands. b-actin was probed as an internal control in relative density analysis of the protein bands. The vehicle control is set as 1.0. Values are averages from seven independent experiments. Each value is expressed as mean ± S.E.M. ##P < 0.05, compared with the control group; **P < 0.05, vs. CCl4-treated group.
crucial mitochondrial event initiating apoptosis is the release of cytochrome c from the inner membrane space of mitochondria (Dhanasekaran et al., 2008; Sinha et al., 2013). After release from mitochondria to the cytosol, cytochrome c binds to apoptosis-activating factor, which in turn stimulates caspase activity to regulate apoptosis (Dhanasekaran et al., 2008; Tien et al., 2011). JNKs also play an essential role in modulating the functions of pro- and anti-apoptotic proteins located in mitochondria. JNK and ROS can stimulate the activities of pro-apoptotic proteins (Bax, Bak, Bok, Bid, Bim, Bik, Bad, Bmf, Hrk, Noxa, Puma, Blk, etc.) and promote apoptosis by inhibiting the anti-apoptotic proteins (Bcl-2, Bcl-XL, Bcl-w, A1, Mcl-1, etc.) to regulate the release of cytochrome c and apoptosis (Tien et al., 2011; Sinha et al., 2013). This study showed that semamin treatment restored the expression levels of cytosol cytochrome c, Bax, Bak and Bcl-2 in the liver of CCl4-trea-
ted mice (Fig. 6), suggesting that sesamin could protect mouse liver by regulating JNK signaling and mitochondrial intrinsic apoptotic pathways. JNK signaling is also involved in the extrinsic apoptotic pathway initiated by death receptors (Dhanasekaran et al., 2008; Sinha et al., 2013). Studies showed that the JNK-c-Jun/Ap1 pathway is involved in the increased expression of pro-apoptotic genes such as TNF-a, Fas-L, and Bak (Fan and Chambers, 2001; Dhanasekaran et al., 2008; Björkblom et al., 2008). Many researches revealed that CCl4 could induce hepatic apoptosis by mitochondrial extrinsic apoptotic pathways (Tien et al., 2011; Lu et al., 2012; Zhang et al., 2013). In the present study, we also found that the expression levels of JNK1/2 phosphorylation, c-Jun phosphorylation, TNF-a and Bak increased in liver of CCl4 treated mice. However, sesamin treatment significantly deceased the expression levels of
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Acknowledgments This work is supported from Startup Project of Doctor scientific research by Sichuan University of Science and Engineering (2013RC14).
References
Fig. 7. Schematic diagram showing protective signaling of sesamin in CCl4-induced liver damage. The ? indicates activation or induction, and indicates inhibition or blockade.
these proteins (Fig. 6), suggesting that sesamin could protect mouse liver by regulating JNK signaling and mitochondrial extrinsic apoptotic pathways. The caspases is a family of proteins that are one of the main executors of the apoptotic process. The caspase pathway is a well-identified downstream target for JNK signaling in apoptosis (Dhanasekaran and Reddy, 2008; Sinha et al., 2013). Caspase-3 is one of the key executioners of apoptosis, capable of cleaving or degrading many key proteins such as nuclear lamins, fodrin, and the nuclear enzyme poly (ADPribose) polymerase (PARP) (Tien et al., 2011; Zhang et al., 2013). The present study showed that CCl4 increased the number of TUNEL-positive cells and the activity of caspase-3 in the liver of mice. However, we found that sesamin markedly decreased the number of TUNEL-positive cells and the activity of caspase-3 in mice treated with CCl4 (Figs. 3 and 6F). Thus, our findings indicate that sesamin might protect liver against CCl4-induced apoptosis by activating the JNK pathway. In summary, this study demonstrates for the first time that sesamin has potent protective effects against CCl4-induced apoptosis by modulating the JNK pathway in mouse liver. We propose a possible protective effect of sesamin (Fig. 7). Here we demonstrated that sesamin administration attenuated CCl4-induced hepatic dysfunction and histopathologic changes. Sesamin attenuated CCl4-induced hepatic oxidative damage by inhibiting ROS generation and increasing liver TAC level. Sesamin treatment could effectively inhibited CCl4-induced apoptosis in liver by decreasing expression levels of phospho-JNK1/2, phospho-c-Jun, Bax, Bak, TNF-a, cytosol cytochrome c and caspase-3 and increasing expression levels of Bcl-2. Sesamin seems to be potent hepatoprotective drug and its use in maintaining a healthy liver and preventing toxic liver damage deserves consideration. However, there are fewer studies on the absorption of sesamin in humans. The question warrants further investigation and further examination.
