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Protection from diclofenac-induced liver injury by Yulangsan polysaccharide in a mouse model
Journal of Ethnopharmacology 193 (2016) 207–213
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Protection from diclofenac-induced liver injury by Yulangsan polysaccharide in a mouse model Jianchun Huang a,1, Vanphuc Nguyen a,1, Xiaojun Tang b, Jinbin Wei a, Xing Lin a, Zefeng Lai a, Vanminh Doan a, Qiuqiao Xie a, Renbin Huang a,n a b
Department of Pharmacology, Guangxi Medical University, Nanning 530021, PR China Department of Laboratory Medicine, Guangxi Medical College, Nanning, Guangxi, China
art ic l e i nf o
a b s t r a c t
Article history: Received 4 February 2016 Received in revised form 9 July 2016 Accepted 3 August 2016 Available online 4 August 2016
Ethnopharmacological relevance: Millettia pulchra Kurz var-laxior (Dunn) Z. Wei, a wild-growing plant of the family Fabaceae is known to possess multifarious medicinal properties. Yulangsan polysaccharide (YLSPS) is a chief ingredient of its root, which has been used in Chinese traditional medicine with a long history for remedy of acute or chronic hepatitis and jaundice. Aim of the study: To investigate the ability of the YLSPS to protect against diclofenac-induced hepatotoxicity in mice. Materials and methods: Mice were orally treated with YLSPS daily 1 h after the injection of diclofenac for 2 weeks. Dimethyl diphenyl bicarboxylate was used as a reference drug. Results: YLSPS effectively reduced the elevated levels of serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase and enhanced the reduction of superoxide dismutase, catalase, and glutathione peroxidase activities in the liver. Moreover, the content of malondialdehyde was reduced by treatment with YLSPS, and histological findings also confirmed the anti-hepatotoxic activity. In addition, YLSPS significantly inhibited proinflammatory mediators, such as tumor necrosis factor-alpha and interleukin 1 beta. YLSPS also enhanced mitochondrial antioxidants and inhibited cell death by preventing the down-regulation of Bcl-2 and the up-regulation and release of Bax along with caspase 9 and 3 activity; thus, these findings confirm the involvement of mitochondria in diclofenac-induced apoptosis. Conclusion: The results indicate that protective effects of YLSPS against diclofenac-induced acute hepatic injury may rely on its effect on reducing oxidative stress, suppressing inflammatory responses, and improving drug-metabolizing enzyme activity in the liver. & 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Yulangsan polysaccharide Diclofenac Hepatoprotection
1. Introduction Non-steroidal anti-inflammatory drugs (NSAIDs) are the centerpiece of pharmacotherapy for most rheumatological disorders and are used in large numbers as analgesics and antipyretics, both as prescription drugs as and over-the-counter purchases (Hau et al., 2009). Diclofenac is an NSAIDs that is used for the treatment of mild-to-moderate pain, fever, and inflammation. Clinical hepatotoxicity is one of the adverse reactions caused by diclofenac. Hepatotoxicity studies of diclofenac showed that it causes a rise in liver function tests (Banks et al., 1995); the histopathological picture suggests hypersensitivity reactions (Boelsterli, 2003), liver n
Corresponding author. E-mail address: [email protected] (R. Huang). 1 They contributed equally to this work.
http://dx.doi.org/10.1016/j.jep.2016.08.012 0378-8741/& 2016 Elsevier Ireland Ltd. All rights reserved.
necrosis, jaundice, fulminate hepatitis with or without jaundice, and liver failure (Aithal and Day, 2007; Banks et al., 1995; O’Connor et al., 2003). Furthermore, there is evidence of clinical and experimental animal studies that diclofenac-induced liver injury involves immune responses (Pohl et al., 1994; Yano et al., 2012). Moreover, previous studies have suggested that diclofenac hepatotoxicity is a result of metabolic activation by CYP2E1 and CYP2C9 and reactive oxygen species (ROS) formation, leading to mitochondrial oxidative stress injury and glutathione depletion. Diclofenac-induced hepatic toxicity was prevented by antioxidants and cytochrome P-450 inhibitors (Pourahmad et al., 2011). In contrast, antioxidants were able to prevent caspase activation by diclofenac (Gómez-Lechón et al., 2003). Millettia pulchra Kurz var-laxior (Dunn) Z.Wei, a wild-growing plant of the family Fabaceae, is known to possess multifarious medicinal properties. Its root, Yulangsan (YLS), has been used in
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Chinese traditional medicine with a long history for remedy of acute or chronic hepatitis and jaundice. In our previous study, the ethanol extract of the root showed anti-inflammatory and antioxidant properties; it has also been used in the treatment of neurological, cardiovascular, and liver diseases (Huang et al., 2003, 2008; Jiao et al., 2004). In addition, YLS polysaccharide (YLSPS) from the root inhibits peroxidation in vitro and suppresses production of excess free radicals in vivo (Duan et al., 2007, 2008). Recently, it was found to have protective effects against hepatic injury induced by carbon tetrachloride, rifampicin, isoniazid, and nimesulide in mice by antioxidant mechanisms (Dong et al., 2014; Fu et al., 2009; Nguyen et al., 2015). Based on the data from our laboratory and others, we suggest that YLSPS may be effective in protecting against diclofenac-induced liver injury. However, reports on the hepatoprotective effects and further exploration of underlying mechanisms of YLSPS are still lacking. To test our hypothesis, a diclofenac-induced liver injury model was chosen to study the hepatoprotective effects of YLSPS in mice. The effect of YLSPS on liver injury was compared with that of dimethyl diphenyl bicarboxylate (DDB) in this study. DDB, an antihepatitis drug, has been widely used in clinical practice for the treatment of liver disease, such as acute or chronic hepatitis and drug-induced liver injury (Park et al., 2005). Various studies indicated that DDB exhibits strong antioxidative activity (Gao et al., 2005; Park et al., 2005). Furthermore, to investigate the hepatoprotective mechanisms of YLSPS, histological examination of hepatic characterization, markers of liver oxidative stress, and antioxidative defense system, we evaluated hepatic proinflammatory mediators (including serum tumor necrosis factor-alpha [TNF-α] and interleukin 1 beta [IL-1β]), key apoptotic events (such as Bcl-2 and Bax), and caspase 9 and 3 activity.
