e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Ursolic acid protects mouse liver against CCl4 -induced oxidative stress and inflammation by the MAPK/NF-B pathway Jie-Qiong Ma a,∗ , Jie Ding a , Li Zhang a , Chan-Min Liu b a
School of Chemistry and Pharmaceutical, Sichuan University of Science and Engineering, 643000 Zigong City, Sichuan Province, PR China b School of Life Science, Jiangsu Normal University, No. 101, Shanghai Road, Tangshan New Area, Xuzhou City 221116, Jiangsu Province, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Ursolic acid (UA), a natural pentacyclic triterpenoid, has been reported to have many benefits
Received 7 January 2014
and medicinal properties. However, its protective effects against carbon tetrachloride (CCl4 )
Received in revised form
induced hepatotoxicity have not been clarified. The aim of the present study was to investi-
12 March 2014
gate the effects of UA on oxidative stress and inflammation in liver of CCl4 treated mice. Male
Accepted 16 March 2014
ICR mice were injected with CCl4 with or without UA co-administration (25 and 50 mg/kg
Available online 22 March 2014
intragastrically once daily) for one week. Our data showed that UA significantly prevented
Keywords:
cators of liver damage (serum aminotransferase activities) and histopathological analysis.
Ursolic acid
Moreover, CCl4 -induced profound elevation of reactive oxygen species (ROS) production and
CCl4 -induced hepatotoxicity in a dose-dependent manner, indicated by both diagnostic indi-
CCl4
oxidative stress, as evidenced by increasing of lipid peroxidation level and depleting of the
MAPKs
total antioxidant capacity (TAC) level in liver, were suppressed by treatment with UA. Fur-
Oxidative stress
thermore, western blot analysis showed that UA significantly decreased CYP2E1 expression
NF-B
levels and production of pro-inflammatory markers including TNF-␣, IL-1 and COX-2 in
Hepatic inflammation
CCl4 -treated mouse liver. In exploring the underlying mechanisms of UA action, we found that UA decreased the activation of mitogen-activated protein kinases (JNK, p38 MAPK, ERK), which in turn inactivated the immunoregulatory transcription factor nuclear factor kappa B (NF-B) in liver of CCl4 treated mice. In conclusion, these results suggested that the inhibition of CCl4 -induced inflammation by UA is due at least in part to its anti-oxidant activity and its ability to modulate the MAPK and NF-B signaling pathway. © 2014 Elsevier B.V. All rights reserved.
Abbreviations: COX-2, cyclooxygenase-2; CYP2E1, cytochrome P450 2E1; ERK, the extracellular-receptor kinases; IL-1, interleukin1beta; JNK, the c-Jun N-terminal kinases; NF-B, nuclear factor-B; MAPKs, mitogen-activated protein kinases; UA, ursolic acid; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substances; TNF-␣, tumor necrosis factor-alpha. ∗ Corresponding author. Tel.: +86 013700950121; fax: +86 516 83500171. E-mail addresses: [email protected], [email protected] (J.-Q. Ma). http://dx.doi.org/10.1016/j.etap.2014.03.011 1382-6689/© 2014 Elsevier B.V. All rights reserved.
976
1.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
Introduction
Liver diseases with severe hepatocyte damage caused by viral infection, non-alcoholic steatohepatitis or other hepatotoxic agent are highly associated with acute or chronic inflammation (Tacke et al., 2009; Tseng et al., 2014). Hepatic inflammation is regarded as a hallmark of early stage fibrosis, which can progress to extensive fibrosis and cirrhosis. Damage associated with hepatic inflammation is mediated by the proinflammatory cytokines tumor necrosis factor-alpha (TNF␣), interleukin-1 (IL-1) and cyclooxygenase-2 (COX-2) (Shin et al., 2013). Carbon tetrachloride (CCl4 ) is a well-known compound for the production of chemical hepatic injury involving in production of inflammatory cytokines and recruitment of inflammatory cells, leading to liver architectural damage and dysfunction (Weber et al., 2003; Ebaid et al., 2013; Hamdy and El-Demerdash, 2012). The chronic liver damage induced by CCl4 in rats produces liver inflammatory and biochemical patterns that resemble rat liver cirrhosis (Laskin and Laskin, 2001; Hamdy and El-Demerdash, 2012; Li et al., 2013). In addition, many researches have suggested that the production of several proinflammatory molecules is associated with the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-B). Both MAPKs and NF-B can be stimulated by CCl4 and are tightly involved in the pathological conditions in the liver (Iida et al., 2009; Kim et al., 2011). Ursolic acid (UA: 3-hydroxy-urs-12-en-28-oic acid), a natural pentacyclic triterpenoid, has been found in various plants including apples, basil, cranberries, peppermint, rosemary, oregano and prunes (Checker et al., 2012; Lv et al., 2012). Many triterpenoids had been used for medicinal purposes for a variety of clinical diseases in many Asian countries (Ikeda et al., 2007). UA has been reported to possess many biological activities, including antioxidant, anti-inflammatory, trypanocidal, antirheumatic, antiviral and antitumoral properties (Ikeda et al., 2007; Wu et al., 2011). UA has also been shown to target multiple proinflammatory transcription factors, cell cycle proteins, growth factors, kinases, cytokines, chemokines, adhesion molecules, and inflammatory enzymes (Shanmugam et al., 2013). Previous researches showed that UA attenuate CCl4 -induced hepatotoxicity by inhibiting oxidative stress (Liu et al., 1994; Martin-Aragon et al., 2001). However, the molecular mechanisms of CCl4 -induced liver injury and anti-inflammatory effects of UA are not yet completely understood. In the present study, we hypothesized that UA can inhibit CCl4 -induced inflammation. We, for the first time, determine whether UA can protect mouse liver from CCl4 induced inflammatory response by modulating the MAPK and NF-B pathway.
2.
Materials and methods
2.1.
