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Astaxanthin pretreatment attenuates acetaminophen-induced liver injury in mice
International Immunopharmacology 45 (2017) 26–33
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Astaxanthin pretreatment attenuates acetaminophen-induced liver injury in mice Jingyao Zhang a,1, Simin Zhang a,1, Jianbin Bi a, Jingxian Gu a, Yan Deng a, Chang Liu a,⁎ a
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, PR China
a r t i c l e
i n f o
Article history: Received 13 June 2016 Received in revised form 20 January 2017 Accepted 21 January 2017 Available online 31 January 2017 Keywords: Liver injury Acetaminophen Astaxanthin Oxidative stress Apoptosis JNK pathway
a b s t r a c t Background: Acetaminophen (APAP) is a conventional drug widely used in the clinic because of its antipyretic-analgesic effects. However, accidental or intentional APAP overdoses induce liver injury and even acute liver failure (ALF). Astaxanthin (ASX) is the strongest antioxidant in nature that shows preventive and therapeutic properties, such as ocular protection, anti-tumor, anti-diabetes, anti-inflammatory, and immunomodulatory effects. The aim of present study was to determine whether ASX pretreatment provides protection against APAP-induced liver failure. Methods: Male C57BL/6 mice were randomly divided into 7 groups, including control, oil, ASX (30 mg/kg or 60 mg/kg), APAP and APAP + ASX (30 mg/kg or 60 mg/kg) groups. Saline, olive oil and ASX were administered for 14 days. The APAP and APAP + ASX groups were given a peritoneal injection of 700 mg/kg or 300 mg/kg APAP to determine the 5-day survival rate and for further observation, respectively. Blood and liver samples were collected to detect alanine transaminase (ALT), aspartate transaminase (AST), inflammation, oxidative stress and antioxidant systems, and to observe histopathologic changes and key proteins in the mitogen-activated protein kinase (MAPK) family. Results: ASX pretreatment before APAP increased the 5-day survival rate in a dose-dependent manner and reduced the ALT, AST, hepatic necrosis, reactive oxygen species (ROS) generation, lipid peroxidation (LPO), oxidative stress and pro-inflammatory factors. ASX protected against APAP toxicity by inhibiting the depletion of glutathione (GSH) and superoxide dismutase (SOD). Administration of ASX did not change the expression of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and P38. However, phosphorylation of JNK, ERK and P38 was reduced, consistent with the level of tumor necrosis factor alpha (TNF-α) and TNF receptor-associated factor 2 (TRAF2). Conclusion: ASX provided protection for the liver against APAP hepatotoxicity by alleviating hepatocyte necrosis, blocking ROS generation, inhibiting oxidative stress, and reducing apoptosis by inhibiting the TNF-α-mediated JNK signal pathway and by phosphorylation of ERK and P38, which made sense in preventing and treating liver damage. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Drug-induced liver injury (DILI) is one of the most familiar and critical adverse drug reactions (ADR) resulting in ALF. DILI has become the leading cause of ALF in the United States and United Kingdom, as well as in some other countries and regions [1]. Based on statistics from the World Health Organization (WHO), DILI has become the fifth fatal cause of death in the worldwide. Acetaminophen (APAP) is a conventional drug widely used in the clinic as antipyretic analgesic. However, accidental or intentional APAP overdoses contribute to liver injury. Recent data suggest that APAP-induced ALF leads to approximately 30,000 cases of hospitalization and ⁎ Corresponding author at: Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, 277 Yanta West Road, Xi'an 710061, PR China. E-mail address: [email protected] (C. Liu). 1 They contribute equally to the paper.
http://dx.doi.org/10.1016/j.intimp.2017.01.028 1567-5769/© 2017 Elsevier B.V. All rights reserved.
500 deaths each year in America [2]. The mechanism of this hepatotoxicity has not been entirely clarified. The ultimate hepatotoxic metabolite, Nacetyl-p-benzoquinoneimine (NAPQI), is the initial step and is detoxified by glutathione (GSH). NAPQI combines with cellular target proteins during APAP overdoses [3]. Evidence suggests that exhaustion of GSH and binding between NAPQI and proteins contribute to cell injury [4,5]. Mitochondrial dysfunction triggered by APAP overdose results in oxidative stress [6,7]. In addition to ROS, increased lipid peroxidation also indicates oxidative stress in APAP-induced liver injury [8]. Recently, it was shown that inflammation, apoptosis and autophagy were also associated with APAP-induced hepatotoxicity [9]. Oxidative stress leads to activation of c-Jun N-terminal kinase (JNK) [10]. Moreover, various studies suggest that pro-inflammatory cytokines, such as TNF-α and interleukin-6 (IL-6), activate JNK and P38, which are related to ERK [11]. Activation of JNK plays an important role in APAP-induced hepatotoxicity [10]. The JNK signaling pathway up-regulates apoptosis and autophagy by activating TNF receptor-associated factor 2 (TRAF2) [12].
J. Zhang et al. / International Immunopharmacology 45 (2017) 26–33
As a type of nature liposoluble carotenoid pigment, astaxanthin is present in aquatic animals, microalgae, flamingoes, and Pfaffia (yeast), and possesses potent antioxidant ability [13]. Additionally, ASX also has many pharmacological properties, such as ocular protection, antitumor, anti-diabetes, anti-inflammatory, and immunomodulatory effects [14]. Previous studies showed that ASX effectively inhibits peroxyl radical-dependent lipid peroxidation, scavenges singlet oxygen, and blocks ROS generation [15]. The antioxidative capacity of ASX makes it a potential pharmacologic resource. ASX pretreatment attenuates hepatic ischemia reperfusion-induced apoptosis and autophagy via the ROS/MAPK pathway in mice [16]. ASX inhibits neural progenitor cellular apoptosis modulated by the P38 and MEK signaling pathways [17]. ASX was found to protect the liver against ConA-induced autoimmune hepatitis by reducing the phosphorylation of Bcl-2 modulated by the JNK signal pathway [18]. According to data, the antioxidant capacity of ASX is 100–500 times greater than vitamin E, and 10 times greater than beta-carotene [19]. Several studies showed that ASX has better capacity in relevant diseases. In N-methyl-N-nitrosourea (MNU)-induced mammary cancer, high concentrations of ASX showed an anti-cancer effect, whereas canthaxanthin did not suppress carcinoma [20]. In ischemia reperfusion (I/R) injury after intestinal transplantation, ASX was effective compared to other antioxidants, such as 4,5-dihydroxy-1,3benzene-disulfonic acid (Tiron) and epigallocatechin gallate (EGCG) [21]. In diet-induced insulin resistance and nonalcoholic steatohepatitis (NASH), ASX played a more effective role in NASH than vitamin E [22]. Moreover, ASX showed no apparent inhibition of seven major human hepatic UGTs so that it is safe for further clinical use [23]. The objectives of the present study were to determine whether ASX provides preventive and therapeutic effects during APAP-induced liver injury. We also assessed the potential mechanism of action. 2. Materials and methods
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olive oil and ASX were administered for 14 days. APAP and ASX were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Experimental protocols Blood samples were collected at selected time points (8 h and 24 h) after APAP administration and centrifuged at 3000× for 15 min to obtain supernatants. Serum samples were stored at −80 °C for further biochemical detection. Sections of liver tissues were fixed in 10% formaldehyde for histology. Blocks of liver tissues were placed in 2.5% glutaraldehyde for scanning electron microscopy (SEM). Additionally, a portion of the remaining liver was immediately homogenized for later analysis. 2.3. Survival The survival study was conducted using the following three groups: control, APAP, and APAP + ASX groups. Mice received an i.p. injection of 700 mg/kg APAP. To observe the impacts of ASX on APAP-induced liver injury, the 5-day survival rate was determined. 2.4. Serum biochemistry Serum ALT and AST were selected as sensitive indicators for hepatotoxicity, and ALT and AST levels were measured with an automated biochemical analyzer in the Department of Inspection, the First Affiliated Hospital of Xi'an Jiaotong University. 2.5. Determination of cytokines Levels of TNF-α and IL-6 were measured using commercial ELISA kits according to the manufacturer's instructions (Dakewe, Shenzhen, China).