2. Materials and methods
Center of Guangxi Medical University (Guangxi, China). The research was conducted according to protocols approved by the Animal Ethics Committee of Guangxi Medical University. All mice were housed under controlled conditions with temperature of 25 72 °C, relative humidity of 60 710%, room air changes 12–18 times/h, and a 12 h light/dark cycle. Feed and water were made available ad libitum. After 1-week acclimatization, the mice were randomly divided into six groups (5 males and 5 females mice /group) and treated for 14 days as follows: in the normal control group, mice were given the same volume of physiological saline; in the diclofenactreated model group, mice were intraperitoneally injected with diclofenac sodium once a day at a dose of 50 mg/kg; in the positive control group, mice were treated with DDB (200 mg/kg) by orally administrating after 1 h with the injection of diclofenac (50 mg/ kg); in the low, medium, and high dosage of YLSPS-treated group, mice were treated with YLSPS (150, 300, and 600 mg/kg, respectively) by oral administration after 1 h, with the injection of diclofenac (50 mg/kg). At the final stage of the experiment, blood samples were collected and serum was separated for assessment of enzyme activity, and then animals were sacrificed by cervical dislocation. Liver samples were dissected out and washed immediately with icecold saline to remove as much blood as possible; one part of the liver samples was immediately stored at 80 °C until analysis, and another part was excised and fixed in 10% formalin solution for histopathologic analysis. 2.4. Calculation of liver index The liver index was calculated according to the formula: (mice liver weight/mice weight) 100% (Huang et al., 2012). 2.5. Estimation of serum marker enzymes and TNF-α and IL-1β levels
2.1. Chemicals Diclofenac was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). The biochemical kits of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TNF-α and IL-1β ELISA kits were purchased from Wuhan Boster Bio-engineering Co. Ltd. (Wuhan, China); all other chemicals were of analytical grade. 2.2. Preparation of YLSPS YLSPS was prepared by a method described previously (Lin et al., 2014a). The root of the Millettia pulchra Kurz var-laxior (Dunn) Z. Wei was dried, powdered, and extracted three times with boiling water. The PS in the filtrate was precipitated fractionally with alcohol. The protein in the product was removed by the Sevag method and further purified using DEAE ion exchange cellulose (DEAE-52). The components of the saccharide were determined by GC and TLC. YLSPS was composed of D-glucose and D-arabinose in a molar ratio of 90.79% and 9.21%, with an average molecular weight of 14,301 Da (Lin et al., 2014a). The maximum tolerable dose (MTD) of LYSPS in mice was 24,000 mg/kg and it was 800 times of adult daily dose. 2.3. Animals and treatment Kunming mice of both sexes, weighing 20–22 g, Specefic pathogen Free (SPF), were provided by the Experimental Animal
The serum AST, ALT, and ALP were determined in accordance with methods provided by the diagnostic kits. TNF-α and IL-1β are important cytokines involved in hepatocyte damage induced by diclofenac. The serum TNF-α and IL-1β levels were measured by commercially available ELISA kits. 2.6. Measurement of SOD, GSH-Px, and CAT in liver homogenate Liver homogenates were prepared in cold Tris–HCl (5 mmol/L containing 2 mmol/L EDTA, pH 7.4) using a homogenizer. The homogenate was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was used immediately for the assays of SOD, GSH-Px, and CAT. All of these enzymes were determined according to the manufacturer's instructions. 2.7. Assay of lipid peroxidation products Lipid peroxidation was assayed by the measurement of the levels of MDA, which was determined following the kit instructions. 2.8. Measurement of caspase 3 and 9 The liver tissue samples were homogenized in lysis buffer. The homogenate was centrifuged for 15 min at 40000g and the resulting supernatant was incubated peotide (DEVD-AFC for caspase 3, LEHD-AFC for caspase 9). The change in fluorescence (excitation at 400 nm and emission at 490 nm) was monitored after 120 min of incubation, according to the procedure reported previously (Lin et al., 2014b).
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2.9. Histological study The liver samples were sectioned and stained with hematoxylin and eosin (H&E) and subsequently examined by light microscopy (IX51, Olympus, Japan) for histopathological examination. Finally, the images were examined and evaluated for pathological changes. 2.10. Evaluation of apoptosis TUNEL staining was performed in representative liver sections to locate apoptotic hepatocytes. Frozen sections (4–5 μm) from the different groups were prepared, fixed in 4% paraformaldehyde, and washed with phosphate-buffered saline (PBS). The tissue sections were then incubated with the TUNEL reaction mixture for 1 h in a humidified atmosphere at 37 °C. The nuclear staining was determined via the green nuclear fluorescence observed by laser scanning confocal microscopy (Leica) with an excitation wavelength of 488 nm and detection at 565 nm.
Fig. 1. Effect of YLSPS on the liver index in mice with diclofenac-induced hepatic injury. Results are presented as the mean 7 SE (%) (n¼ 10). #P o0.05, ##Po 0.01 vs. normal group; *Po 0.05, **Po 0.01 vs. model group.
2.11. Western blot analysis of hepatic Bax and Bcl-2 The electrophoretic separation of the proteins was performed using 10% sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE) and then electrotransferred to nitrocellulose membranes (Schleicher and Schuell, Germany), which was immuno-blotted with Bax, Bcl-2, and β-actin primary antibodies. ALP-labeled goat anti-rabbit IgG or horseradish peroxidase-conjugated anti-rat IgG were used as the secondary antibodies, and the color developed using a mixture of 5-bromo-4-chloro-indolylphosphate and nitroblue tetrazolium. The filter images were captured by the Versa Doc Imaging system (Bio-Rad, USA). β-actin was used as an internal standard.
Fig. 2. Effects of YLSPS on serum ALT, AST, and ALP activities in mice with diclofenac-induced hepatic injury. Results are presented as the means 7 SE. #P o0.05, ## P o 0.01 vs. normal group; *P o 0.05, **Po 0.01 vs. model group.
2.12. Statistical analysis AST, and ALP levels when compared with the model group. The statistical analysis was performed using SPSS version 16 statistical program SPSS Inc. (Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to compare the means among different groups, and Tukey's test was used in the post hoc multiple comparisons. The data were presented as the means 7 SE, and Pvalue o0.05 was considered statistically significant.
3. Results 3.1. Effects of YLSPS on liver index in mice
3.3. Effect of YLSPS on serum TNF-α and IL-1β levels TNF-α and IL-1β are important cytokines involved in hepatocyte damage induced by diclofenac. The levels of TNF-α and IL-1β were low in the normal control animals. However, the levels of the two cytokines increased significantly in the diclofenac-treated mice. This increase was reduced in both YLSPS and DDB supplemented groups (Fig. 3).
The liver index showed a significant increase in the group treated with diclofenac compared with the normal group, indicating that diclofenac induces hypertrophy of liver tissue. Conversely, in the medium and high dosage of YLSPS-treated groups and the DDB-treated group, the liver index decreased more markedly than that of the diclofenac-treated group. The results show that hepatic intumescence had been alleviated after the models were treated with YLSPS (Fig. 1). 3.2. Effect of YLSPS on serum ALT, AST, and ALP The effects of YLSPS pretreatment on the diclofenac-induced modifications in serum ALT, AST, and ALP levels are shown in Fig. 2. A single dose of diclofenac caused hepatotoxicity in mice, as indicated by an increase in serum ALT, AST, and ALP activities after diclofenac administration, whereas mice pretreated with YLSPS exhibited a significant decrease in the activities of the marker enzymes. DDB administration also reversed the alterations of ALT,
Fig. 3. Effects of YLSPS on serum TNF-α and IL-1β levels in mice with diclofenacinduced hepatic injury. Results are presented as the means 7 SE. #P o0.05, ## P o 0.01 vs. normal group; *P o 0.05, **Po 0.01 vs. model group.
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3.4. Effect of YLSPS on hepatic antioxidant enzyme activities GSH-Px, SOD, and CAT showed a significant decrease in animals treated with diclofenac alone compared with the normal group. However, mice treated with diclofenac plus medium- and highdose YLSPS showed a significant promotion towards the normal level. In contrast, YLSPS at the low dose had little influence on the parameters (Figs. 4 and 5). 3.5. Effects of YLSPS on concentration of MDA
Fig. 4. Effects of YLSPS on liver SOD and GSH-Px activities in mice with diclofenacinduced hepatic injury. Results are presented as the means 7 SE. #P o 0.05, ## Po 0.01 vs. normal group; *Po 0.05, **Po 0.01 vs. model group.
The localization of radical formation results in lipid peroxidation, as measured by MDA in mouse liver homogenates. MDA content in the total liver homogenates was increased in the diclofenac-treated group compared with the normal control group. Administration of medium- and high-dose YLSPS to diclofenacintoxicated mice resulted in a significant decrease in MDA compared with the diclofenac-treated group; however, the low treatment dose did not show any significant effect (Fig. 5). 3.6. Histopathological changes To assess histological changes, H&E staining of liver tissue sections from each group was examined. The normal control showed normal lobular architecture with central veins and radiating hepatic cords. Hepatotoxicity induced by diclofenac was confirmed by abnormal histological findings manifested by morphological changes, such as ballooning change of hepatocytes, degeneration of hepatocytes, and inflammatory cell infiltration. In medium- and high-dose YLSPS- and DDB-treated groups, the histopathological abnormalities induced by diclofenac were restored (Fig. 6).
Fig. 5. Effects of YLSPS on liver CAT activities and the MDA contents in mice with diclofenac-induced hepatic injury. Results are presented as the means 7 SE. # P o0.05, ##P o0.01 vs. normal group; *P o0.05, **P o 0.01 vs. model group.