Chemicals and reagents
UA and CCl4 were obtained from Sigma Chemical Co. (St. Louis, MO, USA); IL-1 antibody, COX-2 antibody, and NFB p65 antibody are from Santa Cruz Biotechnology (Santa
Cruz, CA, USA); JNK (total) antibody, ERK1/2 (total) antibody, p38 (total) antibody, phospho-JNK antibody, phospho-ERK1/2 antibody, and phospho-p38 antibody were obtained from Cell Signaling Technology (Beverly, MA, USA); CYP2E1 antibody were obtained from Proteintech Group (Proteintech Group, INC. USA); aminotransferase activities in serum assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and Jingmei Biotech ltd. (Shenzhen, China); protein concentration were measured using the bicinchoninic acid (BCA) assay kit from Pierce Biotechnology, Inc. (Rockford, IL, USA). All other reagents unless indicated were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
2.2.
Animals and treatment
Male ICR mice (20–25 g) were obtained from the Branch of National Breeder Center of Rodents (Beijing) and kept in an environmentally controlled room (23 ± 2 ◦ C, 55 ± 10% humidity) with a 12-h light/dark cycle and allowed free access to food and water. Liver inflammation was induced by intraperitoneal (i.p.) injection of 2 ml of CCl4 in olive oil (1:1, v/v) per kg body weight twice weekly for up to 1 week (Tu et al., 2012). Fifty mice were randomly divided into four groups (10–15 mice/group). Mice in Group 1 were given twice weekly injections of olive oil (vehicle control); mice in Group 2 were injected with CCl4 and received water containing 0.1% Tween 80 by oral gavage; mice in Group 3 were injected with CCl4 , as in Group 2, and received UA in distilled water containing 0.1% Tween 80 at a dose of 25 mg/(kg day) by oral gavage; mice in Group 4 were injected with CCl4 , as in Group 2, and received UA in distilled water containing 0.1% Tween 80 at a dose of 50 mg/(kg day) by oral gavage (Xiang et al., 2012). At the end of treatment, seven mice in each group were used for the biochemical analysis; the others were used for histological evaluations. Mice were sacrificed and about 1 ml of blood samples were drawn by cardiac puncture with heparined tubes. The plasma was collected after centrifugation at 1200 × g for 10 min and stored at −70 ◦ C freezer for further analysis. The liver tissues were immediately collected for experiments and placed in ice-cold 0.9% NaCl solution, perfused with the physiological saline solution to remove blood cells, blotted on filter paper. And then the removed liver was quickly collected for experiments or stored at −70 ◦ C for later use. This research was conducted in accordance with the Chinese laws and NIH publication on the use and care of laboratory animals. Relevant university committees for animal experiments approved these experiments.
2.3. Assay of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity The activities of ALT and AST in serum were estimated spectrophotometrically using commercial diagnostic kits (Jiancheng Institute of Biotechnology, Nanjing, China) (Liu et al., 2013a).
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
2.4.
977
Histological evaluations
The histological changes of liver were evaluated by our previous method (Liu et al., 2012).
2.5.
Assay of ROS level
ROS was measured as described previously, based on the oxidation of 2 7 -dichlorodihy drofluorescein diacetate to 2 7 dichloro-fluorescein (Shinomol and Muralidhara, 2007; Liu et al., 2013b).
2.6. Assay of thiobarbituric acid reactive substances (TBARS) levels in the liver Estimation of lipid peroxidation was performed by previous method (Onkawa et al., 1979). A standard calibration curve was prepared by using 1–10 nM of 1,1,3,3-tetra methoxy propane. The concentration was expressed in terms of nanomoles of TBARS per mg of protein.
2.7.
Assay of the total antioxidant capacity (TAC)
The total antioxidant capacity (TAC) in the liver was assayed with a commercially available assay kit (Jiancheng Biochemical, Inc., Nanjing, China) (Liu et al., 2013b). This method is based on the reduction of iron (III) in acidic medium by intracellular antioxidants. The liberated iron (II) reacts with 1,10-phenanthroline to form a colored complex, which is measured at 520 nm. The TAC of the samples was measured according to the manufacturer’s protocol. One unit of TAC was defined as the capability of increasing 0.01 optical densities (OD520 ) units per mg protein per min at 37 ◦ C.
2.8.
Western blot analysis
Nuclear and cytoplasmic extracts for western blotting were obtained by using a nuclear/cytoplasmic isolation kit (Beyotime Institute of Biotechnology, Bijing, China). Protein levels were determined using the BCA assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Western blot analysis was performed by our previous method (Liu et al., 2012, 2013a,b).
2.9.
Statistic analysis
All statistical analysis were performed using the SPSS software, version 11.5. One-way analysis of variance (ANOVA) followed by Tukey’s test was used to compare the results from different treatments. Student’s t-test was used for comparisons between groups. Data were considered to have statistical differences at P ≤ 0.05.
3.
Fig. 1 – Effect of UA on CCl4 -induced changes in hepatic functional markers. All values are expressed as mean ± S.E.M. (n = 7). ##P < 0.01, compared with the control group; **P < 0.01, vs. CCl4 -treated group. determine whether CCl4 can induce the liver damage, we measured the activities of serum ALT and AST (Fig. 1). In CCl4 -treated mice, the activities of serum ALT and AST significantly increased by 402% and 305% as compared with these of the control group, respectively [FALT (3,24) = 43.306, P < 0.01; FAST (3,24) = 39.729, P < 0.01; CCl4 group vs. control group]. Interestingly, the treatment with low and high dose of UA decreased the activities of ALT (by 30% and 62%0 and AST (by 34% and 64%) as compared with these of the CCl4 -treated mice, respectively (P < 0.01) (Fig. 1).
3.2. UA alleviated CCl4 -induced histology changes in the liver Liver histological study was used to determine the protective effect of UA on CCl4 -induced injury in livers of mice. As shown in Fig. 2, CCl4 treatment caused several visible histology liver changes, including inflammatory cellular infiltrations and hepatic cell necrosis (Fig. 2B). Whereas, UA treatment significantly alleviated the CCl4 -induced damage in livers of mice. This histopathological analysis is in agreement with the observed results in the serum diagnostic indicators.