Conflict of Interest The authors declare that there are no conflicts of interest.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Hepatoprotective properties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathway Jie-Qiong Ma a,⇑, Jie Ding a, Li Zhang a, Chan-Min Liu b a b
School of Chemistry and Pharmaceutical, Sichuan University of Science and Engineering, 643000 Zigong City, Sichuan Province, PR China School of Life Science, Jiangsu Normal University, No. 101, Shanghai Road, Tongshan New Area, Xuzhou City 221116, Jiangsu Province, PR China
a r t i c l e
i n f o
Article history: Received 19 September 2013 Accepted 13 November 2013 Available online 25 November 2013 Keywords: Sesamin CCl4 Oxidative stress Apoptosis JNK Liver
a b s t r a c t Sesamin (Ses), one of the major lignan derived from sesame seeds, has been reported to have many benefits and medicinal properties. However, its protective effects against carbon tetrachloride (CCl4) induced injury in liver have not been clarified. The aim of the present study was to investigate the hepatoprotective effects of sesamin on oxidative stress and apoptosis in mice exposed to CCl4. Our data showed that sesamin significantly prevented CCl4-induced hepatotoxicity in a dose-dependent manner, indicated by both diagnostic indicators of liver damage (serum aminotransferase activities) and histopathological analysis. Moreover, CCl4-induced profound elevation of reactive oxygen species (ROS) production and oxidative stress, as evidenced by increasing of lipid peroxidation level and depleting of the total antioxidant capacity (TAC) in liver, were suppressed by treatment with sesamin. Furthermore, TUNEL assay showed that CCl4-induced apoptosis in mouse liver was significantly inhibited by sesamin. In exploring the underlying mechanisms of sesamin action, we found that activities of caspase-3 were markedly inhibited by the treatment of sesamin in the liver of CCl4 treated mice. Sesamin increased expression levels of phosphorylated Jun N-terminal kinases (JNK) in liver, which in turn inactivated pro-apoptotic signaling events restoring the balance between mitochondrial pro- and anti-apoptotic Bcl-2 proteins and decreasing the release of mitochondrial cytochrome c in liver of CCl4 treated mice. JNK was also involved in the mitochondrial extrinsic apoptotic pathways of sesamin effects against CCl4 induced liver injury by regulating the expression levels of phosphorylated c-Jun proteins, necrosis factor-alpha (TNF-a) and Bak. In conclusion, these results suggested that the inhibition of CCl4-induced apoptosis by sesamin is due at least in part to its anti-oxidant activity and its ability to modulate the JNK signaling pathway. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Oxidative stress is regarded as a mediator of acute and chronic liver injury. Carbon tetrachloride (CCl4) is a well known hepatotoxin that is widely used to induce acute toxic liver injury in a large range of laboratory animals (Kodai et al., 2007; Campo et al., 2008; Leong et al., 2011). A number of studies have shown that CCl4 is metabolized by the P450 enzyme system to yield reactive metabolic products trichloromethyl free radicals, which can initiate the process of lipid peroxidation and ultimately results in the over-
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCl4, carbon tetrachloride; JNK, the c-Jun N-terminal kinases; ROS, reactive oxygen species; Ses, sesamin; TAC, the total antioxidant capacity; TBARS, thiobarbituric acid reactive substances; TNF-a, necrosis factor-alpha; TUNEL, deoxyribonucleotidyl transferase (TdT)-mediated dUTP-fluorescein isothiocyanate (FITC) nick-end labeling.. ⇑ Corresponding author. Tel.: +86 013700950121; fax: +86 516 83500171. E-mail address: [email protected] (J.-Q. Ma). 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.11.017