3.7. Effects of YLSPS on apoptotic cells We measured apoptosis in liver tissue by double staining of
Fig. 6. Histologic results of tissues stained with H&E under light microscopy in mice with diclofenac-induced liver injury ( 400). Hepatotoxicity induced by diclofenac was confirmed by abnormal histological findings manifested by morphological changes, such as inflammation around the portal triad, disorganization of hepatocytes, cell swelling, and congestion of sinusoids. YLSPS and DDB reversed these pathological changes.
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TUNEL. As shown in Fig. 7, numerous TUNEL-positive hepatocytes were observed in liver tissues obtained from the diclofenac group. However, few positive hepatocytes were observed in livers treated with medium- and high-dose of YLSPS or DDB.
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3.8. Effects of YLSPS on activities of caspase 3 and 9 Massive activation of caspases is a crucial process during the induction of apoptosis associated with the pathogenesis of acute
Fig. 7. Effects of YLSPS on the apoptosis of hepatic cells in mice with diclofenac-induced hepatic injury. TUNEL staining was performed in liver tissue sections ( 200). The apoptosis index was defined as the number of apoptotic cells in every 100 cells counted. Results are presented as the means 7 SE. #Po 0.05, ##Po 0.01 vs. normal group; * Po 0.05, **Po 0.01 vs. model group.
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hepatic failure. In this study, the activities of caspase 3 and 9 were found to increase in mice receiving diclofenac treatment as compared with the normal group. There was a decrease in the caspase activity in mice pre-treated with medium- and high-dose of YLSPS or DDB (Fig. 8). These findings suggest that YLSPS is effective enough to combat diclofenac-induced caspase activation. 3.9. Effects of YLSPS on apoptosis-related proteins The proapoptotic Bax and antiapoptotic Bcl-2 proteins are members of the Bcl-2 family. We detected the protein levels of Bax and Bcl-2 in the liver tissue of each group (Fig. 9). Clearly, the levels of Bax were higher in the diclofenac group than those in the normal group, whereas Bax expressions decreased greatly in mice treated with DDB and YLSPS. Conversely, the expression of Bcl-2 was lower in the diclofenac group than that in the normal group, and the down-regulation was markedly inhibited by treatment with DDB and YLSPS.
Fig. 8. Effects of YLSPS on liver caspase 3 and 9 activities in mice with diclofenacinduced hepatic injury. Results are presented as the means 7 SE. #P o 0.05, ## Po 0.01 vs. normal group; *Po 0.05, **Po 0.01 vs. model group.
Fig. 9. Effects of YLSPS on the protein levels of Bax and Bcl-2 in the liver tissue detected by Western blotting. Results are presented as the means 7 SE. #Po 0.05, ## Po 0.01 vs. normal group; *Po 0.05, **Po 0.01 vs. model group.
4. Discussion Clinical findings suggest drug hypersensitivity reaction and direct toxic effects of the drug or its metabolite as the cause for the toxic effect (Cantoni et al., 2003; Galati et al., 2002). Due to diclofenac's high frequency of association with hepatotoxicity, it increases serum aminotransferase activities, liver necrosis, and jaundice (Banks et al., 1995; Boelsterli, 2003). ALT is cytosolic enzyme, AST is cytosolic enzymes or mitochondrial enzymes of the hepatocyte, and increased activities in circulation reflect cell damage and leakage, and ALP is a marker of the relative degree of hepatocellular damage and of obstruction, intrahepatic or extrahepatic, tending to parallel the degree of hyperbilirubinemia (Dong et al., 2014; Labbe et al., 2008). In the present study, the administration of diclofenac caused a dramatic elevation in serum AST, ALT, and ALP activities, indicating subchronic hepatotoxicity induced by administration of diclofenac. Instead, after treatment with DDB and YLSPS, activities of these serum enzymes were significantly decreased, indicating effectiveness of these drugs in liver regeneration after being damaged. ROS is mainly generated in the mitochondria, leading to serious damage to cellular macromolecules, including protein dysfunction, lipid peroxidation, DNA damage, and oxidative stress (Djordjević, 2004; Galati et al., 2002). For this reason, the antioxidative activity and inhibition of free-radical generation are important in terms of protecting the liver from drug-induced damage. Oxidative stress parameters, including SOD, CAT, and GSH-Px, and MDA, were examined in this study; the results on the administration of YLSPS in diclofenac-induced toxicity in the liver revealed decreased production of free radical derivatives, as evidenced by the decreased MDA level. Furthermore, YLSPS increased the activity of endogenous antioxidant enzymes SOD, CAT, and GSH-Px. This indicates that YLSPS may be able to protect against the oxidation of hepatic cellular membrane damage via a free-radical scavenging property. Some evidence suggests that diclofenac-induced hepatic injury involves immune responses (Pohl et al., 1994; Pumford et al., 1993). The pro-inflammatory cytokine TNF-α has been reported to play a key role in the pathogenesis of various liver diseases. IL-1β is a very potent pro-inflammatory cytokine; it increases during drug hepatotoxicity (Holt and Ju, 2006) and is involved in the early onset of diclofenac-induced liver injury (Yano et al., 2012). In this study, the levels of serum IL-1β and TNF-α in the diclofenac group were significantly higher than those in the normal group; however, upregulation of these inflammatory factors was markedly inhibited after treatment with YLSPS. This suggests that YLSPS exerted a therapeutic effect possibly through restriction of the production and release of inflammatory mediators. Histologic examination of rat livers treated with diclofenac showed typical hepatotoxicity characterization, ballooning degeneration of hepatocytes, hepatocellular necrosis, and inflammatory cell infiltration. However, the typical hepatotoxicity characterization was not shown in YLSPS-treated groups, suggesting that YLSPS has potential protective effects against diclofenac-induced liver injury. Mitochondria are regarded as sensors of oxidation damage and may play a major role in apoptosis (Daniel and Korsmeyer, 2004; Djordjević, 2004; Galati et al., 2002). The mitochondrial pathway is involved in diclofenac-induced apoptosis, which is related to cytochrome P-450-mediated metabolism of diclofenac, with the highest apoptotic effect produced by the metabolite 5OH-diclofenac (Gómez-Lechón et al., 2003). In this study, apoptotic hepatocytes were detected using TUNEL staining. Numerous TUNEL-positive hepatocytes were observed in liver tissues obtained from the diclofenac group. However, few positive hepatocytes were observed in livers treated with 600 mg/kg YLSPS or 200 mg/kg DDB.
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Furthermore, we examined the mechanism of apoptosis induced by YLSPS. Measurement of the apoptosis-related proteins, including Bax and Bcl-2, in liver tissues by Western blot immunoassay was then conducted. Diclofenac studies have has revealed that upon translocation of activated Bax to mitochondria, monomers of Bax and Bak can oligomerize to form large channels and induce formation of pores (Siu et al., 2008). Anti-apoptotic proteins, such as Bcl-2, that are localized mainly in the mitochondrial outer membranes block membrane permeability changes (Reed, 1999; Stanley and Korsmeyer, 1999). In this study, immunoblot analysis revealed significant decrease in the level of Bcl-2 and increase in the levels of Bax during diclofenac treatment. YLSPS significant protection against these key control points of apoptosis increased Bcl-2 levels and decreased Bax. Furthermore, the activities of caspases 3 and 9 in the cytosol fraction after diclofenac administration increased rapidly relative to the normal groups. The elevated caspases 3 and 9 were attenuated by treating with YLSPS and DDB, thus confirming the antioxidant efficacy and protective role of YLSPS against diclofenac-induced apoptosis. In conclusion, results from the present study demonstrate that YLSPS had a protective effect against diclofenac-induced hepatic damage in Kunming mice. Contributing to the alleviation of diclofenac triggered typical hepatotoxic characterization as histological finding and serum enzymes relating with liver diseases. The preliminary exploration of the underlying mechanisms indicates its protection against hepatic injury by radical scavenging action, antioxidant activity, and inhibiting critical control points of apoptosis. Therefore, on the basis of our work, YLSPS should be regarded as a new and promising agent with a high potential in the prevention and treatment of drug-induced liver injury and liver disease.