Results
3.1. UA protects against CCl4 -induced liver dysfunction The activities of aminotransferase were considered to be serum biochemical markers of liver damage. In order to
3.3. liver
UA inhibited CCl4 -induced oxidative stress in the
As shown in Fig. 3, CCl4 treatment markedly increased hepatic ROS and TBARS levels by 133% and 71% as compared with those of the control group, respectively [FROS (3,24) = 32.357,
978
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
Fig. 2 – Morphological and histological evaluation of liver in mice. (A) The vehicle control mice; (B) CCl4 -treated mice; (C) CCl4 -treated mice fed with UA (25 mg/kg); (D) CCl4 -treated mice fed with UA (50 mg/kg). The black arrow indicates infiltrating leukocytes. The green arrow indicates hepatic cell necrosis. Original magnification, 20 × 10.
P < 0.01; FTBARS (3,24) = 27.131, P < 0.01; CCl4 group vs. control group]. Interestingly, treatment with low and high dose of UA in CCl4 -treated mice significantly decreased hepatic ROS (by 21% and 45%) and TBARS (by 15% and 27%), respectively (P < 0.01) (Fig. 3). As shown in Fig. 2C, the TAC level in CCl4 -treated mice was markedly decreased by 31% as compared with that of the control [F(3,24) = 22.736, P < 0.01]. However, treatment with low and high dose of UA in CCl4 -treated mice significantly increased the hepatic TAC levels by 27% and 33%, respectively (P < 0.01).
3.4. UA inhibited CCl4 -induced expression of cytochrome P450 2E1 (CYP2E1) in livers In order to determine whether UA can inhibit CCl4 -inducd the liver damage, we examined the expression of CYP2E1 in livers. As shown in Fig. 4, CCl4 treatment caused significant increase in the expression levels of CYP2E1 by 686% in mouse livers as compared with that of the control group [F(3,24) = 46.352, P < 0.01]. Interestingly, treatment with low and high dose of UA in CCl4 -treated mice significantly decreased the expression levels of CYP2E1 by 49% and 70% in the livers, respectively (P < 0.01).
3.5. UA inhibited the CCl4 -induced expression of inflammatory cytokines in livers The expression levels of IL-6, COX-2 and TNF-␣ were extensively studied in terms of their involvement in the acute
and chronic inflammation (Shin et al., 2013; Weber et al., 2003; Wang et al., 2013). As shown in Fig. 5, CCl4 treatment caused significant increase in the expression levels of TNF-␣, IL-6 and COX-2 by 772%, 672% and 920% in mouse livers as compared with those of the control group, respectively [FTNF-␣ (3,24) = 42.332, P < 0.01; FIL-6 (3,24) = 38.642, P < 0.01; FCOX-2 (3,24) = 44.263, P < 0.01, CCl4 group vs. control group]. However, treatment with low and high dose of UA in CCl4 treated mice significantly decreased the expression levels of TNF-␣ (by 49% and 81%), IL-6 (by 72% and 76%) and COX-2 (by 79% and 84%), respectively (P < 0.01).
3.6. UA-mediated protective action involves MAPK and NF-B pathway The accumulated evidence showed that the activation of MAPK and NF-B was tightly associated with inflammation. To further investigate the molecular mechanism of inflammation in mouse liver, we measured the expression levels of ERK1/2, JNK1/2, p38 and NF-B p65 (Fig. 6). The phosphorylation of JNK, p38 and ERK was increased by 639%, 504% and 841% in mice treated with CCl4 alone as compared with the control group [FJNK (3,24) = 39.412, P < 0.01; Fp38 (3,24) = 37.215, P < 0.01; FERK (3,24) = 17.384, P < 0.01, CCl4 group vs. control group]. Interestingly, treatment with low and high dose of UA in CCl4 -treated mice significantly decreased the expression levels of phosphorylated JNK (by 39% and 73%) and phosphorylated p38 (by 56% and 61%), respectively (P < 0.01) (Fig. 6C). Treatment with high dose (50 mg/(kg day)) of UA also
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
979
Fig. 4 – Western blot analysis of the expression of CYP2E1 in liver. -Actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Values are averages from seven independent experiments. Each value is expressed as mean ± S.E.M. **P < 0.01, compared with the control group; ##P < 0.01, vs. CCl4 -treated group.
of UA in CCl4 -treated mice strongly inhibited the translocation of NF-B p65 from the cytosol to the nuclear fraction by 40% and 67% (P < 0.01).
4.
Fig. 3 – Effect of UA on the oxidative stress markers in CCl4 -treated mouse liver. (A) Level of ROS; (B) Level of TBARS; (E) The total antioxidant capacity (TAC). Each value is expressed as mean ± S.E.M. (n = 7). ##P < 0.01, compared with the control group; **P < 0.01, vs. CCl4 -treated group.