production of reactive oxygen species (ROS) and hepatocyte injuries (Kodai et al., 2007; Tien et al., 2011). Jun N-terminal kinases (JNKs), which belong to the superfamily of MAP-kinases, play a critical role in death receptor-initiated extrinsic as well as mitochondrial intrinsic apoptotic pathways (Dhanasekaran et al., 2008). JNKs activate apoptotic signaling by the upregulation pro-apoptotic genes via the transactivation of specific transcription factors or by directly modulating the activities of mitochondrial pro- and anti-apoptotic proteins through distinct phosphorylation events. Many researches showed that ROS can induce apoptosis by activating JNK signaling pathway (Dhanasekaran et al., 2008; Sinha et al., 2013). Many studies also revealed that CCl4 induced hepatic apoptosis by mitochondrial intrinsic and extrinsic apoptotic pathways (Huang et al., 2008; Leong et al., 2011; Borkham-Kamphorst et al., 2013; Monga et al., 2013). Sesame seeds (Sesamun indicum L.) are widely used as dietary supplements. Sesame seeds and their oil have been used in human diets for thousands of years and are believed to provide health ben-
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efits. Sesamin (Ses) is one of the major lignan in sesame seeds and oil. Several studies have shown that sesamin exerts antioxidative (Nakai et al., 2003; Liu et al., 2013) anti-inflammatory (Lee et al., 2009, 2011; Bournival et al., 2012), anti-apoptosis (Bournival et al., 2012; Liu et al., 2013), antihypertensive (Kong et al., 2009; Wu et al., 2012), cholesterol-lowering (Peñalvo et al., 2006), anticarcinogen (Hirose et al., 1992; Lee et al., 2011), hepatoprotective (Chang et al., 2009; Liu et al., 2013), renoprotective (Kong et al., 2009; Wu et al., 2012) and neuroprotective effects (Lee et al., 2011). Reports from our laboratory and others had shown that sesamin can protect liver from injury induced by hepatotoxins (Hemalatha et al., 2004; Chang et al., 2009; Liu et al., 2013). Despite those pharmacological benefits, the molecular mechanisms by which sesamin elicits hepatoprotective effects are still unclear. In the present study, we, for the first time, aimed to determine whether sesamin can protect mouse liver from CCl4-induced oxidative stress and apoptosis via JNK pathway.
2.5. Deoxyribonucleotidyl transferase (TdT)-mediated dUTP-fluorescein isothiocy -anate (FITC) nick-end labeling (TUNEL) assay For the TUNEL staining, the standard protocol for frozen sections was followed (BD ApoAlertTM DNA Fragmentation assay kit, BD Biosciences Clontech, Palo Alto, CA, USA). Apoptosis was assayed by TUNNEL staining using our previous method (Liu et al., 2013). 2.6. Assay of ROS level ROS was measured as described previously, which is based on the oxidation of 20 70 -dichlorodihydrofluorescein diacetate to 20 70 -dichloro-fluorescein (Shinomol and Muralidhara, 2007; Liu et al., 2012). 2.7. Assay of thiobarbituric acid reactive substances (TBARS) levels in liver Tissue lipid peroxidation was measured by our previous method (Liu et al., 2012, 2013). Liver homogenate was incubated with 8.1% SDS (w/v) for 10 min followed by addition of 20% acetic acid (pH 3.5). Reaction mixture was incubated with 0.6% TBA (w/v) for 1 h in boiling water bath. Pink color chromogen was extracted in butanol-pyridine solution (15:1) and read at 532 nm.