2.1. Preparation of animals and reagents 2.6. Histology Male C57BL/6 mice were purchased from the Animal Feeding Center of Xi'an Jiaotong University Health Science Center. Experimental animals included in this study were 4–5 weeks old and weighed 23.0 ± 1.4 g. Mice were housed in an air-conditioned room under a 12-h light/dark cycle (lights on 6:00 AM to 6:00 PM) and allowed food and water ad libitum. All experimental protocols followed the criteria of the Ethics Committee of Xi'an Jiaotong University Health Science Center. Mice were randomly allocated into the following groups: control (saline intraperitoneal (i.p.) injection); oil (olive oil by gavage); ASX (30 mg/kg/d or 60 mg/kg/d by gavage, dissolved in olive oil) [16]; APAP (700 mg/kg or 300 mg/kg i.p. injection), and ASX administrated after APAP (APAP + ASX; doses same as described above). Saline,
Liver samples were fixed in 10% formaldehyde and prepare to be embedded in paraffin. Serial liver sections (5 μm thick) were stained with H&E or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)\(Roche Molecular Biochemicals, Indianapolis, IN) kits and were observed under a microscope (Olympus Optical Co., Tokyo, Japan). Portions of liver tissues were prefixed in 2.5% glutaraldehyde and washed with 0.1 M phosphate buffer. The samples were post-fixed with 1% OsO4 and again washed with 0.1 M phosphate solution. The fixed tissues were dehydrated with graded ethanol and dried using the critical point drying method. After conductive treatment, ultrathin
Fig. 1. Olive oil and ASX cause no damage to normal liver tissues. (A) Serum ALT levels were measured in four groups of male C57BL/6 mice respectively treated with 0.9% saline, olive oil, ASX (30 mg/kg/d), and ASX (60 mg/kg/d) for 14 days; (B) Serum AST levels were detected as in (A). Data are presented as the mean ± SD.
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Fig. 2. ASX improves liver function in hepatic injury induced by APAP. (A) Serum ALT values were measured in four groups of male C57BL/6 mice treated with 0.9% saline, APAP (300 mg/kg), APAP (300 mg/kg) + ASX (30 mg/kg/d), and APAP (300 mg/kg) + ASX (60 mg/kg/d). Saline and ASX were administered for 14 days; (B) Serum AST values were consistent with (A). ALT and AST are expressed as the mean ± SD. *P b 0.05 for APAP + ASX group vs APAP group, #P b 0.01 for APAP + ASX group vs APAP group.
sections (50-nm thick) were studied with a scanning electron microscope.
3. Results 3.1. ASX improves liver function in hepatic injury induced by APAP
2.7. Detection of ROS activation Liver samples were fixed with 10% formaldehyde and dehydrated using 30% sucrose solution. The sections (5 μm thick) were incubated with dihydroethidium (DHE; 10 μM). After 60 min in the dark, the specimens were washed with adequate volumes of PBS. DHE oxidized by ROS in the cells showed red emission under a fluorescence microscope using green wavelength excitation.
In C57BL/6 mice treated with olive oil or ASX (30 mg/kg/d or 60 mg/kg/d) for 14 days, the ALT and AST activity values did not significantly differ from the controls (Fig. 1A and B), indicating that neither ASX nor olive oil had hepatic toxicity. In Fig. 2A and B, serum ALT and AST levels decreased at 8 h and 24 h (P b 0.05 or P b 0.01) in APAP mice treated with ASX. 3.2. Effects of ASX on survival after APAP overdose
2.8. Measurement of lipid peroxidation Tissue malondiadehyde (MDA) in liver homogenates were determined to analyze the lipid peroxidation (LPO) level using a kit (Jiancheng Institute of Biotechnology) as described by the manufacturer. 2.9. Determination of enzymatic activities Liver tissue was homogenized to be centrifuged at 4000 rpm for 20 min. The supernatants were prepared to measure the level of myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) based on the kit instructions from Jiancheng Institute of Biotechnology. 2.10. Western blotting Homogenates were centrifuged and proteins were fractionated by SDS-PAGE on 4% gels under reducing conditions. Separated proteins were electrotransferred onto polyvinylidine difluoride membranes (PVDF, Immobilin-P, Millipore, Bedford, MA) and blocked with 5% to 10% milk for 2 h at room temperature, followed by incubation with the appropriate primary antibodies overnight at 4 °C. After washing with PBST 3 times for 10 min each, the membranes were incubated with secondary antibodies for 2 h at room temperature. Protein bands were visualized using the Immobilon Western Chemilum HRP substrate (Millipore, Bedford, MA) and Image Lab Software (Bio-Rad Laboratories, Hercules, CA, USA).
Animals were given 700 mg/kg APAP to monitor the effects of ASX for 5 days in the APAP, APAP + ASX (30) and APAP + ASX (60) groups. The survival rates were calculated at 5 time points (24 h, 48 h, 72 h, 96 h, and 120 h). At each time point, the survival rate was higher in the APAP + ASX group than the APAP group. We found a low survival rate in each group at 120 h. Administration of ASX significantly increased the 5-day survival rate of mice (Fig. 3), indicating that ASX significantly reduced the mortality caused by APAP overdose. 3.3. ASX ameliorates histopathologic changes Mice were randomly divided into four groups, including control, APAP, APAP + ASX (30), and APAP + ASX (60). The latter three groups were given APAP 300 mg/kg. Sections from liver samples were stained with H&E and observed. The disorganized liver form, hemorrhage, and leukocyte infiltration in the APAP + ASX groups were ameliorated compared to the APAP group at 8 h and 24 h. This was more apparent in the APAP + ASX (60) group than the APAP + ASX (30) group. ASX clearly reduced the hepatocyte necrosis caused by APAP (Fig. 4A and B). As shown in Fig. 4C, there was no significant decrease of the liver index
2.11. Statistical analysis Analysis was performed with the SPSS statistical package, and values are reported as the mean ± SE. Differences between multiple groups were assessed by ANOVA. Values of P b 0.05 were considered statistically significant.