decreased phosphorylated ERK as compared with the CCl4 treated group (P < 0.01), while UA (25 mg/(kg day)) did not reach the effect (P > 0.05). As shown in Fig. 6, NF-B p65 levels in nuclear fractions were significantly increased in CCl4 -treated mice as compared with vehicle controls. Accordingly, NF-B p65 levels in cytoplasma fractions were significantly reduced by 704% in CCl4 -treated mice as compared with the control group [F(3,24) = 42.432, P < 0.01]. Treatment with low and high dose
Discussion
UA is an isoflavone abundant in many kinds of plants. Recent evidences have supported the beneficial effects of UA in a variety of human diseases (Checker et al., 2012; Shanmugam et al., 2013). In this study, we showed that UA possessed the protective effects on CCl4 -induced liver injury mice. These results indicated that UA inhibited liver CCl4 -induced inflammation probably through regulating MAPK/NF-B pathway. The liver is the main site of drug and toxicant metabolism. In most cases, the metabolic process is accomplished without injury to the liver itself, whereas many inorganic or organic compounds are toxic that can cause liver injury (Lv et al., 2012). Many studies have demonstrated CCl4 can induce dysfunction and histopathologic changes in liver (Kim et al., 2011; Wang et al., 2013). The current study showed that CCl4 treatment caused the increase in serum levels of aminotransferases (ALT and AST) and histopathologic alterations, such as leukocyte infiltration and hepatic cell necrosis (Figs. 1 and 2). In this study, the results showed that UA markedly decreased the levels of aminotransferases in the CCl4 -treated group (Fig. 1) and reduced the CCl4 -induced histopathologic changes in livers (Fig. 2). These results suggest that UA could protect mouse liver against CCl4 -induced damage. CCl4 is metabolized to produce highly toxic trichloromethyl free radical (• CCl3 ) and/or trichloro methyl peroxyl (• OOCCl3 ) free radicals by cytochrome P450 enzyme and causes damage to hepatocytes within the body (Singh et al., 2008; Shim et al., 2010). Both trichloromethyl and its peroxy radical are capable of binding to proteins or lipids, or of abstracting a hydrogen atom from an unsaturated lipid, initiating lipid peroxidation
980
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
Fig. 5 – Western blot analysis of inflammatory cytokine levels in liver. -Actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Values are averages from seven independent experiments. Each value is expressed as mean ± S.E.M. **P < 0.01, compared with the control group; ##P < 0.01, vs. CCl4 -treated group.
and liver damage. Reactive oxygen species (ROS) generated by metabolic intermediates of xenobiotics via induction of CYP450 families as well as activated inflammatory cells through NADPH oxidases promote the accumulation of lipidderived oxidation products that cause liver injury, resulting in cell necrosis (Weber et al., 2003). These free radicals combine with polyunsaturated fatty acids of hepatic and testicular cell membranes, cause elevation of thiobarbituric acid reactive substances (TBARS) concentration with subsequent necrosis and increase lysosomal enzymes activities (Weber et al., 2003). In the present study, we revealed CCl4 treatment induced over-production of ROS, TBARS and decreased the total antioxidant capacity (TAC) in mice, which could lead to liver oxidative damage. However, UA markedly inhibit CCl4 -induced oxidative stress in livers of mice (Fig. 3), which may be due to the powerful antioxidant and free radical scavenging activities (Xiang et al., 2012; Saravanan et al., 2006; Gayathri et al., 2009). CYP2E1 is an important hepatic P450 enzyme, which plays important role in CCl4 metabolism and ROS production (Shim et al., 2010; Ebaid et al., 2013). Our study demonstrated that CCl4 increased CYP2E1 expression in mouse liver. Interestingly, treatment with UA showed dosedependent inhibition in this elevation (Fig. 4). Consistent with this result, the experiments showed that UA decreased the CCl4 -induced production of ROS and TBARS (Fig. 3). Our finding suggests that UA could at least partly attenuate CCl4 -induced hepatotoxicity by inhibiting oxidative stress. Accumulating evidences have revealed that CCl4 and excessive ROS induced by CCl4 can stimulate circulating monocytes and tissue macrophages, which lead to the synthesis and release of a variety of proinflammatory cytokines (Wang et al., 2013; Tu et al., 2012; Shim et al., 2010). TNF-␣ and IL-1 are among the best characterized early response
proinflammatory cytokines. Many studies also demonstrated that IL-1 and TNF-␣ play a key role in the development and maintenance of inflammatory and those cytokines elevation is associated with many liver diseases (Shin et al., 2013; Weber et al., 2003; Shim et al., 2010). Cyclooxygenase-2 (COX-2) is a crucial enzyme in the biosynthesis of prostaglandins. COX-2 is inducible by a variety of pro-inflammatory stimuli. In the liver, COX-2 and prostaglandins production has been implicated in inflammation, matrix remodeling, fibrosis progress and development of hepatocellular carcinoma (Martinez et al., 2003; Ma et al., 2011). The present study showed that CCl4 treatment significantly up-regulated the expression of IL-1, TNF-␣ and COX-2 expression in the mouse liver. However, UA markedly inhibited this up-regulation (Fig. 5). These results suggested that UA could alleviate liver injury caused by CCl4 through suppressing inflammatory response. Moreover, leucocytes and macrophages respond non-specifically to foreign substances in immune system. Once tissue damage occurs, leucocytes rapidly migrate to sites of injury initiating an inflammatory response. Therefore leukocyte infiltration was considered as a marker of inflammatory response (Ma et al., 2011). As previously described, we observed that CCl4 markedly induced leukocyte infiltration in the mouse livers. Whereas, UA efficaciously suppressed the leukocyte infiltration induced by CCl4 liver injury in mice (Fig. 2). So the results of histological analysis also substantiated that UA could suppress inflammatory response induced by CCl4 in mouse livers. The MAP kinase family plays important roles in regulation of cell proliferation and cell death in response to various cellular stresses. To date, at least three major MAPK cascades have been described that involve the activation of the extracellularreceptor kinases (ERK), the c-Jun N-terminal kinases/stressactivated protein kinases (JNK/SAPK) and the p38 MAPKinases.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
981
Fig. 6 – Western blot analysis of the proteins which in association with the MAPK and NF-B pathway in liver. (A) Effect of UA on the expression of the proteins in association with the MAPK pathway in mouse liver; (B) effect of UA on the expression of the proteins in association with the NF-B pathway in mouse liver; -actin was probed as an internal control in relative density analysis. The relative density is expressed as the ratio (Phospho-p38MAPK/Total-p38MAPK, PhosphoJNK/Total-JNK and Phospho-ERK/Total-ERK). The relative density of the NF-B p65 protein bands is expressed as the ratio in cytosol and nucleus. The vehicle control is set as 1.0. Values are averages from seven independent experiments. Each value is expressed as mean ± S.E.M. ##P < 0.01, compared with the control group; **P < 0.01, vs. CCl4 -treated group.