2. Materials and methods
2.8. Measurement of the total antioxidant capacity (TAC)
2.1. Chemicals and reagents
The total antioxidant capacity (TAC) in liver was assayed with a commercially available assay kit (Jiancheng Biochemical, Inc., Nanjing, China). This method is based on the oxidation of intracellular antioxidants with iron (III) in acidic medium. The liberated iron (II) reacts with 1,10-phenanthroline to form a colored complex, which is measured at 520 nm. The TAC of the samples was measured according to the manufacturer’s protocol. One unit of TAC was defined as the capability of increasing 0.01 optical densities (OD) value per mg protein per min at 37 °C.
Sesamin (>95% purity) and Carbon tetrachloride (CCl4) were obtained from Sigma Chemical Co. (St. Louis, MO, USA); anti-Bcl-2 antibody, anti-cleaved caspase-3, anti-Bax antibody, anti-Bak antibody, anti-JNK1/2 antibody, anti-phospho-JNK1/2 antibody, anti-phospho-c-Jun antibody, anti-c-Jun antibody, anti-cytochrome c antibody and goat anti-rabbit IgG-HRP from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (Beverly, MA, USA); Reagents and kits used in the assays of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); BCA assay kit from Pierce Biotechnology, Inc. (Rockford, IL, USA); TUNEL apoptosis detection kit from GenScript Corporation (Piscataway, NJ, USA). All other reagents unless indicated were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
2.9. Western blot analyses Nuclear and cytoplasmic extracts for western blotting were obtained by using a nuclear/cytoplasmic isolation kit (Beyotime Institute of Biotechnology, Bijing, China). Protein levels were determined using the BCA assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Western blot analysis was performed as previously described by us (Liu et al., 2012, 2013).
2.2. Animals and treatment 2.10. Statistic analysis Male ICR mice (20–25 g) were obtained from the Branch of National Breeder Center of Rodents (Shanghai) and kept in an environmentally controlled room (23 ± 2 °C, 55 ± 10% humidity) with a 12-h light/dark cycle and allowed free access to food and water. After acclimation for 1 week, the animals were randomly divided into four groups (n = 10). Group I (normal control) was given distilled water (10 ml/ kg body weight). Group II (model) was given distilled water. Groups III and IV were administrated UA (60 and 120 mg/kg body weight, suspended in 0.5% carboxymethylcellulose sodium, daily). The choice of sesamin dose is based on previous findings, which showed that sesamin has protective effects on tissue damage (Chang et al., 2009; Liu et al., 2013). After the oral administration for 7 days, the animals were treated as described previously (Zhang et al., 2013). Two hours after the final administration, mice in Groups II–VI were injected intraperitoneally with 0.3% (v/ v) CCl4 (10 ml/kg, dissolved in olive oil), while the mice in Groups I received appropriate vehicle (olive oil, i.p.). Twenty-four hours after the CCl4 challenge followed by fasting, the animals were anesthetized for obtaining the blood and sacrificed to collect the livers. The right lobe of the liver was fixed in 10% formalin to prepare paraffin sections. The present research reported in this paper was conducted in accordance with the Chinese legislation and NIH publication on the use and care of laboratory animals and were approved by the respective university committees for animal experiments.
2.3. Measurement of serum aminotransferase activities The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were estimated spectrophotometrically using commercial diagnostic kits (Jiancheng Institute of Biotechnology, Nanjing, China) (Liu et al., 2013).
2.4. Histological evaluations The histological changes of liver were evaluated as described previously (Liu et al., 2013). Cryosections were collected on 3-aminopropyl-trimethoxysilanecoated slides (Sigma–Aldrich). The extent of hepatic damage was evaluated on H&E slides. The histological changes were scored according to the following criteria: 0, absent; 1, mild; 2, moderate; and 3, severe (Kim et al., 2011).
All statistical analyses were performed using the SPSS software, version 11.5. A one-way analysis of variance (ANOVA; P < 0.05) was used to determine significant differences between groups and the individual comparisons were obtained by Turkey’s HSD Post Hoc test. Statistical significance was set at P < 0.05.