Fig. 3. Effects of ASX on survival after APAP overdose. Experimental and control groups were given 700 mg/kg APAP and the survival rates were monitored for 5 days. *P b 0.05 for APAP + ASX group vs APAP group.
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Fig. 4. ASX ameliorates histopathologic changes. (A) Serial liver sections (5 μm thick) stained with HE were observed under a microscope at 8 h. These sections were obtained from normal liver tissues, the tissues of livers with APAP-induced injury and the tissues of livers pretreated with ASX (30 mg/kg/d or 60 mg/kg/d) before APAP. Magnifications: 100× and 200×. (B) Serial liver sections stained with H&E were observed at 24 h. The sections, microscope and magnifications are the same as those in (A). (C) The liver index was calculated at 8 h and 24 h to assess the effect of ASX against APAP. (D) The percentage of liver necrosis was measured. (E) Ultrathin liver sections (50 nm thick) were studied at 24 h with a scanning electron microscope. Magnifications: 20,000×. *P b 0.05 for APAP + ASX group vs APAP group, #P b 0.01 for APAP + ASX group vs APAP group.
at 8 h. The liver index decreased (P b 0.05) in the APAP + ASX (60) group at 24 h. Moreover, the percentage necrosis was significantly reduced in the APAP + ASX groups (Fig. 4D). In APAP mice administered ASX, the disordered lobular structure disappeared in a dose-dependent manner (Fig. 4E). It is unknown whether apoptosis exists as another method of causing hepatic injury in APAP-induced hepatotoxicity. TUNEL results showed that the stained zones were decreased (P b 0.05) in the APAP + ASX group (Fig. 5).
3.4. Effect of ASX on oxidative stress and antioxidant system Oxidative stress is an early event in APAP-induced liver injury [8]. The product of oxidative stress related to radical generation mainly consists of ROS and MPO. More ROS presented as bright red dot-like substances in the APAP group than in the APAP + ASX groups at 8 h and 24 h (Fig. 6A and B), demonstrating that ASX protected against APAP toxicity by inhibiting ROS generation. The level of MPO in liver
Fig. 5. Detection of ROS activation. Hepatic apoptosis was observed with photography and quantitation of stained sections under a standard fluorescence microscope. #P b 0.01 for APAP + ASX group vs APAP group.
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Fig. 6. Effect of ASX on ROS generation, oxidative stress, lipid peroxidation and antioxidant systems. (A) ROS was stained with DHE and presented as bright red dot-like substances and was observed at 8 h in experimental and control groups. Magnification: 200×. (B) Presentation of ROS obtained at 24 h. (C) MDA levels in the liver. (D) MPO levels in the liver. (E) SOD levels in the liver. *P b 0.05 for APAP + ASX group vs APAP group, #P b 0.01 for APAP + ASX group vs APAP group.
homogenates was detected and was significantly decreased in the APAP + ASX groups compared to the APAP group (Fig. 6D). LPO played an important role during APAP-induced liver injury [24]. MDA was the indicator during LPO. In contrast to the high level of MDA in the APAP group, the level significantly decreased in the APAP + ASX groups at 8 h and 24 h (Fig. 6C). SOD and the glutathione redox system are major cellular antioxidant system that combats and neutralizes free radicals [25]. The activities of SOD, GSH and GSH-Px were measured in liver homogenates at 8 h and 24 h after APAP treatment. Depletion of SOD and GSH decreased significantly when mice were treated with ASX at 8 h and 24 h (Figs. 6E and 7A), suggesting that ASX prevents the depletion of SOD and GSH. The function of GSH-Px was to neutralize lipid hydroperoxides. The level of GSH-Px was recovered
(P b 0.01) in the APAP + ASX (30) group at 24 h (Fig. 7B), indicating that ASX also reduced the depletion of GSH-Px. 3.5. ASX reduces pro-inflammatory cytokines Excessive pro-inflammatory cytokines, such as TNF-α and IL-6, activate the innate immune system to cause severe liver damage with a toxic dose of APAP [26]. The levels of TNF-α and IL-6 in serum were detected at 8 h and 24 h after APAP administration. TNF-α was reduced (P b 0.01) in the APAP + ASX (60) group at 24 h (Fig. 8A). Treatment with ASX resulted in a significantly decrease in IL-6 in the APAP + ASX groups compared to the APAP group at 8 h and 24 h (Fig. 8B). In the present report, ASX inhibited the release of pro-inflammatory cytokines, such as IL-6, and showed a potential ability to reduce TNF-α.
Fig. 7. ASX attenuates the depletion of GSH. (A) The depletion and levels of GSH in liver were observed at 8 h and 24 h in mice pretreated with or without ASX. (B) The GSH-Px levels in the liver. *P b 0.05 for APAP + ASX group vs APAP group, #P b 0.01 for APAP + ASX group vs APAP group.
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Fig. 8. ASX reduces pro-inflammatory cytokines. (A) Serum TNF-α was measured in four groups of mice (6 male C57BL/6 mice) treated with 0.9% saline, APAP (300 mg/kg), APAP (300 mg/kg) + ASX (30 mg/kg/d) and APAP (300 mg/kg) + ASX (60 mg/kg/d). (B) Serum IL-6 was obtained from four different groups described above. *P b 0.05 for APAP + ASX group vs APAP group, #P b 0.01 for APAP + ASX group vs APAP group.
3.6. ASX protects liver from APAP by reducing the activation of JNK, ERK and P38 To assess key proteins in the signaling pathway for apoptosis, the levels of TRAF2, TNF-α, JNK, p-JNK, ERK, p-ERK, P38 and p-P38 were measured in liver tissues. As shown in Fig. 9, administration of ASX
did not change the expression of ERK and P38 at 8 h in APAP-induced liver injury. The phosphorylation of ERK (p-ERK), P38 (p-P38), and JNK (p-JNK) was reduced. This suggests that ASX inhibits the phosphorylation of ERK and P38. High expression of TNF-α was detected in the APAP group compared to the APAP + ASX groups at 8 h and 24 h (Fig. 8). Similar changes of protein expression were found for TRAF2
Fig. 9. ASX protects liver from APAP by reducing phosphorylation of JNK, ERK and P38. (A) The levels of proteins ERK, p-ERK, P38, and p-P38 at 8 h in APAP-induced liver tissue were shown as Western blot bands. The levels of proteins TNF-α, TRAF2 and JNK signaling pathway at 8 h and 24 h in APAP-induced liver tissue were detected with Western blotting. (B) The relative band densities were calculated. *P b 0.05 for APAP + ASX group vs APAP group, #P b 0.01 for APAP + ASX group vs APAP group.