The ERK cascade is mostly responsive to mitogenic and differentiation stimuli, where as the JNK and p38 MAPK pathways are preferentially activated by pro-inflammatory cytokines and extracellular stress (Ikeda et al., 2005). One type of stress that induces potential activation of MAPK pathways is the oxidative stress caused by ROS. ROS activated the MAPK signaling pathways, which further activates several inflammatory cytokines (Ikeda et al., 2005). Our results also showed that CCl4 exposure induced activation of MAPKs in mouse liver (Fig. 6). In vivo and in vitro studies have demonstrated that UA can protect cell against inflammatory responses by MAPK signaling pathway (Ikeda et al., 2007; Wu et al., 2011; Checker et al., 2012). In the present study, our data showed that UA inhibited CCl4 -induced JNK, ERK and p38 phosphorylation (particular JNK and p38) and verified the proinflammatory nature of the MAPK pathway in mouse liver (Fig. 6). It demonstrates, further, an additional anti-inflammatory property of UA. Based on these results, we ensure that MAPK pathways have important roles in inflammatory processes in
CCl4 -stimulated mouse liver. UA may exert its anti-inflammatory effect on rat liver by inhibiting the phosphorylation of MAPKs. NF-B is a nuclear transcription factor that regulates expression of a large number of genes that are critical for the regulation of apoptosis, viral replication, tumorigenesis, inflammation, and various autoimmune diseases (Ma et al., 2011). In liver, NF-B is activated by a variety of cytokines and stimuli. CCl4 can activate numerous intracellular signaling pathways (such as MAPK, TGF-1), which may converge on NFB (Ma et al., 2011; Wang et al., 2013). Excessive ROS induced by CCl4 intermediates can also function as signaling messengers to NF-B, and ultimately lead to increased cytokine production, such as COX-2, IL-6 and TNF-␣ (Li et al., 2013). Previous studies had demonstrated that UA can protect cell against inflammatory responses by inhibiting NF-B activity (Checker et al., 2012; Xiang et al., 2012). In the present study, we found that the translocation of activated NF-B to the nucleus was markedly increased in mouse liver under CCl4 treatment.
982
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
However, this NF-B activation was inhibited by UA, implying that inhibition of NF-B activation was tightly involved in the anti-inflammatory action of UA (Fig. 6). So the results in vitro also substantiated that UA could suppress inflammatory response through by MAPK and NFB pathway. In conclusion, this study demonstrates for the first time that UA has potent protective effects against CCl4 -induced inflammation through inhibiting TNF-␣, IL-1 and COX-2 protein expression, at least in part, by suppressing the MAPKs and NF-B activation. Here we demonstrated that UA administration attenuated CCl4 -induced hepatic dysfunction and histopathologic changes. UA attenuated CCl4 -induced oxidative damage by inhibiting ROS generation in liver. UA seems to be potent hepatoprotective drug and its use in maintaining a healthy liver and preventing toxic liver damage deserves consideration and further examination.
Conflict of interest statement The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgments This work is supported from Startup Project of Doctor scientific research by Sichuan University of Science and Engineering (2013RC14).
references
Checker, R., Sandur, S.K., Sharma, D., Patwardhan, R.S., Jayakumar, S., Kohli, V., Sethi, G., Aggarwal, B.B., Sainis, K.B., 2012. Potent anti-inflammatory activity of ursolic acid, a triterpenoid antioxidant, is mediated through suppression of NF-kB, AP-1 and NF-AT. Plos One 7, e31318. Ebaid, H., Bashandy, S.A., Alhazza, I.M., Rady, A., EI-Shehry, S., 2013. Folic acid and melatonin ameliorate carbon tetrachloride-induced hepatic injury, oxidative stress and inflammation in rats. Nutr. Metab. 10, 20–31. Gayathri, R., Priya, D.K., Gunassekaran, G.R., Sakthisekaran, D., 2009. Ursolic acid attenuates oxidative stress-mediated hepatocellular carcinoma induction by diethylnitrosamine in male Wistar rats. Asian Pac. J. Cancer Prev. 10, 933–938. Hamdy, N., El-Demerdash, E., 2012. New therapeutic aspect for carvedilol: anti fibrotic effects of carvedilol in chronic carbon tetrachloride-induced liver damage. Toxicol. Appl. Pharmacol. 261, 292–299. Iida, C., Fujii, K., Koga, E., Washino, Y., Kitamura, Y., Ichi, I., Abe, K., Matsura, T., Kojo, S., 2009. Effect of ␣-tocopherol on carbon tetrachloride intoxication in the rat liver. Arch. Toxicol. 83, 477–483. Ikeda, Y., Murakami, A., Ohigashi, H., 2005. Ursolic acid promotes the release of macrophag emigration inhibitory factor via ERK2 activation in resting mouse macrophages. Biochem. Pharmacol. 70, 1497–1505. Ikeda, Y., Murakami, A., Fujimura, Y., Tachibana, H., Yamada, K., Masuda, D., Hirano, K., Yamashita, S., Ohigashi, H., 2007.