3. Results 3.1. Sesamin protects against CCl4-induced hepatic dysfunction Serum aminotransferase (such as AST and ALT) is commonly used as an indicator of liver disease (Kodai et al., 2007; Campo et al., 2008; Liu et al., 2013). In order to determine whether sesamin can attenuate the liver damage in the CCl4-treated mouse, we measured the activities of serum ALT and AST (Fig. 1). In CCl4-treated mice, the activities of serum ALT and AST significantly increased by 332% and 608% as compared with these of the controls, respectively (P < 0.05). Interestingly, the treatment with sesamin on the other hand showed dose-dependent inhibition in this elevation (P < 0.05) (Fig. 1). 3.2. Sesamin alleviated CCl4-induced histology changes in liver Liver histological study was used to determine the protective effect of sesamin on CCl4-induced injury. As shown in Fig. 2, the results of histopathological evaluation showed that sesamin exhibited protective effect against CCl4-induced liver injury. CCl4 treatment caused visible histology changes including structure damage including large areas of extensive hepatocellular necrosis, leukocyte infiltration in mouse liver. Moreover, the score for liver injury was significantly higher in the CCl4-treated mice as com-
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pared with vehicle controls. Whereas, sesamin treatment significantly alleviated the CCl4-induced damage in mouse liver (Fig. 2). 3.3. Sesamin inhibited CCl4-induced apoptosis in liver We used the TUNEL assay to investigate the effect of sesamin on the CCl4-induced apoptosis (Fig. 3). The number of TUNEL-positive cells in the liver of CCl4-treated mice was significantly increased (P < 0.05). Whereas, sesamin markedly decreased the liver TUNEL-positive cells of mice treated with CCl4 in a dose-dependent manner (Fig. 3). 3.4. Sesamin inhibited CCl4-induced oxidative stress in liver
Fig. 1. Effect of sesamin on hepatic functional markers in CCl4 treated mice. (A) ALT activity; (B) AST activity. All values are expressed as mean ± S.E.M. (n = 7). ## P < 0.05, compared with the control group; **P < 0.05, vs. nickel-treated group.
Many studies suggested that the levels of ROS, TBARS and TAC might be indicators of oxidative stress (Li et al., 2004, 2012, 2013).The results showed that sesamin could decrease CCl4-induced ROS and TBARS levels (Fig. 4). CCl4 treatment markedly increased hepatic ROS and TBARS levels by 121% and 72% as compared with those of the controls, respectively (P < 0.05). However, the treatment with sesamin on the other hand showed dosedependent inhibition in this elevation (P < 0.05) (Fig. 4). As shown in Fig. 4C, the TAC level in CCl4-treated mice was markedly decreased by 37% as compared with that of the control (P < 0.05). However, treatment with semamin in CCl4-treated mice significantly increased the hepatic TAC level in a dose-dependent manner (P < 0.05).
Fig. 2. Morphological and histological evaluation of liver in mice. (A) The control group; (B) Mice treated with CCl4; (C) Mice treated with CCl4 and fed with sesamin (60 mg / kg); (D) Mice treated with CCl4 and fed with sesamin (120 mg /kg). The black arrow indicates infiltrating leukocytes. The white arrow indicates hepatic cell necrosis. Original magnification, 20 10.