5. Conflict of Interest The authors declare that there are no conflicts of interest.
Acknowledgments This work was supported by the Natural Science Foundation of China (No. 81560587); the Fok Ying-Tong Education Foundation of China (No. 151107); the Guangxi Natural Science Foundation (No. 2013GXNSFAA019175); the Science and Technology Research Projects of Guangxi colleges and universities (No.2013YB343); Science and Technology foundation platform construction project of Guangxi Province (No.12-97-20).
References Aithal, G.P., Day, C.P., 2007. Nonsteroidal anti-inflammatory drug-induced hepatotoxicity vi-vii. Clin. Liver Dis. 11, 563–575. Banks, A.T., Zimmerman, H.J., Ishak, K.G., Harter, J.G., 1995. Diclofenac-associated hepatotoxicity: analysis of 180 cases reported to the Food and Drug Administration as adverse reactions. Hepatology 22, 820–827. Boelsterli, U.A., 2003. Diclofenac-induced liver injury: a paradigm of idiosyncratic drug toxicity. Toxicol. Appl. Pharmacol. 192, 307–322. Cantoni, L., Valaperta, R., Ponsoda, X., Castell, J.V., Barelli, D., Rizzardini, M., Mangolini, A., Hauri, L., Villa, P., 2003. Induction of hepatic heme oxygenase-1 by diclofenac in rodents: role of oxidative stress and cytochrome P-450 activity. J. Hepatol. 38, 776–783. Daniel, N.N., Korsmeyer, S.J., 2004. Cell death: critical control points. Cell 116,
213
205–219. Djordjević, V.B., 2004. Free radicals in cell biology. Int. Rev. Cytol. 237, 57–89. Dong, Y., Huang, J., Lin, X., Zhang, S., Jiao, Y., Liang, T., Chen, Z., Huang, R., 2014. Hepatoprotective effects of Yulangsan polysaccharide against isoniazid and rifampicin-induced liver injury in mice. J. Ethnopharmacol. 152, 201–206. Duan, X., Jiao, Y., Zhang, S., Huang, R., 2007. Protective effects of Yulangsan polysaccharide (YLS) on primary cultured rat hepatocytes injury induced by H2O2. LiShiZhen Med. Mater. Med. Res., 1592–1593. Duan, X., Jiao, Y., Zhang, S., Lu, X., Huang, R., 2008. Effects of Yulangsan polysaccharide on proliferation and collagen I production of hepatic stellate cells . China J. Mod. Med., 423–425. Fu, S., Huang, J., Wang, N., Huang, R., 2009. Protective effect of Yulangsan polysaccharide on acute alcohol hepatic injury in mice . China Pharm., 406–408. Galati, G., Tafazoli, S., Sabzevari, O., Chan, T.S., O’Brien, P.J., 2002. Idiosyncratic NSAID drug induced oxidative stress. Chem. Biol. Interact. 142, 25–41. Gao, M., Zhang, J., Liu, G., 2005. Effect of diphenyl dimethyl bicarboxylate on concanavalin A-induced liver injury in mice. Liver Int. : Off. J. Int. Assoc. Study Liver 25, 904–912. Gómez-Lechón, M.J., Ponsoda, X., O’Connor, E., Donato, T., Castell, J.V., Jover, R., 2003. Diclofenac induces apoptosis in hepatocytes by alteration of mitochondrial function and generation of ROS. Biochem. Pharmacol. 66, 2155–2167. Hau, D.K.P., Roberto, G., Wong, R.S.M., Yuen, M.C.W., Cheng, G.Y.M., Tong, C.S.W., Guo Yuan, Z., Leung, A.K.M., Lai, P.B.S., Fung, Yi, L., 2009. Phyllanthus urinaria extract attenuates acetaminophen induced hepatotoxicity: involvement of cytochrome P450 CYP2E1. Phytomedicine Int. J. Phytother. Phytopharm. 16, 751–760. Holt, M.P., Ju, C., 2006. Mechanisms of Drug-Induced Liver Injury. Am. Assoc. Pharma. Sci. J. 2006 (8), E48–E54. Huang, Q., Zhang, S., Zheng, L., He, M., Huang, R., Lin, X., 2012. Hepatoprotective effects of total saponins isolated from Taraphochlamys affinis against carbon tetrachloride induced liver injury in rats. Food Chem. Toxicol. 50 (3), 713–718. Huang, R., Jiao, Y., Jiang, W., 2003. Effect of Yulangsan extract on the cardiac hemodynamics and the coronary flow in rats. Chin. J. Hosp. Pharm., 321–322. Huang, Y., Chen, J., Huang, R., Lin, X., Wang, N., 2008. Anti-inflammatory effect of Yulangsan extracts and the mechanisms Chinese. Chin. J. New Drugs 20, 1764–1767. Jiao, Y., Duan, X., Huang, R., 2004. Scavenging and inhibiting effect of Yulangsan extract on superoxide anion free radical and hydroxy free radical. Guangxi Med. Univ., 22–23. Labbe, G., Pessayre, D., Fromenty, B., 2008. Drug-induced liver injury through mitochondrial dysfunction : mechanisms and detection during preclinical safety studies. Fund. Clin. Pharmacol. 22, 335–353. Lin, X., Huang, Z., Chen, X., Rong, Y., Zhang, S., Jiao, Y., Huang, Q., Huang, R., 2014a. Protective effect of Millettia pulchra polysaccharide on cognitive impairment induced by D-galactose in mice. Carbohydr. Polym., 533–543. Lin, X., Zhang, S., Huang, R., Tan, S., Liang, S., Wu, X., Zhuo, L., Huang, Q., 2014b. Protective effect of tormentic acid from Potentilla chinensis against lipopolysaccharide/D-galactosamine induced fulminant hepatic failure in mice. Int. Immunopharmacol. 19, 365–372. Nguyen, V., Huang, J., Doan, V., Lin, X., Tang, X., Huang, Y., Tang, A., Yang, X., Huang, R., 2015. Hepatoprotective effects of Yulangsan polysaccharide against nimesulide-induced liver injury in mice. J. Ethnopharmacol. 172, 273–280. O’Connor, N., Dargan, P.I., Jones, A.L., 2003. Hepatocellular damage from non-steroidal anti-inflammatory drugs. QJM : Mon. J. Assoc. Physicians 96, 787–791. Park, E.Y., Ki, S.H., Ko, M.S., Kim, C.W., Lee, M.H., Lee, Y.S., Kim, S.G., 2005. Garlic oil and DDB, comprised in a pharmaceutical composition for the treatment of patients with viral hepatitis, prevents acute liver injuries potentiated by glutathione deficiency in rats. Chem. Biol. Interact. 155, 82–96. Pohl, L.R., Martin, J.L., Hargus, S.J., 1994. Immunochemical methods of studying the mechanism of diclofenac-induced hepatitis [corrected]. Arthritis Rheum. 37, 1112. Pourahmad, J., Mortada, Y., Eskandari, M.R., Shahraki, J., 2011. Involvement of lysosomal labilisation and lysosomal/mitochondrial cross-talk in diclofenac induced hepatotoxicity. Iran. J. Pharm. Res. 10, 877–887. Pumford, N.R., Myers, T.G., Davila, J.C., Highet, R.J., Pohl, L.R., 1993. Immunochemical detection of liver protein adducts of the nonsteroidal antiinflammatory drug diclofenac. Chem. Res. Toxicol. 6, 147–150. Reed, J.C., 1999. Double identity for proteins of the Bcl-2 family. Nature 387, 773–776. Siu, W.P., Pun, P.B.L., Latchoumycandane, C., Boelsterli, U.A., 2008. Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: multiple protective roles of cyclosporin A. Toxicol. Appl. Pharmacol. 227, 451–461. Stanley, J., Korsmeyer, 1999. BCL-2 Gene Family and the Regulation of Programmed Cell Death. Cancer Res. 59, 1693–1700. Yano, A., Higuchi, S., Tsuneyama, K., Fukami, T., Nakajima, M., Yokoi, T., 2012. Involvement of immune-related factors in diclofenac-induced acute liver injury in mice. Toxicology 293, 107–114.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Protection from diclofenac-induced liver injury by Yulangsan polysaccharide in a mouse model Jianchun Huang a,1, Vanphuc Nguyen a,1, Xiaojun Tang b, Jinbin Wei a, Xing Lin a, Zefeng Lai a, Vanminh Doan a, Qiuqiao Xie a, Renbin Huang a,n a b