Aggregated ursolic acid, a natural triterpenoid, induces IL-1 release from murine peritoneal macrophages: role of CD36. J. Immunol. 178, 4854–4864. Kim, H.Y., Park, J., Lee, K.H., Lee, D.U., Kwak, J.H., Kim, Y.S., Lee, S.M., 2011. Ferulic acid protects against carbon tetrachlorideinduced liver injury in mice. Toxicology 282, 104–111. Laskin, D.L., Laskin, J.D., 2001. Role of macrophages and inflammatory mediators in chemically induced toxicity. Toxicology 160, 111–118. Li, R., Xu, L., Liang, T., Li, Y., Zhang, S., Duan, X., 2013. Puerarin mediates hepatoprotection against CCl4 -induced hepatic fibrosis rats through attenuation of inflammation response and amelioration of metabolic function. Food Chem. Toxicol. 52, 69–75. Liu, J., Liu, Y., Mao, Q., Klaassen, C.D., 1994. The effects of 10 triterpenoid compounds on experimental liver injury in mice. Fundam. Appl. Toxicol. 22, 34–40. Liu, C.M., Ma, J.Q., Sun, Y.Z., 2012. Puerarin protects rat kidney from lead-induced apoptosis by modulating the PI3K/Akt/eNOS pathway. Toxicol. Appl. Pharmacol. 258, 330–342. Liu, C.M., Zheng, G.H., Ming, Q.L., Chao, C., Sun, J.M., 2013a. Sesamin protects mouse liver against nickel-induced oxidative DNA damage and apoptosis by the PI3K-Akt pathway. J. Agric. Food Chem. 61, 1146–1154. Liu, C.M., Zheng, G.H., Ming, Q.L., Sun, J.M., Cheng, C., 2013b. Protective effect of quercetin on lead-induced oxidative stress and endoplasmic reticulum stress in rat liver via the IRE1/JNK and PI3K/Akt pathway. Free Radic. Res. 47, 192–201. Lv, Y.Y., Jin, Y., Han, G.Z., Liu, Y.X., Wu, T., Liu, P., Zhou, Q., Liu, K.X., Sun, H.J., 2012. Ursolic acid suppresses IL-6 induced C-reactive protein expression in HepG2 and protects HUVECs from injury induced by CRP. Eur. J. Pharm. Sci. 45, 190–194. Ma, J.Q., Liu, C.M., Qin, Z.H., Jiang, J.H., Sun, Y.Z., 2011. Ganoderma applanatum terpenes protect mouse liver against benzo(␣)pyren-induced oxidative stress and inflammation. Environ. Toxicol. Pharmacol. 31, 460–468. Martin-Aragon, S., de las Heras, B., Sanchez-Reus, M.I., Benedi, J., 2001. Pharmacological modification of endogenous antioxidant enzymes by ursolic acid on tetrachloride-induced liver damage in rats and primary cultures of rat hepatocytes. Exp. Toxicol. Pathol. 53, 199–206. Martinez, O.N., Ricote, G.C., Monzon, C.G., 2003. Role of cyclooxygenase-2 in the pathogenesis of chronic liver diseases. Med. Clin. 121, 743–748. Onkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Saravanan, R., Viswanathan, P., Pugalendi, K.V., 2006. Protective effect of ursolic acid on ethanol-mediated experimental liver damage in rats. Life Sci. 78, 713–718. Shanmugam, M.K., Dai, X., Kumar, A.P., Tan, B.K.H., Sethi, G., Bishayee, A., 2013. Ursolic acid in cancer prevention and treatment: molecular targets, pharmacokinetics and clinical studies. Biochem. Pharmacol. 85, 1579–1587. Shim, J.Y., Kim, M.H., Kim, H.D., Ahn, J.Y., Yun, Y.S., Song, J.Y., 2010. Protective action of the immunomodulator ginsan against carbon tetrachloride-induced liver injury via control of oxidative stress and the in flammatory response. Toxicol. Appl. Pharmacol. 242, 318–325. Shin, D.S., Kim, K.W., Chung, H.Y., Yoon, S., Moon, J.O., 2013. Effect of sinapic acid against carbon tetrachloride-induced acute hepatic injury in rats. Arch. Pharm. Res. 36, 626–633. Shinomol, G.K., Muralidhara, 2007. Differential induction of oxidative impairments in brain regions of male mice following subchronic consumption of Khesari dhal (Lathyrus sativus) and detoxified Khesari dhal. Neurotoxicology 28, 798–806. Singh, N., Kamath, V., Narasimhamurthy, K., Rajini, P.S., 2008. Protective effect of potato peel extract against carbon
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
tetrachloride-induced liver injury in rats. Environ. Toxicol. Pharmacol. 26, 241–246. Tacke, F., Luedde, T., Trautwein, C., 2009. Inflammatory pathways in liver homeostasis and liver injury. Clin. Rev. Allergy Immunol. 36, 4–12. Tseng, C.K., Lin, C.K., Chang, H.W., Wu, Y.H., Yen, F.L., Chang, F.R., Chen, W.C., Yeh, C.C., Lee, J.C., 2014. Aqueous extract of Gracilaria tenuistipitata suppresses LPS-Induced NF-kB and MAPK Activation in RAW 264.7 and rat peritoneal macrophages and exerts hepatoprotective effects on carbon tetrachloride treated rat. PLoS One 9, e86557. Tu, C.T., Yao, Q.Y., Xu, B.L., Wang, J.Y., Zhou, C.H., Zhang, S.C., 2012. Protective effects of curcumin against hepatic fibrosis induced by carbon tetrachloride: modulation of high-mobility group box 1. Toll-like receptor 4 and 2 expression. Food Chem. Toxicol. 50, 3343–3351.
983
Wang, J.Q., Chen, X., Zhang, C., Tao, L., Zhang, Z.H., Liu, X.Q., Xu, Y.B., Wang, H., Li, J., Xu, D.X., 2013. Phenylbutyric acid protects against carbon tetrachloride-induced hepatic fibrogenesis in mice. Toxicol. Appl. Pharmacol. 266, 307–316. Weber, L.W., Boll, M., Stampfl, A., 2003. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit. Rev. Toxicol. 33, 105–136. Wu, H.Y., Chang, C.I., Lin, B.W., Yu, F.L., Lin, P.Y., Hsu, J.L., Yen, C.H., Liao, M.H., Shih, W.L., 2011. Suppression of hepatitis B virus X protein-mediated tumorigenic effects by ursolic acid. J. Agric. Food Chem. 59, 1713–1722. Xiang, M., Wang, J., Zhang, Y., Ling, J., Xu, X., 2012. Attenuation of aortic injury by ursolic acid through RAGE-Nox-NFB pathway in streptozocin-induced diabetic rats. Arch. Pharm. Res. 35, 877–886.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Ursolic acid protects mouse liver against CCl4 -induced oxidative stress and inflammation by the MAPK/NF-B pathway Jie-Qiong Ma a,∗ , Jie Ding a , Li Zhang a , Chan-Min Liu b a
School of Chemistry and Pharmaceutical, Sichuan University of Science and Engineering, 643000 Zigong City, Sichuan Province, PR China b School of Life Science, Jiangsu Normal University, No. 101, Shanghai Road, Tangshan New Area, Xuzhou City 221116, Jiangsu Province, PR China
a r t i c l e
i n f o
a b s t r a c t
Article history:
Ursolic acid (UA), a natural pentacyclic triterpenoid, has been reported to have many benefits
Received 7 January 2014
and medicinal properties. However, its protective effects against carbon tetrachloride (CCl4 )
Received in revised form
induced hepatotoxicity have not been clarified. The aim of the present study was to investi-
12 March 2014
gate the effects of UA on oxidative stress and inflammation in liver of CCl4 treated mice. Male
Accepted 16 March 2014
ICR mice were injected with CCl4 with or without UA co-administration (25 and 50 mg/kg
Available online 22 March 2014
intragastrically once daily) for one week. Our data showed that UA significantly prevented
Keywords:
cators of liver damage (serum aminotransferase activities) and histopathological analysis.