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3.5. Sesamin-mediated protective action involves JNK and c-Jun activation Activation of JNK is known to promote apoptosis (Dhanasekaran et al., 2008; Sinha et al., 2013). To investigate whether JNK signaling was involved in the action of sesamin, we determined the effects of sesamin on JNK pathway in mouse liver. The results showed the level of phospho-JNK was markedly increased in the livers of CCl4-treated mice as compared with the vehicle controls (Fig. 5). However, the up-regulation of phospho-JNK was markedly suppressed in the sesamin and CCl4 co-treated mice (P < 0.05). Activation of c-Jun was involved in mitochondrial extrinsic apoptotic pathways. As shown in Fig. 5, the level of phospho-c-Jun was markedly increased in the livers of CCl4-treated mice as compared with the vehicle controls (P < 0.05). However, the treatment with sesamin on the other hand showed dose-dependent inhibition in this elevation (P < 0.05). 3.6. Sesamin modulates CCl4-induced expression of pro-apoptotic proteins JNK has been shown to regulate the expression of pro-apoptotic and anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bax (Dhanasekaran et al., 2008; Sinha et al., 2013). Therefore, we examined the effects of sesamin on JNK regulated intrinsic proapoptotic proteins. As shown in Fig. 6, CCl4 treatment increased the expression of Bax, Bak, TNF-a and cytosol cytochrome c and
caused a reduction in the expression of the anti-apoptotic protein Bcl-2 (P < 0.05). However, sesamin treatment abolished the CCl4evoked pro-apoptotic signaling events in the liver of mice (P < 0.05) (Fig. 6). 3.7. Sesamin reduced CCl4-induced caspase-3 activation In response to both the extrinsic and intrinsic apoptotic stimuli, JNK plays an essential role through its ability to interact and modulate the activities of caspase proteins (Dhanasekaran et al., 2008; Sinha et al., 2013). Caspase-3 is one of the key executioners of apoptosis, capable of cleaving or degrading many key proteins such as nuclear lamins, fodrin, and the nuclear enzyme poly (ADPribose) polymerase (PARP) (Sinha et al., 2013; Liu et al., 2013). In order to determine whether sesamin can attenuate apoptosis in the liver of CCl4-treated mice, the activity of caspase-3 was also examined. As shown in Fig. 6F, cleaved caspase-3 levels were significantly elevated as compared with that of the controls in the CCl4-treated mouse liver (P < 0.05). Interestingly, the treatment with sesamin on the other hand inhibited this elevation in a dose-dependent manner (P < 0.05). 4. Discussion The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemicals necessary for
Fig. 3. In situ detection of fragmented DNA [deoxyribonucleotidyl transferase-mediated dUTP-FITC nick-end labeling (TUNEL) assay] in the liver of mice. The merged images (down) show that apoptotic cells appear yellow and non-apoptotic cells appear red. Scale bars = 100 mm. The histogram showed the relative proportion of TUNEL-positive cells in the liver of mice. All values are expressed as mean ± S.E.M. **P < 0.05, compared with the control group; ##P < 0.05, vs. nickel-treated group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Effect of sesamin on the oxidative stress in liver of CCl4-treated mice. (A) Level of ROS; (B) Level of TBARS; (C) TAC. Each value is expressed as mean ± S.E.M. (n = 7). ##P < 0.05, compared with the control group; **P < 0.05, vs. nickel-treated group.