Department of Pharmacology, Guangxi Medical University, Nanning 530021, PR China Department of Laboratory Medicine, Guangxi Medical College, Nanning, Guangxi, China
art ic l e i nf o
a b s t r a c t
Article history: Received 4 February 2016 Received in revised form 9 July 2016 Accepted 3 August 2016 Available online 4 August 2016
Ethnopharmacological relevance: Millettia pulchra Kurz var-laxior (Dunn) Z. Wei, a wild-growing plant of the family Fabaceae is known to possess multifarious medicinal properties. Yulangsan polysaccharide (YLSPS) is a chief ingredient of its root, which has been used in Chinese traditional medicine with a long history for remedy of acute or chronic hepatitis and jaundice. Aim of the study: To investigate the ability of the YLSPS to protect against diclofenac-induced hepatotoxicity in mice. Materials and methods: Mice were orally treated with YLSPS daily 1 h after the injection of diclofenac for 2 weeks. Dimethyl diphenyl bicarboxylate was used as a reference drug. Results: YLSPS effectively reduced the elevated levels of serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase and enhanced the reduction of superoxide dismutase, catalase, and glutathione peroxidase activities in the liver. Moreover, the content of malondialdehyde was reduced by treatment with YLSPS, and histological findings also confirmed the anti-hepatotoxic activity. In addition, YLSPS significantly inhibited proinflammatory mediators, such as tumor necrosis factor-alpha and interleukin 1 beta. YLSPS also enhanced mitochondrial antioxidants and inhibited cell death by preventing the down-regulation of Bcl-2 and the up-regulation and release of Bax along with caspase 9 and 3 activity; thus, these findings confirm the involvement of mitochondria in diclofenac-induced apoptosis. Conclusion: The results indicate that protective effects of YLSPS against diclofenac-induced acute hepatic injury may rely on its effect on reducing oxidative stress, suppressing inflammatory responses, and improving drug-metabolizing enzyme activity in the liver. & 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Yulangsan polysaccharide Diclofenac Hepatoprotection
1. Introduction Non-steroidal anti-inflammatory drugs (NSAIDs) are the centerpiece of pharmacotherapy for most rheumatological disorders and are used in large numbers as analgesics and antipyretics, both as prescription drugs as and over-the-counter purchases (Hau et al., 2009). Diclofenac is an NSAIDs that is used for the treatment of mild-to-moderate pain, fever, and inflammation. Clinical hepatotoxicity is one of the adverse reactions caused by diclofenac. Hepatotoxicity studies of diclofenac showed that it causes a rise in liver function tests (Banks et al., 1995); the histopathological picture suggests hypersensitivity reactions (Boelsterli, 2003), liver n
Corresponding author. E-mail address: [email protected] (R. Huang). 1 They contributed equally to this work.
http://dx.doi.org/10.1016/j.jep.2016.08.012 0378-8741/& 2016 Elsevier Ireland Ltd. All rights reserved.
necrosis, jaundice, fulminate hepatitis with or without jaundice, and liver failure (Aithal and Day, 2007; Banks et al., 1995; O’Connor et al., 2003). Furthermore, there is evidence of clinical and experimental animal studies that diclofenac-induced liver injury involves immune responses (Pohl et al., 1994; Yano et al., 2012). Moreover, previous studies have suggested that diclofenac hepatotoxicity is a result of metabolic activation by CYP2E1 and CYP2C9 and reactive oxygen species (ROS) formation, leading to mitochondrial oxidative stress injury and glutathione depletion. Diclofenac-induced hepatic toxicity was prevented by antioxidants and cytochrome P-450 inhibitors (Pourahmad et al., 2011). In contrast, antioxidants were able to prevent caspase activation by diclofenac (Gómez-Lechón et al., 2003). Millettia pulchra Kurz var-laxior (Dunn) Z.Wei, a wild-growing plant of the family Fabaceae, is known to possess multifarious medicinal properties. Its root, Yulangsan (YLS), has been used in
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Chinese traditional medicine with a long history for remedy of acute or chronic hepatitis and jaundice. In our previous study, the ethanol extract of the root showed anti-inflammatory and antioxidant properties; it has also been used in the treatment of neurological, cardiovascular, and liver diseases (Huang et al., 2003, 2008; Jiao et al., 2004). In addition, YLS polysaccharide (YLSPS) from the root inhibits peroxidation in vitro and suppresses production of excess free radicals in vivo (Duan et al., 2007, 2008). Recently, it was found to have protective effects against hepatic injury induced by carbon tetrachloride, rifampicin, isoniazid, and nimesulide in mice by antioxidant mechanisms (Dong et al., 2014; Fu et al., 2009; Nguyen et al., 2015). Based on the data from our laboratory and others, we suggest that YLSPS may be effective in protecting against diclofenac-induced liver injury. However, reports on the hepatoprotective effects and further exploration of underlying mechanisms of YLSPS are still lacking. To test our hypothesis, a diclofenac-induced liver injury model was chosen to study the hepatoprotective effects of YLSPS in mice. The effect of YLSPS on liver injury was compared with that of dimethyl diphenyl bicarboxylate (DDB) in this study. DDB, an antihepatitis drug, has been widely used in clinical practice for the treatment of liver disease, such as acute or chronic hepatitis and drug-induced liver injury (Park et al., 2005). Various studies indicated that DDB exhibits strong antioxidative activity (Gao et al., 2005; Park et al., 2005). Furthermore, to investigate the hepatoprotective mechanisms of YLSPS, histological examination of hepatic characterization, markers of liver oxidative stress, and antioxidative defense system, we evaluated hepatic proinflammatory mediators (including serum tumor necrosis factor-alpha [TNF-α] and interleukin 1 beta [IL-1β]), key apoptotic events (such as Bcl-2 and Bax), and caspase 9 and 3 activity.