Ursolic acid
Moreover, CCl4 -induced profound elevation of reactive oxygen species (ROS) production and
CCl4 -induced hepatotoxicity in a dose-dependent manner, indicated by both diagnostic indi-
CCl4
oxidative stress, as evidenced by increasing of lipid peroxidation level and depleting of the
MAPKs
total antioxidant capacity (TAC) level in liver, were suppressed by treatment with UA. Fur-
Oxidative stress
thermore, western blot analysis showed that UA significantly decreased CYP2E1 expression
NF-B
levels and production of pro-inflammatory markers including TNF-␣, IL-1 and COX-2 in
Hepatic inflammation
CCl4 -treated mouse liver. In exploring the underlying mechanisms of UA action, we found that UA decreased the activation of mitogen-activated protein kinases (JNK, p38 MAPK, ERK), which in turn inactivated the immunoregulatory transcription factor nuclear factor kappa B (NF-B) in liver of CCl4 treated mice. In conclusion, these results suggested that the inhibition of CCl4 -induced inflammation by UA is due at least in part to its anti-oxidant activity and its ability to modulate the MAPK and NF-B signaling pathway. © 2014 Elsevier B.V. All rights reserved.
Abbreviations: COX-2, cyclooxygenase-2; CYP2E1, cytochrome P450 2E1; ERK, the extracellular-receptor kinases; IL-1, interleukin1beta; JNK, the c-Jun N-terminal kinases; NF-B, nuclear factor-B; MAPKs, mitogen-activated protein kinases; UA, ursolic acid; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substances; TNF-␣, tumor necrosis factor-alpha. ∗ Corresponding author. Tel.: +86 013700950121; fax: +86 516 83500171. E-mail addresses: [email protected], [email protected] (J.-Q. Ma). http://dx.doi.org/10.1016/j.etap.2014.03.011 1382-6689/© 2014 Elsevier B.V. All rights reserved.
976
1.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
Introduction
Liver diseases with severe hepatocyte damage caused by viral infection, non-alcoholic steatohepatitis or other hepatotoxic agent are highly associated with acute or chronic inflammation (Tacke et al., 2009; Tseng et al., 2014). Hepatic inflammation is regarded as a hallmark of early stage fibrosis, which can progress to extensive fibrosis and cirrhosis. Damage associated with hepatic inflammation is mediated by the proinflammatory cytokines tumor necrosis factor-alpha (TNF␣), interleukin-1 (IL-1) and cyclooxygenase-2 (COX-2) (Shin et al., 2013). Carbon tetrachloride (CCl4 ) is a well-known compound for the production of chemical hepatic injury involving in production of inflammatory cytokines and recruitment of inflammatory cells, leading to liver architectural damage and dysfunction (Weber et al., 2003; Ebaid et al., 2013; Hamdy and El-Demerdash, 2012). The chronic liver damage induced by CCl4 in rats produces liver inflammatory and biochemical patterns that resemble rat liver cirrhosis (Laskin and Laskin, 2001; Hamdy and El-Demerdash, 2012; Li et al., 2013). In addition, many researches have suggested that the production of several proinflammatory molecules is associated with the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-B). Both MAPKs and NF-B can be stimulated by CCl4 and are tightly involved in the pathological conditions in the liver (Iida et al., 2009; Kim et al., 2011). Ursolic acid (UA: 3-hydroxy-urs-12-en-28-oic acid), a natural pentacyclic triterpenoid, has been found in various plants including apples, basil, cranberries, peppermint, rosemary, oregano and prunes (Checker et al., 2012; Lv et al., 2012). Many triterpenoids had been used for medicinal purposes for a variety of clinical diseases in many Asian countries (Ikeda et al., 2007). UA has been reported to possess many biological activities, including antioxidant, anti-inflammatory, trypanocidal, antirheumatic, antiviral and antitumoral properties (Ikeda et al., 2007; Wu et al., 2011). UA has also been shown to target multiple proinflammatory transcription factors, cell cycle proteins, growth factors, kinases, cytokines, chemokines, adhesion molecules, and inflammatory enzymes (Shanmugam et al., 2013). Previous researches showed that UA attenuate CCl4 -induced hepatotoxicity by inhibiting oxidative stress (Liu et al., 1994; Martin-Aragon et al., 2001). However, the molecular mechanisms of CCl4 -induced liver injury and anti-inflammatory effects of UA are not yet completely understood. In the present study, we hypothesized that UA can inhibit CCl4 -induced inflammation. We, for the first time, determine whether UA can protect mouse liver from CCl4 induced inflammatory response by modulating the MAPK and NF-B pathway.
2.
Materials and methods
2.1.
Chemicals and reagents
UA and CCl4 were obtained from Sigma Chemical Co. (St. Louis, MO, USA); IL-1 antibody, COX-2 antibody, and NFB p65 antibody are from Santa Cruz Biotechnology (Santa
Cruz, CA, USA); JNK (total) antibody, ERK1/2 (total) antibody, p38 (total) antibody, phospho-JNK antibody, phospho-ERK1/2 antibody, and phospho-p38 antibody were obtained from Cell Signaling Technology (Beverly, MA, USA); CYP2E1 antibody were obtained from Proteintech Group (Proteintech Group, INC. USA); aminotransferase activities in serum assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and Jingmei Biotech ltd. (Shenzhen, China); protein concentration were measured using the bicinchoninic acid (BCA) assay kit from Pierce Biotechnology, Inc. (Rockford, IL, USA). All other reagents unless indicated were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
2.2.