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(Fig. 9). At the protein level, with 60 mg/kg ASX treatment, the amount of p-JNK decreased more than p-ERK, p-P38, or TRAF2. Taken together, these results suggest that the JNK/p-JNK pathway is greatly attenuated by blocking both TNF-α and TRAF2 when treated with ASX. 4. Discussion Over 1100 types of classical drugs induce hepatotoxicity [27]. APAPinduced liver injury is one of the most studied drug-induced diseases [28]. APAP overdoses cause serious hepatotoxic effects, with ALF as the worst outcome [29]. We used APAP for overdoses in male C57BL/6 mice to determine whether ASX exerts a dose-dependent hepatoprotective effect. We observed a short 5-day survival rate and remarkable elevations of ALT and AST levels in the APAP group, which showed our successful APAP-induced liver injury model. In APAP hepatotoxicity, NAPQI covalently combines with cellular proteins, leading to hepatocyte death and the release of ALT and AST into plasma [30]. ALT and AST were decreased, we initially predicted that ASX protected the structure of hepatic cells from damage. We determined the mechanism by which ASX exerts protective effects on APAP-induced liver injury. The metabolite NAPQI depletes GSH and generates mitochondrial dysfunction, oxidative stress and necrosis [31]. To further confirm our findings, the morphology, cytokines, enzymes, chemokines and pro-inflammatory factors were examined. In the histological observation, severe hepatic necrosis and tumid structure caused by APAP were visually ameliorated by ASX. The percentage necrosis was consistent with H&E and SEM changes. We demonstrated that ASX protected the liver from necrosis. In our APAP-induced liver injury model, GSH performed poorly because of depletion. With ASX, the reduction of GSH was decreased. Furthermore, oxidative stress has an important contribution to liver injury, starting with LPO and the accumulation of ROS [8]. During this progress, superoxide, ROS generation, and hydroxyl and hydrogen peroxide cause oxidative stress and damage to DNA, proteins and lipids [32]. ASX reduces the production of ROS and MPO because of its potent antioxidant properties. Because of its unique molecular structure, ASX has a hydroxyl on one ionone ring and a keto on the other ring to scavenge radicals by combining with the two ionone moieties [33,34]. A commercial kit for MDA showed that LPO was significantly attenuated. Data indicated that ASX reduced cell damage by inhibiting lipid peroxidation (LPO). Moreover, it is thought to be antioxidant defense system along with oxidative stress [35]. Antioxidant enzymes, such as SOD and GSH-Px, are massively depleted with APAP treatment [36]. The depletion of SOD and GSH-Px was reduced after ASX administration, demonstrating the protective impact of ASX on APAP toxicity. At the same time, many events in the liver culminate in centrilobular necrosis [37]. Necrotic hepatocytes release many damage-associated molecular patterns (DAMPs), resulting in immune activation in the noninfectious inflammatory response [38–40]. DAMPs facilitate the release of IL-6, TNF-α, MCP-1 and IL-1β, resulting in further infiltration of leukocytes that augments liver injury [41]. A previous study showed that IL-6 and TNF-α cause drug-induced hepatic injury. TNF-α can activate TRAF2, thereby up-regulating apoptosis through the JNK/p-JNK pathway in liver injury [12]. Apoptosis can activate numerous liver diseases [42]. Several previous studies showed that apoptosis contributes to APAP-induced liver injury [43,44]. In our study, ASX reduced the production of pro-inflammatory cytokines, including IL-6 and TNF-α, consistent with a decrease in the levels of TRAF2 and p-JNK, which mediate apoptosis. The activation of ERK/p-ERK and P38/p-P38 is consistent with JNK. As indicated by TUNEL, a clear reduction of DNA fragmentation and nuclear staining around centrilobular areas was observed with ASX compared to APAP-induced liver failure, correlating with the Western blot values. It demonstrates that ASX provides protection by TNF-α mediated apoptotic cell death through the JNK/p-JNK pathway and other MAPK family members, including the ERK and P38 signaling pathway.
5. Conclusion In conclusion, we discussed the potent protective capacity of ASX, which showed preventive and therapeutic potential in APAP-induced liver injury in mice. The results above, for the first time, indicate that ASX protects the liver against APAP hepatotoxicity by alleviating necrosis, blocking ROS generation, inhibiting lipid peroxidation and oxidative stress, and mediating TNF-α-induced apoptosis through the JNK/p-JNK pathway and other MAPK family members, including the ERK and P38 signal pathway. Further studies are necessary to investigate the mechanism by which ASX is metabolized to protect the liver. Funding This study was supported by funding from “The Project of Innovative Research Team for Key Science and Technology in Shaanxi Province” (Grant No. 2013KCJ-23), “The Fundamental Research Funds for the Central Universities” (Grant No. 1191320114) and “the National Natural Science Foundation of China” (Grant No. 81601672). Author contributions Zhang JY and Zhang SM participated in the research design and in the writing of the paper; they contributed equally to the work. Bi JB participated in the IHC performance. Gu JX participated in the literature searches and IHC performance. Deng Y participated in the paper revisions. Liu C provided substantial advice in designing the study and assisting in the division of labor. Conflicts of interest The authors declare no conflict of interest. Acknowledgements We are indebted to all individuals who participated in or helped with this research project. References [1] A.M. Larson, J. Polson, R.J. Fontana, T.J. Davern, E. Lalani, L.S. Hynan, et al., Acetaminophen-induced acute liver failure: results of a United States multicenter, prospective study, Hepatology 42 (2005) 1364–1372. [2] J.D. Perkins, Acetaminophen sets records in the United States: Number 1 analgesic and number 1 cause of acute liver failure - Acetaminophen-induced acute liver failure: Results of a United States multicenter, prospective study. Larson AM, Polson J, Fontana RJ, Davern TJ, Lalani E, Hynan LS, Reisch JS, Schiodt FV, Ostapowicz G, Shakil AO, Lee WW, Acute Liver Failure Study Group. Hepatology 2005;42: 1364–1372, Liver Transpl. 12 (2006) 682–683. [3] D.C. Dahlin, G.T. Miwa, A.Y. Lu, S.D. Nelson, N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen, Proc. Natl. Acad. Sci. U. S. A. 81 (1984) 1327–1331. [4] J.R. Mitchell, D.J. Jollow, W.Z. Potter, J.R. Gillette, B.B. Brodie, Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione, J. Pharmacol. Exp. Ther. 187 (1973) 211–217. [5] D.J. Jollow, J.R. Mitchell, W.Z. Potter, D.C. Davis, J.R. Gillette, B.B. Brodie, Acetaminophen-induced hepatic necrosis. II. Role of covalent binding in vivo, J. Pharmacol. Exp. Ther. 187 (1973) 195–202. [6] H. Jaeschke, T.R. Knight, M.L. Bajt, The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity, Toxicol. Lett. 144 (2003) 279–288. [7] H. Jaeschke, M.R. McGill, A. Ramachandran, Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity, Drug Metab. Rev. 44 (2012) 88–106. [8] K. Du, A. Ramachandran, H. Jaeschke, Oxidative stress during acetaminophen hepatotoxicity: sources, pathophysiological role and therapeutic potential, Redox Biol. 10 (2016) 148–156. [9] D. Dong, L. Xu, X. Han, Y. Qi, Y. Xu, L. Yin, et al., Effects of the total saponins from Rosa laevigata Michx fruit against acetaminophen-induced liver damage in mice via induction of autophagy and suppression of inflammation and apoptosis, Molecules 19 (2014) 7189–7206. [10] B.K. Gunawan, Z.X. Liu, D. Han, N. Hanawa, W.A. Gaarde, N. Kaplowitz, c-Jun N-terminal kinase plays a major role in murine acetaminophen hepatotoxicity, Gastroenterology 131 (2006) 165–178.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Astaxanthin pretreatment attenuates acetaminophen-induced liver injury in mice Jingyao Zhang a,1, Simin Zhang a,1, Jianbin Bi a, Jingxian Gu a, Yan Deng a, Chang Liu a,⁎ a
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, PR China
a r t i c l e