2. Materials and methods
Center of Guangxi Medical University (Guangxi, China). The research was conducted according to protocols approved by the Animal Ethics Committee of Guangxi Medical University. All mice were housed under controlled conditions with temperature of 25 72 °C, relative humidity of 60 710%, room air changes 12–18 times/h, and a 12 h light/dark cycle. Feed and water were made available ad libitum. After 1-week acclimatization, the mice were randomly divided into six groups (5 males and 5 females mice /group) and treated for 14 days as follows: in the normal control group, mice were given the same volume of physiological saline; in the diclofenactreated model group, mice were intraperitoneally injected with diclofenac sodium once a day at a dose of 50 mg/kg; in the positive control group, mice were treated with DDB (200 mg/kg) by orally administrating after 1 h with the injection of diclofenac (50 mg/ kg); in the low, medium, and high dosage of YLSPS-treated group, mice were treated with YLSPS (150, 300, and 600 mg/kg, respectively) by oral administration after 1 h, with the injection of diclofenac (50 mg/kg). At the final stage of the experiment, blood samples were collected and serum was separated for assessment of enzyme activity, and then animals were sacrificed by cervical dislocation. Liver samples were dissected out and washed immediately with icecold saline to remove as much blood as possible; one part of the liver samples was immediately stored at 80 °C until analysis, and another part was excised and fixed in 10% formalin solution for histopathologic analysis. 2.4. Calculation of liver index The liver index was calculated according to the formula: (mice liver weight/mice weight) 100% (Huang et al., 2012). 2.5. Estimation of serum marker enzymes and TNF-α and IL-1β levels
2.1. Chemicals Diclofenac was purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). The biochemical kits of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TNF-α and IL-1β ELISA kits were purchased from Wuhan Boster Bio-engineering Co. Ltd. (Wuhan, China); all other chemicals were of analytical grade. 2.2. Preparation of YLSPS YLSPS was prepared by a method described previously (Lin et al., 2014a). The root of the Millettia pulchra Kurz var-laxior (Dunn) Z. Wei was dried, powdered, and extracted three times with boiling water. The PS in the filtrate was precipitated fractionally with alcohol. The protein in the product was removed by the Sevag method and further purified using DEAE ion exchange cellulose (DEAE-52). The components of the saccharide were determined by GC and TLC. YLSPS was composed of D-glucose and D-arabinose in a molar ratio of 90.79% and 9.21%, with an average molecular weight of 14,301 Da (Lin et al., 2014a). The maximum tolerable dose (MTD) of LYSPS in mice was 24,000 mg/kg and it was 800 times of adult daily dose. 2.3. Animals and treatment Kunming mice of both sexes, weighing 20–22 g, Specefic pathogen Free (SPF), were provided by the Experimental Animal
The serum AST, ALT, and ALP were determined in accordance with methods provided by the diagnostic kits. TNF-α and IL-1β are important cytokines involved in hepatocyte damage induced by diclofenac. The serum TNF-α and IL-1β levels were measured by commercially available ELISA kits. 2.6. Measurement of SOD, GSH-Px, and CAT in liver homogenate Liver homogenates were prepared in cold Tris–HCl (5 mmol/L containing 2 mmol/L EDTA, pH 7.4) using a homogenizer. The homogenate was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was used immediately for the assays of SOD, GSH-Px, and CAT. All of these enzymes were determined according to the manufacturer's instructions. 2.7. Assay of lipid peroxidation products Lipid peroxidation was assayed by the measurement of the levels of MDA, which was determined following the kit instructions. 2.8. Measurement of caspase 3 and 9 The liver tissue samples were homogenized in lysis buffer. The homogenate was centrifuged for 15 min at 40000g and the resulting supernatant was incubated peotide (DEVD-AFC for caspase 3, LEHD-AFC for caspase 9). The change in fluorescence (excitation at 400 nm and emission at 490 nm) was monitored after 120 min of incubation, according to the procedure reported previously (Lin et al., 2014b).
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2.9. Histological study The liver samples were sectioned and stained with hematoxylin and eosin (H&E) and subsequently examined by light microscopy (IX51, Olympus, Japan) for histopathological examination. Finally, the images were examined and evaluated for pathological changes. 2.10. Evaluation of apoptosis TUNEL staining was performed in representative liver sections to locate apoptotic hepatocytes. Frozen sections (4–5 μm) from the different groups were prepared, fixed in 4% paraformaldehyde, and washed with phosphate-buffered saline (PBS). The tissue sections were then incubated with the TUNEL reaction mixture for 1 h in a humidified atmosphere at 37 °C. The nuclear staining was determined via the green nuclear fluorescence observed by laser scanning confocal microscopy (Leica) with an excitation wavelength of 488 nm and detection at 565 nm.
Fig. 1. Effect of YLSPS on the liver index in mice with diclofenac-induced hepatic injury. Results are presented as the mean 7 SE (%) (n¼ 10). #P o0.05, ##Po 0.01 vs. normal group; *Po 0.05, **Po 0.01 vs. model group.
2.11. Western blot analysis of hepatic Bax and Bcl-2 The electrophoretic separation of the proteins was performed using 10% sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE) and then electrotransferred to nitrocellulose membranes (Schleicher and Schuell, Germany), which was immuno-blotted with Bax, Bcl-2, and β-actin primary antibodies. ALP-labeled goat anti-rabbit IgG or horseradish peroxidase-conjugated anti-rat IgG were used as the secondary antibodies, and the color developed using a mixture of 5-bromo-4-chloro-indolylphosphate and nitroblue tetrazolium. The filter images were captured by the Versa Doc Imaging system (Bio-Rad, USA). β-actin was used as an internal standard.
Fig. 2. Effects of YLSPS on serum ALT, AST, and ALP activities in mice with diclofenac-induced hepatic injury. Results are presented as the means 7 SE. #P o0.05, ## P o 0.01 vs. normal group; *P o 0.05, **Po 0.01 vs. model group.
2.12. Statistical analysis AST, and ALP levels when compared with the model group. The statistical analysis was performed using SPSS version 16 statistical program SPSS Inc. (Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to compare the means among different groups, and Tukey's test was used in the post hoc multiple comparisons. The data were presented as the means 7 SE, and Pvalue o0.05 was considered statistically significant.
3. Results 3.1. Effects of YLSPS on liver index in mice
3.3. Effect of YLSPS on serum TNF-α and IL-1β levels TNF-α and IL-1β are important cytokines involved in hepatocyte damage induced by diclofenac. The levels of TNF-α and IL-1β were low in the normal control animals. However, the levels of the two cytokines increased significantly in the diclofenac-treated mice. This increase was reduced in both YLSPS and DDB supplemented groups (Fig. 3).
The liver index showed a significant increase in the group treated with diclofenac compared with the normal group, indicating that diclofenac induces hypertrophy of liver tissue. Conversely, in the medium and high dosage of YLSPS-treated groups and the DDB-treated group, the liver index decreased more markedly than that of the diclofenac-treated group. The results show that hepatic intumescence had been alleviated after the models were treated with YLSPS (Fig. 1). 3.2. Effect of YLSPS on serum ALT, AST, and ALP The effects of YLSPS pretreatment on the diclofenac-induced modifications in serum ALT, AST, and ALP levels are shown in Fig. 2. A single dose of diclofenac caused hepatotoxicity in mice, as indicated by an increase in serum ALT, AST, and ALP activities after diclofenac administration, whereas mice pretreated with YLSPS exhibited a significant decrease in the activities of the marker enzymes. DDB administration also reversed the alterations of ALT,
Fig. 3. Effects of YLSPS on serum TNF-α and IL-1β levels in mice with diclofenacinduced hepatic injury. Results are presented as the means 7 SE. #P o0.05, ## P o 0.01 vs. normal group; *P o 0.05, **Po 0.01 vs. model group.
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3.4. Effect of YLSPS on hepatic antioxidant enzyme activities GSH-Px, SOD, and CAT showed a significant decrease in animals treated with diclofenac alone compared with the normal group. However, mice treated with diclofenac plus medium- and highdose YLSPS showed a significant promotion towards the normal level. In contrast, YLSPS at the low dose had little influence on the parameters (Figs. 4 and 5). 3.5. Effects of YLSPS on concentration of MDA
Fig. 4. Effects of YLSPS on liver SOD and GSH-Px activities in mice with diclofenacinduced hepatic injury. Results are presented as the means 7 SE. #P o 0.05, ## Po 0.01 vs. normal group; *Po 0.05, **Po 0.01 vs. model group.
The localization of radical formation results in lipid peroxidation, as measured by MDA in mouse liver homogenates. MDA content in the total liver homogenates was increased in the diclofenac-treated group compared with the normal control group. Administration of medium- and high-dose YLSPS to diclofenacintoxicated mice resulted in a significant decrease in MDA compared with the diclofenac-treated group; however, the low treatment dose did not show any significant effect (Fig. 5). 3.6. Histopathological changes To assess histological changes, H&E staining of liver tissue sections from each group was examined. The normal control showed normal lobular architecture with central veins and radiating hepatic cords. Hepatotoxicity induced by diclofenac was confirmed by abnormal histological findings manifested by morphological changes, such as ballooning change of hepatocytes, degeneration of hepatocytes, and inflammatory cell infiltration. In medium- and high-dose YLSPS- and DDB-treated groups, the histopathological abnormalities induced by diclofenac were restored (Fig. 6).
Fig. 5. Effects of YLSPS on liver CAT activities and the MDA contents in mice with diclofenac-induced hepatic injury. Results are presented as the means 7 SE. # P o0.05, ##P o0.01 vs. normal group; *P o0.05, **P o 0.01 vs. model group.