digestion. However, the liver is the most common target organ for chemically induced injuries (Liu et al., 2013). CCl4 is considered as a direct hepatotoxin which produces centrilobular necrosis and steatosis in liver (Kodai et al., 2007; Tien et al., 2011; Kim et al., 2011). The present investigation reveals that the activities of AST and ALT in the serum of CCl4 treated mice were markedly increased (Fig. 1), which in response to CCl4 have been attributed to hepatic structural damage because these enzymes are normally localized to the cytoplasm and are released into the circulation after cellular damage has occurred (Ozturk et al., 2009; Liu et al., 2013). Moreover, the histological changes of liver, such as structure damage, hepatocellular necrosis and leukocyte infiltration, had been observed in CCl4-treated animals (Fig. 2). Our previous study had demonstrated that sesamin has hepatoprotective effects (Liu et al., 2013). In this study, treatment with sesamin effectively protected mice against CCl4-induced liver damage by reducing elevated serum ALT & AST activities (Fig. 1) and alleviating hepatic histological changes (Fig. 2). These results suggest that sesamin could protect mice against CCl4-induced hepatic dysfunction and histopathologic damage. Many studies suggest that one possible molecular mechanism involved in CCl4 hepatotoxicity is the disruption of delicate oxidant/antioxidant balance, which can lead to liver injury via oxidative damage (Kodai et al., 2007; Chang et al., 2009; Leong et al., 2011). Accumulating evidence has also shown that CCl4 is metabolized to produce highly toxic trichloromethyl free radical (CCl3) and/or trichloro methyl peroxyl (OOCCl3) free radicals by cytochrome P450 enzyme and causes damage (Kodai et al., 2007; Tien et al., 2011; Sinha et al., 2013). Both trichloromethyl and its peroxy radical are capable of binding to proteins or lipids, or of abstracting
Fig. 5. The protective effect of sesamin against CCl4-induced apoptosis depends on the JNK pathway. (A) Relative density analysis of the phospho-JNK protein bands; (B) Relative density analysis of the phospho-c-Jun protein bands; b-actin was probed as an internal control in relative density analysis of the JNK and c-Jun protein bands. The relative density is expressed as the ratio (phospho-JNK/totalJNK, phospho-c-Jun/total-c-Jun). The vehicle control is set as 1.0. Values are averages from seven independent experiments. Each value is expressed as mean ± S.E.M. ##P < 0.05, compared with the control group; **P < 0.05, vs. CCl4treated group.
a hydrogen atom from an unsaturated lipid, initiating lipid peroxidation and liver damage (Weber et al., 2003; Chang et al., 2009; Sinha et al., 2013). These free radicals combine with polyunsaturated fatty acids of hepatic and testicular cell membranes, cause elevation of thiobarbituric acid reactive substances (TBARS) concentration with subsequent necrosis and increase lysosomal enzymes activities (Yen et al., 2009; Shim et al., 2010; Zhang et al., 2013). In the present research, levels of ROS and TBARS were remarkably increased in CCl4-treated mouse liver as compared with these of the control, indicating that CCl4 exposure induced oxidative stress (Fig. 4). Sesamin has been reported to have many benefits and medicinal properties (Nakai et al., 2003; Bournival et al., 2012). In this study, sesamin decreased the ROS and TBARS productions in the CCl4-treated mouse liver (Fig. 4), which may be due to the powerful antioxidant and free radical scavenging activities (Hemalatha et al., 2004; Shinomol and Muralidhara, 2007). Our finding suggests that sesamin could at least partly attenuate oxidative stress by decreasing levels of ROS and lipid peroxide in CCl4-treated mouse liver. JNKs have been shown to be have involved in stimulating apoptotic signaling. Oxidative stress can activate JNK to cause apoptosis by receptor-initiated extrinsic and mitochondrial intrinsic apoptotic pathways (Dhanasekaran et al., 2008; Sinha et al., 2013). The
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Fig. 6. Immunoblotting analysis of apoptosis-provoking proteins in response to CCl4 and sesamin. (A) Relative density analysis of the Bax protein bands; (B) Relative density analysis of the Bcl-2 protein bands; (C) Relative density analysis of the cytosol cytochrome c bands; (D) Relative density analysis of the Bak protein bands; (E) Relative density analysis of the TNF-a protein bands; (F) Relative density analysis of the cleaved caspase-3 protein bands. b-actin was probed as an internal control in relative density analysis of the protein bands. The vehicle control is set as 1.0. Values are averages from seven independent experiments. Each value is expressed as mean ± S.E.M. ##P < 0.05, compared with the control group; **P < 0.05, vs. CCl4-treated group.