i n f o
Article history: Received 13 June 2016 Received in revised form 20 January 2017 Accepted 21 January 2017 Available online 31 January 2017 Keywords: Liver injury Acetaminophen Astaxanthin Oxidative stress Apoptosis JNK pathway
a b s t r a c t Background: Acetaminophen (APAP) is a conventional drug widely used in the clinic because of its antipyretic-analgesic effects. However, accidental or intentional APAP overdoses induce liver injury and even acute liver failure (ALF). Astaxanthin (ASX) is the strongest antioxidant in nature that shows preventive and therapeutic properties, such as ocular protection, anti-tumor, anti-diabetes, anti-inflammatory, and immunomodulatory effects. The aim of present study was to determine whether ASX pretreatment provides protection against APAP-induced liver failure. Methods: Male C57BL/6 mice were randomly divided into 7 groups, including control, oil, ASX (30 mg/kg or 60 mg/kg), APAP and APAP + ASX (30 mg/kg or 60 mg/kg) groups. Saline, olive oil and ASX were administered for 14 days. The APAP and APAP + ASX groups were given a peritoneal injection of 700 mg/kg or 300 mg/kg APAP to determine the 5-day survival rate and for further observation, respectively. Blood and liver samples were collected to detect alanine transaminase (ALT), aspartate transaminase (AST), inflammation, oxidative stress and antioxidant systems, and to observe histopathologic changes and key proteins in the mitogen-activated protein kinase (MAPK) family. Results: ASX pretreatment before APAP increased the 5-day survival rate in a dose-dependent manner and reduced the ALT, AST, hepatic necrosis, reactive oxygen species (ROS) generation, lipid peroxidation (LPO), oxidative stress and pro-inflammatory factors. ASX protected against APAP toxicity by inhibiting the depletion of glutathione (GSH) and superoxide dismutase (SOD). Administration of ASX did not change the expression of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and P38. However, phosphorylation of JNK, ERK and P38 was reduced, consistent with the level of tumor necrosis factor alpha (TNF-α) and TNF receptor-associated factor 2 (TRAF2). Conclusion: ASX provided protection for the liver against APAP hepatotoxicity by alleviating hepatocyte necrosis, blocking ROS generation, inhibiting oxidative stress, and reducing apoptosis by inhibiting the TNF-α-mediated JNK signal pathway and by phosphorylation of ERK and P38, which made sense in preventing and treating liver damage. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Drug-induced liver injury (DILI) is one of the most familiar and critical adverse drug reactions (ADR) resulting in ALF. DILI has become the leading cause of ALF in the United States and United Kingdom, as well as in some other countries and regions [1]. Based on statistics from the World Health Organization (WHO), DILI has become the fifth fatal cause of death in the worldwide. Acetaminophen (APAP) is a conventional drug widely used in the clinic as antipyretic analgesic. However, accidental or intentional APAP overdoses contribute to liver injury. Recent data suggest that APAP-induced ALF leads to approximately 30,000 cases of hospitalization and ⁎ Corresponding author at: Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, 277 Yanta West Road, Xi'an 710061, PR China. E-mail address: [email protected] (C. Liu). 1 They contribute equally to the paper.
http://dx.doi.org/10.1016/j.intimp.2017.01.028 1567-5769/© 2017 Elsevier B.V. All rights reserved.
500 deaths each year in America [2]. The mechanism of this hepatotoxicity has not been entirely clarified. The ultimate hepatotoxic metabolite, Nacetyl-p-benzoquinoneimine (NAPQI), is the initial step and is detoxified by glutathione (GSH). NAPQI combines with cellular target proteins during APAP overdoses [3]. Evidence suggests that exhaustion of GSH and binding between NAPQI and proteins contribute to cell injury [4,5]. Mitochondrial dysfunction triggered by APAP overdose results in oxidative stress [6,7]. In addition to ROS, increased lipid peroxidation also indicates oxidative stress in APAP-induced liver injury [8]. Recently, it was shown that inflammation, apoptosis and autophagy were also associated with APAP-induced hepatotoxicity [9]. Oxidative stress leads to activation of c-Jun N-terminal kinase (JNK) [10]. Moreover, various studies suggest that pro-inflammatory cytokines, such as TNF-α and interleukin-6 (IL-6), activate JNK and P38, which are related to ERK [11]. Activation of JNK plays an important role in APAP-induced hepatotoxicity [10]. The JNK signaling pathway up-regulates apoptosis and autophagy by activating TNF receptor-associated factor 2 (TRAF2) [12].
J. Zhang et al. / International Immunopharmacology 45 (2017) 26–33
As a type of nature liposoluble carotenoid pigment, astaxanthin is present in aquatic animals, microalgae, flamingoes, and Pfaffia (yeast), and possesses potent antioxidant ability [13]. Additionally, ASX also has many pharmacological properties, such as ocular protection, antitumor, anti-diabetes, anti-inflammatory, and immunomodulatory effects [14]. Previous studies showed that ASX effectively inhibits peroxyl radical-dependent lipid peroxidation, scavenges singlet oxygen, and blocks ROS generation [15]. The antioxidative capacity of ASX makes it a potential pharmacologic resource. ASX pretreatment attenuates hepatic ischemia reperfusion-induced apoptosis and autophagy via the ROS/MAPK pathway in mice [16]. ASX inhibits neural progenitor cellular apoptosis modulated by the P38 and MEK signaling pathways [17]. ASX was found to protect the liver against ConA-induced autoimmune hepatitis by reducing the phosphorylation of Bcl-2 modulated by the JNK signal pathway [18]. According to data, the antioxidant capacity of ASX is 100–500 times greater than vitamin E, and 10 times greater than beta-carotene [19]. Several studies showed that ASX has better capacity in relevant diseases. In N-methyl-N-nitrosourea (MNU)-induced mammary cancer, high concentrations of ASX showed an anti-cancer effect, whereas canthaxanthin did not suppress carcinoma [20]. In ischemia reperfusion (I/R) injury after intestinal transplantation, ASX was effective compared to other antioxidants, such as 4,5-dihydroxy-1,3benzene-disulfonic acid (Tiron) and epigallocatechin gallate (EGCG) [21]. In diet-induced insulin resistance and nonalcoholic steatohepatitis (NASH), ASX played a more effective role in NASH than vitamin E [22]. Moreover, ASX showed no apparent inhibition of seven major human hepatic UGTs so that it is safe for further clinical use [23]. The objectives of the present study were to determine whether ASX provides preventive and therapeutic effects during APAP-induced liver injury. We also assessed the potential mechanism of action. 2. Materials and methods
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olive oil and ASX were administered for 14 days. APAP and ASX were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Experimental protocols Blood samples were collected at selected time points (8 h and 24 h) after APAP administration and centrifuged at 3000× for 15 min to obtain supernatants. Serum samples were stored at −80 °C for further biochemical detection. Sections of liver tissues were fixed in 10% formaldehyde for histology. Blocks of liver tissues were placed in 2.5% glutaraldehyde for scanning electron microscopy (SEM). Additionally, a portion of the remaining liver was immediately homogenized for later analysis. 2.3. Survival The survival study was conducted using the following three groups: control, APAP, and APAP + ASX groups. Mice received an i.p. injection of 700 mg/kg APAP. To observe the impacts of ASX on APAP-induced liver injury, the 5-day survival rate was determined. 2.4. Serum biochemistry Serum ALT and AST were selected as sensitive indicators for hepatotoxicity, and ALT and AST levels were measured with an automated biochemical analyzer in the Department of Inspection, the First Affiliated Hospital of Xi'an Jiaotong University. 2.5. Determination of cytokines Levels of TNF-α and IL-6 were measured using commercial ELISA kits according to the manufacturer's instructions (Dakewe, Shenzhen, China).