3.7. Effects of YLSPS on apoptotic cells We measured apoptosis in liver tissue by double staining of
Fig. 6. Histologic results of tissues stained with H&E under light microscopy in mice with diclofenac-induced liver injury ( 400). Hepatotoxicity induced by diclofenac was confirmed by abnormal histological findings manifested by morphological changes, such as inflammation around the portal triad, disorganization of hepatocytes, cell swelling, and congestion of sinusoids. YLSPS and DDB reversed these pathological changes.
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TUNEL. As shown in Fig. 7, numerous TUNEL-positive hepatocytes were observed in liver tissues obtained from the diclofenac group. However, few positive hepatocytes were observed in livers treated with medium- and high-dose of YLSPS or DDB.
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3.8. Effects of YLSPS on activities of caspase 3 and 9 Massive activation of caspases is a crucial process during the induction of apoptosis associated with the pathogenesis of acute
Fig. 7. Effects of YLSPS on the apoptosis of hepatic cells in mice with diclofenac-induced hepatic injury. TUNEL staining was performed in liver tissue sections ( 200). The apoptosis index was defined as the number of apoptotic cells in every 100 cells counted. Results are presented as the means 7 SE. #Po 0.05, ##Po 0.01 vs. normal group; * Po 0.05, **Po 0.01 vs. model group.
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hepatic failure. In this study, the activities of caspase 3 and 9 were found to increase in mice receiving diclofenac treatment as compared with the normal group. There was a decrease in the caspase activity in mice pre-treated with medium- and high-dose of YLSPS or DDB (Fig. 8). These findings suggest that YLSPS is effective enough to combat diclofenac-induced caspase activation. 3.9. Effects of YLSPS on apoptosis-related proteins The proapoptotic Bax and antiapoptotic Bcl-2 proteins are members of the Bcl-2 family. We detected the protein levels of Bax and Bcl-2 in the liver tissue of each group (Fig. 9). Clearly, the levels of Bax were higher in the diclofenac group than those in the normal group, whereas Bax expressions decreased greatly in mice treated with DDB and YLSPS. Conversely, the expression of Bcl-2 was lower in the diclofenac group than that in the normal group, and the down-regulation was markedly inhibited by treatment with DDB and YLSPS.
Fig. 8. Effects of YLSPS on liver caspase 3 and 9 activities in mice with diclofenacinduced hepatic injury. Results are presented as the means 7 SE. #P o 0.05, ## Po 0.01 vs. normal group; *Po 0.05, **Po 0.01 vs. model group.
Fig. 9. Effects of YLSPS on the protein levels of Bax and Bcl-2 in the liver tissue detected by Western blotting. Results are presented as the means 7 SE. #Po 0.05, ## Po 0.01 vs. normal group; *Po 0.05, **Po 0.01 vs. model group.
4. Discussion Clinical findings suggest drug hypersensitivity reaction and direct toxic effects of the drug or its metabolite as the cause for the toxic effect (Cantoni et al., 2003; Galati et al., 2002). Due to diclofenac's high frequency of association with hepatotoxicity, it increases serum aminotransferase activities, liver necrosis, and jaundice (Banks et al., 1995; Boelsterli, 2003). ALT is cytosolic enzyme, AST is cytosolic enzymes or mitochondrial enzymes of the hepatocyte, and increased activities in circulation reflect cell damage and leakage, and ALP is a marker of the relative degree of hepatocellular damage and of obstruction, intrahepatic or extrahepatic, tending to parallel the degree of hyperbilirubinemia (Dong et al., 2014; Labbe et al., 2008). In the present study, the administration of diclofenac caused a dramatic elevation in serum AST, ALT, and ALP activities, indicating subchronic hepatotoxicity induced by administration of diclofenac. Instead, after treatment with DDB and YLSPS, activities of these serum enzymes were significantly decreased, indicating effectiveness of these drugs in liver regeneration after being damaged. ROS is mainly generated in the mitochondria, leading to serious damage to cellular macromolecules, including protein dysfunction, lipid peroxidation, DNA damage, and oxidative stress (Djordjević, 2004; Galati et al., 2002). For this reason, the antioxidative activity and inhibition of free-radical generation are important in terms of protecting the liver from drug-induced damage. Oxidative stress parameters, including SOD, CAT, and GSH-Px, and MDA, were examined in this study; the results on the administration of YLSPS in diclofenac-induced toxicity in the liver revealed decreased production of free radical derivatives, as evidenced by the decreased MDA level. Furthermore, YLSPS increased the activity of endogenous antioxidant enzymes SOD, CAT, and GSH-Px. This indicates that YLSPS may be able to protect against the oxidation of hepatic cellular membrane damage via a free-radical scavenging property. Some evidence suggests that diclofenac-induced hepatic injury involves immune responses (Pohl et al., 1994; Pumford et al., 1993). The pro-inflammatory cytokine TNF-α has been reported to play a key role in the pathogenesis of various liver diseases. IL-1β is a very potent pro-inflammatory cytokine; it increases during drug hepatotoxicity (Holt and Ju, 2006) and is involved in the early onset of diclofenac-induced liver injury (Yano et al., 2012). In this study, the levels of serum IL-1β and TNF-α in the diclofenac group were significantly higher than those in the normal group; however, upregulation of these inflammatory factors was markedly inhibited after treatment with YLSPS. This suggests that YLSPS exerted a therapeutic effect possibly through restriction of the production and release of inflammatory mediators. Histologic examination of rat livers treated with diclofenac showed typical hepatotoxicity characterization, ballooning degeneration of hepatocytes, hepatocellular necrosis, and inflammatory cell infiltration. However, the typical hepatotoxicity characterization was not shown in YLSPS-treated groups, suggesting that YLSPS has potential protective effects against diclofenac-induced liver injury. Mitochondria are regarded as sensors of oxidation damage and may play a major role in apoptosis (Daniel and Korsmeyer, 2004; Djordjević, 2004; Galati et al., 2002). The mitochondrial pathway is involved in diclofenac-induced apoptosis, which is related to cytochrome P-450-mediated metabolism of diclofenac, with the highest apoptotic effect produced by the metabolite 5OH-diclofenac (Gómez-Lechón et al., 2003). In this study, apoptotic hepatocytes were detected using TUNEL staining. Numerous TUNEL-positive hepatocytes were observed in liver tissues obtained from the diclofenac group. However, few positive hepatocytes were observed in livers treated with 600 mg/kg YLSPS or 200 mg/kg DDB.
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Furthermore, we examined the mechanism of apoptosis induced by YLSPS. Measurement of the apoptosis-related proteins, including Bax and Bcl-2, in liver tissues by Western blot immunoassay was then conducted. Diclofenac studies have has revealed that upon translocation of activated Bax to mitochondria, monomers of Bax and Bak can oligomerize to form large channels and induce formation of pores (Siu et al., 2008). Anti-apoptotic proteins, such as Bcl-2, that are localized mainly in the mitochondrial outer membranes block membrane permeability changes (Reed, 1999; Stanley and Korsmeyer, 1999). In this study, immunoblot analysis revealed significant decrease in the level of Bcl-2 and increase in the levels of Bax during diclofenac treatment. YLSPS significant protection against these key control points of apoptosis increased Bcl-2 levels and decreased Bax. Furthermore, the activities of caspases 3 and 9 in the cytosol fraction after diclofenac administration increased rapidly relative to the normal groups. The elevated caspases 3 and 9 were attenuated by treating with YLSPS and DDB, thus confirming the antioxidant efficacy and protective role of YLSPS against diclofenac-induced apoptosis. In conclusion, results from the present study demonstrate that YLSPS had a protective effect against diclofenac-induced hepatic damage in Kunming mice. Contributing to the alleviation of diclofenac triggered typical hepatotoxic characterization as histological finding and serum enzymes relating with liver diseases. The preliminary exploration of the underlying mechanisms indicates its protection against hepatic injury by radical scavenging action, antioxidant activity, and inhibiting critical control points of apoptosis. Therefore, on the basis of our work, YLSPS should be regarded as a new and promising agent with a high potential in the prevention and treatment of drug-induced liver injury and liver disease.