crucial mitochondrial event initiating apoptosis is the release of cytochrome c from the inner membrane space of mitochondria (Dhanasekaran et al., 2008; Sinha et al., 2013). After release from mitochondria to the cytosol, cytochrome c binds to apoptosis-activating factor, which in turn stimulates caspase activity to regulate apoptosis (Dhanasekaran et al., 2008; Tien et al., 2011). JNKs also play an essential role in modulating the functions of pro- and anti-apoptotic proteins located in mitochondria. JNK and ROS can stimulate the activities of pro-apoptotic proteins (Bax, Bak, Bok, Bid, Bim, Bik, Bad, Bmf, Hrk, Noxa, Puma, Blk, etc.) and promote apoptosis by inhibiting the anti-apoptotic proteins (Bcl-2, Bcl-XL, Bcl-w, A1, Mcl-1, etc.) to regulate the release of cytochrome c and apoptosis (Tien et al., 2011; Sinha et al., 2013). This study showed that semamin treatment restored the expression levels of cytosol cytochrome c, Bax, Bak and Bcl-2 in the liver of CCl4-trea-
ted mice (Fig. 6), suggesting that sesamin could protect mouse liver by regulating JNK signaling and mitochondrial intrinsic apoptotic pathways. JNK signaling is also involved in the extrinsic apoptotic pathway initiated by death receptors (Dhanasekaran et al., 2008; Sinha et al., 2013). Studies showed that the JNK-c-Jun/Ap1 pathway is involved in the increased expression of pro-apoptotic genes such as TNF-a, Fas-L, and Bak (Fan and Chambers, 2001; Dhanasekaran et al., 2008; Björkblom et al., 2008). Many researches revealed that CCl4 could induce hepatic apoptosis by mitochondrial extrinsic apoptotic pathways (Tien et al., 2011; Lu et al., 2012; Zhang et al., 2013). In the present study, we also found that the expression levels of JNK1/2 phosphorylation, c-Jun phosphorylation, TNF-a and Bak increased in liver of CCl4 treated mice. However, sesamin treatment significantly deceased the expression levels of
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Acknowledgments This work is supported from Startup Project of Doctor scientific research by Sichuan University of Science and Engineering (2013RC14).
References
Fig. 7. Schematic diagram showing protective signaling of sesamin in CCl4-induced liver damage. The ? indicates activation or induction, and indicates inhibition or blockade.
these proteins (Fig. 6), suggesting that sesamin could protect mouse liver by regulating JNK signaling and mitochondrial extrinsic apoptotic pathways. The caspases is a family of proteins that are one of the main executors of the apoptotic process. The caspase pathway is a well-identified downstream target for JNK signaling in apoptosis (Dhanasekaran and Reddy, 2008; Sinha et al., 2013). Caspase-3 is one of the key executioners of apoptosis, capable of cleaving or degrading many key proteins such as nuclear lamins, fodrin, and the nuclear enzyme poly (ADPribose) polymerase (PARP) (Tien et al., 2011; Zhang et al., 2013). The present study showed that CCl4 increased the number of TUNEL-positive cells and the activity of caspase-3 in the liver of mice. However, we found that sesamin markedly decreased the number of TUNEL-positive cells and the activity of caspase-3 in mice treated with CCl4 (Figs. 3 and 6F). Thus, our findings indicate that sesamin might protect liver against CCl4-induced apoptosis by activating the JNK pathway. In summary, this study demonstrates for the first time that sesamin has potent protective effects against CCl4-induced apoptosis by modulating the JNK pathway in mouse liver. We propose a possible protective effect of sesamin (Fig. 7). Here we demonstrated that sesamin administration attenuated CCl4-induced hepatic dysfunction and histopathologic changes. Sesamin attenuated CCl4-induced hepatic oxidative damage by inhibiting ROS generation and increasing liver TAC level. Sesamin treatment could effectively inhibited CCl4-induced apoptosis in liver by decreasing expression levels of phospho-JNK1/2, phospho-c-Jun, Bax, Bak, TNF-a, cytosol cytochrome c and caspase-3 and increasing expression levels of Bcl-2. Sesamin seems to be potent hepatoprotective drug and its use in maintaining a healthy liver and preventing toxic liver damage deserves consideration. However, there are fewer studies on the absorption of sesamin in humans. The question warrants further investigation and further examination.
Conflict of Interest The authors declare that there are no conflicts of interest.
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