2.1. Preparation of animals and reagents 2.6. Histology Male C57BL/6 mice were purchased from the Animal Feeding Center of Xi'an Jiaotong University Health Science Center. Experimental animals included in this study were 4–5 weeks old and weighed 23.0 ± 1.4 g. Mice were housed in an air-conditioned room under a 12-h light/dark cycle (lights on 6:00 AM to 6:00 PM) and allowed food and water ad libitum. All experimental protocols followed the criteria of the Ethics Committee of Xi'an Jiaotong University Health Science Center. Mice were randomly allocated into the following groups: control (saline intraperitoneal (i.p.) injection); oil (olive oil by gavage); ASX (30 mg/kg/d or 60 mg/kg/d by gavage, dissolved in olive oil) [16]; APAP (700 mg/kg or 300 mg/kg i.p. injection), and ASX administrated after APAP (APAP + ASX; doses same as described above). Saline,
Liver samples were fixed in 10% formaldehyde and prepare to be embedded in paraffin. Serial liver sections (5 μm thick) were stained with H&E or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)\(Roche Molecular Biochemicals, Indianapolis, IN) kits and were observed under a microscope (Olympus Optical Co., Tokyo, Japan). Portions of liver tissues were prefixed in 2.5% glutaraldehyde and washed with 0.1 M phosphate buffer. The samples were post-fixed with 1% OsO4 and again washed with 0.1 M phosphate solution. The fixed tissues were dehydrated with graded ethanol and dried using the critical point drying method. After conductive treatment, ultrathin
Fig. 1. Olive oil and ASX cause no damage to normal liver tissues. (A) Serum ALT levels were measured in four groups of male C57BL/6 mice respectively treated with 0.9% saline, olive oil, ASX (30 mg/kg/d), and ASX (60 mg/kg/d) for 14 days; (B) Serum AST levels were detected as in (A). Data are presented as the mean ± SD.
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J. Zhang et al. / International Immunopharmacology 45 (2017) 26–33
Fig. 2. ASX improves liver function in hepatic injury induced by APAP. (A) Serum ALT values were measured in four groups of male C57BL/6 mice treated with 0.9% saline, APAP (300 mg/kg), APAP (300 mg/kg) + ASX (30 mg/kg/d), and APAP (300 mg/kg) + ASX (60 mg/kg/d). Saline and ASX were administered for 14 days; (B) Serum AST values were consistent with (A). ALT and AST are expressed as the mean ± SD. *P b 0.05 for APAP + ASX group vs APAP group, #P b 0.01 for APAP + ASX group vs APAP group.
sections (50-nm thick) were studied with a scanning electron microscope.
3. Results 3.1. ASX improves liver function in hepatic injury induced by APAP
2.7. Detection of ROS activation Liver samples were fixed with 10% formaldehyde and dehydrated using 30% sucrose solution. The sections (5 μm thick) were incubated with dihydroethidium (DHE; 10 μM). After 60 min in the dark, the specimens were washed with adequate volumes of PBS. DHE oxidized by ROS in the cells showed red emission under a fluorescence microscope using green wavelength excitation.
In C57BL/6 mice treated with olive oil or ASX (30 mg/kg/d or 60 mg/kg/d) for 14 days, the ALT and AST activity values did not significantly differ from the controls (Fig. 1A and B), indicating that neither ASX nor olive oil had hepatic toxicity. In Fig. 2A and B, serum ALT and AST levels decreased at 8 h and 24 h (P b 0.05 or P b 0.01) in APAP mice treated with ASX. 3.2. Effects of ASX on survival after APAP overdose
2.8. Measurement of lipid peroxidation Tissue malondiadehyde (MDA) in liver homogenates were determined to analyze the lipid peroxidation (LPO) level using a kit (Jiancheng Institute of Biotechnology) as described by the manufacturer. 2.9. Determination of enzymatic activities Liver tissue was homogenized to be centrifuged at 4000 rpm for 20 min. The supernatants were prepared to measure the level of myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) based on the kit instructions from Jiancheng Institute of Biotechnology. 2.10. Western blotting Homogenates were centrifuged and proteins were fractionated by SDS-PAGE on 4% gels under reducing conditions. Separated proteins were electrotransferred onto polyvinylidine difluoride membranes (PVDF, Immobilin-P, Millipore, Bedford, MA) and blocked with 5% to 10% milk for 2 h at room temperature, followed by incubation with the appropriate primary antibodies overnight at 4 °C. After washing with PBST 3 times for 10 min each, the membranes were incubated with secondary antibodies for 2 h at room temperature. Protein bands were visualized using the Immobilon Western Chemilum HRP substrate (Millipore, Bedford, MA) and Image Lab Software (Bio-Rad Laboratories, Hercules, CA, USA).
Animals were given 700 mg/kg APAP to monitor the effects of ASX for 5 days in the APAP, APAP + ASX (30) and APAP + ASX (60) groups. The survival rates were calculated at 5 time points (24 h, 48 h, 72 h, 96 h, and 120 h). At each time point, the survival rate was higher in the APAP + ASX group than the APAP group. We found a low survival rate in each group at 120 h. Administration of ASX significantly increased the 5-day survival rate of mice (Fig. 3), indicating that ASX significantly reduced the mortality caused by APAP overdose. 3.3. ASX ameliorates histopathologic changes Mice were randomly divided into four groups, including control, APAP, APAP + ASX (30), and APAP + ASX (60). The latter three groups were given APAP 300 mg/kg. Sections from liver samples were stained with H&E and observed. The disorganized liver form, hemorrhage, and leukocyte infiltration in the APAP + ASX groups were ameliorated compared to the APAP group at 8 h and 24 h. This was more apparent in the APAP + ASX (60) group than the APAP + ASX (30) group. ASX clearly reduced the hepatocyte necrosis caused by APAP (Fig. 4A and B). As shown in Fig. 4C, there was no significant decrease of the liver index
2.11. Statistical analysis Analysis was performed with the SPSS statistical package, and values are reported as the mean ± SE. Differences between multiple groups were assessed by ANOVA. Values of P b 0.05 were considered statistically significant.
Fig. 3. Effects of ASX on survival after APAP overdose. Experimental and control groups were given 700 mg/kg APAP and the survival rates were monitored for 5 days. *P b 0.05 for APAP + ASX group vs APAP group.