5. Conflict of Interest The authors declare that there are no conflicts of interest.
Acknowledgments This work was supported by the Natural Science Foundation of China (No. 81560587); the Fok Ying-Tong Education Foundation of China (No. 151107); the Guangxi Natural Science Foundation (No. 2013GXNSFAA019175); the Science and Technology Research Projects of Guangxi colleges and universities (No.2013YB343); Science and Technology foundation platform construction project of Guangxi Province (No.12-97-20).
References Aithal, G.P., Day, C.P., 2007. Nonsteroidal anti-inflammatory drug-induced hepatotoxicity vi-vii. Clin. Liver Dis. 11, 563–575. Banks, A.T., Zimmerman, H.J., Ishak, K.G., Harter, J.G., 1995. Diclofenac-associated hepatotoxicity: analysis of 180 cases reported to the Food and Drug Administration as adverse reactions. Hepatology 22, 820–827. Boelsterli, U.A., 2003. Diclofenac-induced liver injury: a paradigm of idiosyncratic drug toxicity. Toxicol. Appl. Pharmacol. 192, 307–322. Cantoni, L., Valaperta, R., Ponsoda, X., Castell, J.V., Barelli, D., Rizzardini, M., Mangolini, A., Hauri, L., Villa, P., 2003. Induction of hepatic heme oxygenase-1 by diclofenac in rodents: role of oxidative stress and cytochrome P-450 activity. J. Hepatol. 38, 776–783. Daniel, N.N., Korsmeyer, S.J., 2004. Cell death: critical control points. Cell 116,
213
205–219. Djordjević, V.B., 2004. Free radicals in cell biology. Int. Rev. Cytol. 237, 57–89. Dong, Y., Huang, J., Lin, X., Zhang, S., Jiao, Y., Liang, T., Chen, Z., Huang, R., 2014. Hepatoprotective effects of Yulangsan polysaccharide against isoniazid and rifampicin-induced liver injury in mice. J. Ethnopharmacol. 152, 201–206. Duan, X., Jiao, Y., Zhang, S., Huang, R., 2007. Protective effects of Yulangsan polysaccharide (YLS) on primary cultured rat hepatocytes injury induced by H2O2. LiShiZhen Med. Mater. Med. Res., 1592–1593. Duan, X., Jiao, Y., Zhang, S., Lu, X., Huang, R., 2008. Effects of Yulangsan polysaccharide on proliferation and collagen I production of hepatic stellate cells . China J. Mod. Med., 423–425. Fu, S., Huang, J., Wang, N., Huang, R., 2009. Protective effect of Yulangsan polysaccharide on acute alcohol hepatic injury in mice . China Pharm., 406–408. Galati, G., Tafazoli, S., Sabzevari, O., Chan, T.S., O’Brien, P.J., 2002. Idiosyncratic NSAID drug induced oxidative stress. Chem. Biol. Interact. 142, 25–41. Gao, M., Zhang, J., Liu, G., 2005. Effect of diphenyl dimethyl bicarboxylate on concanavalin A-induced liver injury in mice. Liver Int. : Off. J. Int. Assoc. Study Liver 25, 904–912. Gómez-Lechón, M.J., Ponsoda, X., O’Connor, E., Donato, T., Castell, J.V., Jover, R., 2003. Diclofenac induces apoptosis in hepatocytes by alteration of mitochondrial function and generation of ROS. Biochem. Pharmacol. 66, 2155–2167. Hau, D.K.P., Roberto, G., Wong, R.S.M., Yuen, M.C.W., Cheng, G.Y.M., Tong, C.S.W., Guo Yuan, Z., Leung, A.K.M., Lai, P.B.S., Fung, Yi, L., 2009. Phyllanthus urinaria extract attenuates acetaminophen induced hepatotoxicity: involvement of cytochrome P450 CYP2E1. Phytomedicine Int. J. Phytother. Phytopharm. 16, 751–760. Holt, M.P., Ju, C., 2006. Mechanisms of Drug-Induced Liver Injury. Am. Assoc. Pharma. Sci. J. 2006 (8), E48–E54. Huang, Q., Zhang, S., Zheng, L., He, M., Huang, R., Lin, X., 2012. Hepatoprotective effects of total saponins isolated from Taraphochlamys affinis against carbon tetrachloride induced liver injury in rats. Food Chem. Toxicol. 50 (3), 713–718. Huang, R., Jiao, Y., Jiang, W., 2003. Effect of Yulangsan extract on the cardiac hemodynamics and the coronary flow in rats. Chin. J. Hosp. Pharm., 321–322. Huang, Y., Chen, J., Huang, R., Lin, X., Wang, N., 2008. Anti-inflammatory effect of Yulangsan extracts and the mechanisms Chinese. Chin. J. New Drugs 20, 1764–1767. Jiao, Y., Duan, X., Huang, R., 2004. Scavenging and inhibiting effect of Yulangsan extract on superoxide anion free radical and hydroxy free radical. Guangxi Med. Univ., 22–23. Labbe, G., Pessayre, D., Fromenty, B., 2008. Drug-induced liver injury through mitochondrial dysfunction : mechanisms and detection during preclinical safety studies. Fund. Clin. Pharmacol. 22, 335–353. Lin, X., Huang, Z., Chen, X., Rong, Y., Zhang, S., Jiao, Y., Huang, Q., Huang, R., 2014a. Protective effect of Millettia pulchra polysaccharide on cognitive impairment induced by D-galactose in mice. Carbohydr. Polym., 533–543. Lin, X., Zhang, S., Huang, R., Tan, S., Liang, S., Wu, X., Zhuo, L., Huang, Q., 2014b. Protective effect of tormentic acid from Potentilla chinensis against lipopolysaccharide/D-galactosamine induced fulminant hepatic failure in mice. Int. Immunopharmacol. 19, 365–372. Nguyen, V., Huang, J., Doan, V., Lin, X., Tang, X., Huang, Y., Tang, A., Yang, X., Huang, R., 2015. Hepatoprotective effects of Yulangsan polysaccharide against nimesulide-induced liver injury in mice. J. Ethnopharmacol. 172, 273–280. O’Connor, N., Dargan, P.I., Jones, A.L., 2003. Hepatocellular damage from non-steroidal anti-inflammatory drugs. QJM : Mon. J. Assoc. Physicians 96, 787–791. Park, E.Y., Ki, S.H., Ko, M.S., Kim, C.W., Lee, M.H., Lee, Y.S., Kim, S.G., 2005. Garlic oil and DDB, comprised in a pharmaceutical composition for the treatment of patients with viral hepatitis, prevents acute liver injuries potentiated by glutathione deficiency in rats. Chem. Biol. Interact. 155, 82–96. Pohl, L.R., Martin, J.L., Hargus, S.J., 1994. Immunochemical methods of studying the mechanism of diclofenac-induced hepatitis [corrected]. Arthritis Rheum. 37, 1112. Pourahmad, J., Mortada, Y., Eskandari, M.R., Shahraki, J., 2011. Involvement of lysosomal labilisation and lysosomal/mitochondrial cross-talk in diclofenac induced hepatotoxicity. Iran. J. Pharm. Res. 10, 877–887. Pumford, N.R., Myers, T.G., Davila, J.C., Highet, R.J., Pohl, L.R., 1993. Immunochemical detection of liver protein adducts of the nonsteroidal antiinflammatory drug diclofenac. Chem. Res. Toxicol. 6, 147–150. Reed, J.C., 1999. Double identity for proteins of the Bcl-2 family. Nature 387, 773–776. Siu, W.P., Pun, P.B.L., Latchoumycandane, C., Boelsterli, U.A., 2008. Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: multiple protective roles of cyclosporin A. Toxicol. Appl. Pharmacol. 227, 451–461. Stanley, J., Korsmeyer, 1999. BCL-2 Gene Family and the Regulation of Programmed Cell Death. Cancer Res. 59, 1693–1700. Yano, A., Higuchi, S., Tsuneyama, K., Fukami, T., Nakajima, M., Yokoi, T., 2012. Involvement of immune-related factors in diclofenac-induced acute liver injury in mice. Toxicology 293, 107–114.