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Fig. 4. ASX ameliorates histopathologic changes. (A) Serial liver sections (5 μm thick) stained with HE were observed under a microscope at 8 h. These sections were obtained from normal liver tissues, the tissues of livers with APAP-induced injury and the tissues of livers pretreated with ASX (30 mg/kg/d or 60 mg/kg/d) before APAP. Magnifications: 100× and 200×. (B) Serial liver sections stained with H&E were observed at 24 h. The sections, microscope and magnifications are the same as those in (A). (C) The liver index was calculated at 8 h and 24 h to assess the effect of ASX against APAP. (D) The percentage of liver necrosis was measured. (E) Ultrathin liver sections (50 nm thick) were studied at 24 h with a scanning electron microscope. Magnifications: 20,000×. *P b 0.05 for APAP + ASX group vs APAP group, #P b 0.01 for APAP + ASX group vs APAP group.
at 8 h. The liver index decreased (P b 0.05) in the APAP + ASX (60) group at 24 h. Moreover, the percentage necrosis was significantly reduced in the APAP + ASX groups (Fig. 4D). In APAP mice administered ASX, the disordered lobular structure disappeared in a dose-dependent manner (Fig. 4E). It is unknown whether apoptosis exists as another method of causing hepatic injury in APAP-induced hepatotoxicity. TUNEL results showed that the stained zones were decreased (P b 0.05) in the APAP + ASX group (Fig. 5).
3.4. Effect of ASX on oxidative stress and antioxidant system Oxidative stress is an early event in APAP-induced liver injury [8]. The product of oxidative stress related to radical generation mainly consists of ROS and MPO. More ROS presented as bright red dot-like substances in the APAP group than in the APAP + ASX groups at 8 h and 24 h (Fig. 6A and B), demonstrating that ASX protected against APAP toxicity by inhibiting ROS generation. The level of MPO in liver
Fig. 5. Detection of ROS activation. Hepatic apoptosis was observed with photography and quantitation of stained sections under a standard fluorescence microscope. #P b 0.01 for APAP + ASX group vs APAP group.
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Fig. 6. Effect of ASX on ROS generation, oxidative stress, lipid peroxidation and antioxidant systems. (A) ROS was stained with DHE and presented as bright red dot-like substances and was observed at 8 h in experimental and control groups. Magnification: 200×. (B) Presentation of ROS obtained at 24 h. (C) MDA levels in the liver. (D) MPO levels in the liver. (E) SOD levels in the liver. *P b 0.05 for APAP + ASX group vs APAP group, #P b 0.01 for APAP + ASX group vs APAP group.
homogenates was detected and was significantly decreased in the APAP + ASX groups compared to the APAP group (Fig. 6D). LPO played an important role during APAP-induced liver injury [24]. MDA was the indicator during LPO. In contrast to the high level of MDA in the APAP group, the level significantly decreased in the APAP + ASX groups at 8 h and 24 h (Fig. 6C). SOD and the glutathione redox system are major cellular antioxidant system that combats and neutralizes free radicals [25]. The activities of SOD, GSH and GSH-Px were measured in liver homogenates at 8 h and 24 h after APAP treatment. Depletion of SOD and GSH decreased significantly when mice were treated with ASX at 8 h and 24 h (Figs. 6E and 7A), suggesting that ASX prevents the depletion of SOD and GSH. The function of GSH-Px was to neutralize lipid hydroperoxides. The level of GSH-Px was recovered
(P b 0.01) in the APAP + ASX (30) group at 24 h (Fig. 7B), indicating that ASX also reduced the depletion of GSH-Px. 3.5. ASX reduces pro-inflammatory cytokines Excessive pro-inflammatory cytokines, such as TNF-α and IL-6, activate the innate immune system to cause severe liver damage with a toxic dose of APAP [26]. The levels of TNF-α and IL-6 in serum were detected at 8 h and 24 h after APAP administration. TNF-α was reduced (P b 0.01) in the APAP + ASX (60) group at 24 h (Fig. 8A). Treatment with ASX resulted in a significantly decrease in IL-6 in the APAP + ASX groups compared to the APAP group at 8 h and 24 h (Fig. 8B). In the present report, ASX inhibited the release of pro-inflammatory cytokines, such as IL-6, and showed a potential ability to reduce TNF-α.
Fig. 7. ASX attenuates the depletion of GSH. (A) The depletion and levels of GSH in liver were observed at 8 h and 24 h in mice pretreated with or without ASX. (B) The GSH-Px levels in the liver. *P b 0.05 for APAP + ASX group vs APAP group, #P b 0.01 for APAP + ASX group vs APAP group.
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Fig. 8. ASX reduces pro-inflammatory cytokines. (A) Serum TNF-α was measured in four groups of mice (6 male C57BL/6 mice) treated with 0.9% saline, APAP (300 mg/kg), APAP (300 mg/kg) + ASX (30 mg/kg/d) and APAP (300 mg/kg) + ASX (60 mg/kg/d). (B) Serum IL-6 was obtained from four different groups described above. *P b 0.05 for APAP + ASX group vs APAP group, #P b 0.01 for APAP + ASX group vs APAP group.
3.6. ASX protects liver from APAP by reducing the activation of JNK, ERK and P38 To assess key proteins in the signaling pathway for apoptosis, the levels of TRAF2, TNF-α, JNK, p-JNK, ERK, p-ERK, P38 and p-P38 were measured in liver tissues. As shown in Fig. 9, administration of ASX
did not change the expression of ERK and P38 at 8 h in APAP-induced liver injury. The phosphorylation of ERK (p-ERK), P38 (p-P38), and JNK (p-JNK) was reduced. This suggests that ASX inhibits the phosphorylation of ERK and P38. High expression of TNF-α was detected in the APAP group compared to the APAP + ASX groups at 8 h and 24 h (Fig. 8). Similar changes of protein expression were found for TRAF2
Fig. 9. ASX protects liver from APAP by reducing phosphorylation of JNK, ERK and P38. (A) The levels of proteins ERK, p-ERK, P38, and p-P38 at 8 h in APAP-induced liver tissue were shown as Western blot bands. The levels of proteins TNF-α, TRAF2 and JNK signaling pathway at 8 h and 24 h in APAP-induced liver tissue were detected with Western blotting. (B) The relative band densities were calculated. *P b 0.05 for APAP + ASX group vs APAP group, #P b 0.01 for APAP + ASX group vs APAP group.
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(Fig. 9). At the protein level, with 60 mg/kg ASX treatment, the amount of p-JNK decreased more than p-ERK, p-P38, or TRAF2. Taken together, these results suggest that the JNK/p-JNK pathway is greatly attenuated by blocking both TNF-α and TRAF2 when treated with ASX. 4. Discussion Over 1100 types of classical drugs induce hepatotoxicity [27]. APAPinduced liver injury is one of the most studied drug-induced diseases [28]. APAP overdoses cause serious hepatotoxic effects, with ALF as the worst outcome [29]. We used APAP for overdoses in male C57BL/6 mice to determine whether ASX exerts a dose-dependent hepatoprotective effect. We observed a short 5-day survival rate and remarkable elevations of ALT and AST levels in the APAP group, which showed our successful APAP-induced liver injury model. In APAP hepatotoxicity, NAPQI covalently combines with cellular proteins, leading to hepatocyte death and the release of ALT and AST into plasma [30]. ALT and AST were decreased, we initially predicted that ASX protected the structure of hepatic cells from damage. We determined the mechanism by which ASX exerts protective effects on APAP-induced liver injury. The metabolite NAPQI depletes GSH and generates mitochondrial dysfunction, oxidative stress and necrosis [31]. To further confirm our findings, the morphology, cytokines, enzymes, chemokines and pro-inflammatory factors were examined. In the histological observation, severe hepatic necrosis and tumid structure caused by APAP were visually ameliorated by ASX. The percentage necrosis was consistent with H&E and SEM changes. We demonstrated that ASX protected the liver from necrosis. In our APAP-induced liver injury model, GSH performed poorly because of depletion. With ASX, the reduction of GSH was decreased. Furthermore, oxidative stress has an important contribution to liver injury, starting with LPO and the accumulation of ROS [8]. During this progress, superoxide, ROS generation, and hydroxyl and hydrogen peroxide cause oxidative stress and damage to DNA, proteins and lipids [32]. ASX reduces the production of ROS and MPO because of its potent antioxidant properties. Because of its unique molecular structure, ASX has a hydroxyl on one ionone ring and a keto on the other ring to scavenge radicals by combining with the two ionone moieties [33,34]. A commercial kit for MDA showed that LPO was significantly attenuated. Data indicated that ASX reduced cell damage by inhibiting lipid peroxidation (LPO). Moreover, it is thought to be antioxidant defense system along with oxidative stress [35]. Antioxidant enzymes, such as SOD and GSH-Px, are massively depleted with APAP treatment [36]. The depletion of SOD and GSH-Px was reduced after ASX administration, demonstrating the protective impact of ASX on APAP toxicity. At the same time, many events in the liver culminate in centrilobular necrosis [37]. Necrotic hepatocytes release many damage-associated molecular patterns (DAMPs), resulting in immune activation in the noninfectious inflammatory response [38–40]. DAMPs facilitate the release of IL-6, TNF-α, MCP-1 and IL-1β, resulting in further infiltration of leukocytes that augments liver injury [41]. A previous study showed that IL-6 and TNF-α cause drug-induced hepatic injury. TNF-α can activate TRAF2, thereby up-regulating apoptosis through the JNK/p-JNK pathway in liver injury [12]. Apoptosis can activate numerous liver diseases [42]. Several previous studies showed that apoptosis contributes to APAP-induced liver injury [43,44]. In our study, ASX reduced the production of pro-inflammatory cytokines, including IL-6 and TNF-α, consistent with a decrease in the levels of TRAF2 and p-JNK, which mediate apoptosis. The activation of ERK/p-ERK and P38/p-P38 is consistent with JNK. As indicated by TUNEL, a clear reduction of DNA fragmentation and nuclear staining around centrilobular areas was observed with ASX compared to APAP-induced liver failure, correlating with the Western blot values. It demonstrates that ASX provides protection by TNF-α mediated apoptotic cell death through the JNK/p-JNK pathway and other MAPK family members, including the ERK and P38 signaling pathway.
5. Conclusion In conclusion, we discussed the potent protective capacity of ASX, which showed preventive and therapeutic potential in APAP-induced liver injury in mice. The results above, for the first time, indicate that ASX protects the liver against APAP hepatotoxicity by alleviating necrosis, blocking ROS generation, inhibiting lipid peroxidation and oxidative stress, and mediating TNF-α-induced apoptosis through the JNK/p-JNK pathway and other MAPK family members, including the ERK and P38 signal pathway. Further studies are necessary to investigate the mechanism by which ASX is metabolized to protect the liver. Funding This study was supported by funding from “The Project of Innovative Research Team for Key Science and Technology in Shaanxi Province” (Grant No. 2013KCJ-23), “The Fundamental Research Funds for the Central Universities” (Grant No. 1191320114) and “the National Natural Science Foundation of China” (Grant No. 81601672). Author contributions Zhang JY and Zhang SM participated in the research design and in the writing of the paper; they contributed equally to the work. Bi JB participated in the IHC performance. Gu JX participated in the literature searches and IHC performance. Deng Y participated in the paper revisions. Liu C provided substantial advice in designing the study and assisting in the division of labor. Conflicts of interest The authors declare no conflict of interest. Acknowledgements We are indebted to all individuals who participated in or helped with this research project. References [1] A.M. Larson, J. Polson, R.J. Fontana, T.J. Davern, E. Lalani, L.S. Hynan, et al., Acetaminophen-induced acute liver failure: results of a United States multicenter, prospective study, Hepatology 42 (2005) 1364–1372. [2] J.D. Perkins, Acetaminophen sets records in the United States: Number 1 analgesic and number 1 cause of acute liver failure - Acetaminophen-induced acute liver failure: Results of a United States multicenter, prospective study. Larson AM, Polson J, Fontana RJ, Davern TJ, Lalani E, Hynan LS, Reisch JS, Schiodt FV, Ostapowicz G, Shakil AO, Lee WW, Acute Liver Failure Study Group. Hepatology 2005;42: 1364–1372, Liver Transpl. 12 (2006) 682–683. [3] D.C. Dahlin, G.T. Miwa, A.Y. Lu, S.D. Nelson, N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen, Proc. Natl. Acad. Sci. U. S. A. 81 (1984) 1327–1331. [4] J.R. Mitchell, D.J. Jollow, W.Z. Potter, J.R. Gillette, B.B. Brodie, Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione, J. Pharmacol. Exp. Ther. 187 (1973) 211–217. [5] D.J. Jollow, J.R. Mitchell, W.Z. Potter, D.C. Davis, J.R. Gillette, B.B. Brodie, Acetaminophen-induced hepatic necrosis. II. Role of covalent binding in vivo, J. Pharmacol. Exp. Ther. 187 (1973) 195–202. [6] H. Jaeschke, T.R. Knight, M.L. Bajt, The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity, Toxicol. Lett. 144 (2003) 279–288. [7] H. Jaeschke, M.R. McGill, A. Ramachandran, Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity, Drug Metab. Rev. 44 (2012) 88–106. [8] K. Du, A. Ramachandran, H. Jaeschke, Oxidative stress during acetaminophen hepatotoxicity: sources, pathophysiological role and therapeutic potential, Redox Biol. 10 (2016) 148–156. [9] D. Dong, L. Xu, X. Han, Y. Qi, Y. Xu, L. Yin, et al., Effects of the total saponins from Rosa laevigata Michx fruit against acetaminophen-induced liver damage in mice via induction of autophagy and suppression of inflammation and apoptosis, Molecules 19 (2014) 7189–7206. [10] B.K. Gunawan, Z.X. Liu, D. Han, N. Hanawa, W.A. Gaarde, N. Kaplowitz, c-Jun N-terminal kinase plays a major role in murine acetaminophen hepatotoxicity, Gastroenterology 131 (2006) 165–178.
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