Animals and treatment
Male ICR mice (20–25 g) were obtained from the Branch of National Breeder Center of Rodents (Beijing) and kept in an environmentally controlled room (23 ± 2 ◦ C, 55 ± 10% humidity) with a 12-h light/dark cycle and allowed free access to food and water. Liver inflammation was induced by intraperitoneal (i.p.) injection of 2 ml of CCl4 in olive oil (1:1, v/v) per kg body weight twice weekly for up to 1 week (Tu et al., 2012). Fifty mice were randomly divided into four groups (10–15 mice/group). Mice in Group 1 were given twice weekly injections of olive oil (vehicle control); mice in Group 2 were injected with CCl4 and received water containing 0.1% Tween 80 by oral gavage; mice in Group 3 were injected with CCl4 , as in Group 2, and received UA in distilled water containing 0.1% Tween 80 at a dose of 25 mg/(kg day) by oral gavage; mice in Group 4 were injected with CCl4 , as in Group 2, and received UA in distilled water containing 0.1% Tween 80 at a dose of 50 mg/(kg day) by oral gavage (Xiang et al., 2012). At the end of treatment, seven mice in each group were used for the biochemical analysis; the others were used for histological evaluations. Mice were sacrificed and about 1 ml of blood samples were drawn by cardiac puncture with heparined tubes. The plasma was collected after centrifugation at 1200 × g for 10 min and stored at −70 ◦ C freezer for further analysis. The liver tissues were immediately collected for experiments and placed in ice-cold 0.9% NaCl solution, perfused with the physiological saline solution to remove blood cells, blotted on filter paper. And then the removed liver was quickly collected for experiments or stored at −70 ◦ C for later use. This research was conducted in accordance with the Chinese laws and NIH publication on the use and care of laboratory animals. Relevant university committees for animal experiments approved these experiments.
2.3. Assay of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity The activities of ALT and AST in serum were estimated spectrophotometrically using commercial diagnostic kits (Jiancheng Institute of Biotechnology, Nanjing, China) (Liu et al., 2013a).
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
2.4.
977
Histological evaluations
The histological changes of liver were evaluated by our previous method (Liu et al., 2012).
2.5.
Assay of ROS level
ROS was measured as described previously, based on the oxidation of 2 7 -dichlorodihy drofluorescein diacetate to 2 7 dichloro-fluorescein (Shinomol and Muralidhara, 2007; Liu et al., 2013b).
2.6. Assay of thiobarbituric acid reactive substances (TBARS) levels in the liver Estimation of lipid peroxidation was performed by previous method (Onkawa et al., 1979). A standard calibration curve was prepared by using 1–10 nM of 1,1,3,3-tetra methoxy propane. The concentration was expressed in terms of nanomoles of TBARS per mg of protein.
2.7.
Assay of the total antioxidant capacity (TAC)
The total antioxidant capacity (TAC) in the liver was assayed with a commercially available assay kit (Jiancheng Biochemical, Inc., Nanjing, China) (Liu et al., 2013b). This method is based on the reduction of iron (III) in acidic medium by intracellular antioxidants. The liberated iron (II) reacts with 1,10-phenanthroline to form a colored complex, which is measured at 520 nm. The TAC of the samples was measured according to the manufacturer’s protocol. One unit of TAC was defined as the capability of increasing 0.01 optical densities (OD520 ) units per mg protein per min at 37 ◦ C.
2.8.
Western blot analysis
Nuclear and cytoplasmic extracts for western blotting were obtained by using a nuclear/cytoplasmic isolation kit (Beyotime Institute of Biotechnology, Bijing, China). Protein levels were determined using the BCA assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Western blot analysis was performed by our previous method (Liu et al., 2012, 2013a,b).
2.9.
Statistic analysis
All statistical analysis were performed using the SPSS software, version 11.5. One-way analysis of variance (ANOVA) followed by Tukey’s test was used to compare the results from different treatments. Student’s t-test was used for comparisons between groups. Data were considered to have statistical differences at P ≤ 0.05.
3.
Fig. 1 – Effect of UA on CCl4 -induced changes in hepatic functional markers. All values are expressed as mean ± S.E.M. (n = 7). ##P < 0.01, compared with the control group; **P < 0.01, vs. CCl4 -treated group. determine whether CCl4 can induce the liver damage, we measured the activities of serum ALT and AST (Fig. 1). In CCl4 -treated mice, the activities of serum ALT and AST significantly increased by 402% and 305% as compared with these of the control group, respectively [FALT (3,24) = 43.306, P < 0.01; FAST (3,24) = 39.729, P < 0.01; CCl4 group vs. control group]. Interestingly, the treatment with low and high dose of UA decreased the activities of ALT (by 30% and 62%0 and AST (by 34% and 64%) as compared with these of the CCl4 -treated mice, respectively (P < 0.01) (Fig. 1).
3.2. UA alleviated CCl4 -induced histology changes in the liver Liver histological study was used to determine the protective effect of UA on CCl4 -induced injury in livers of mice. As shown in Fig. 2, CCl4 treatment caused several visible histology liver changes, including inflammatory cellular infiltrations and hepatic cell necrosis (Fig. 2B). Whereas, UA treatment significantly alleviated the CCl4 -induced damage in livers of mice. This histopathological analysis is in agreement with the observed results in the serum diagnostic indicators.
Results
3.1. UA protects against CCl4 -induced liver dysfunction The activities of aminotransferase were considered to be serum biochemical markers of liver damage. In order to
3.3. liver
UA inhibited CCl4 -induced oxidative stress in the
As shown in Fig. 3, CCl4 treatment markedly increased hepatic ROS and TBARS levels by 133% and 71% as compared with those of the control group, respectively [FROS (3,24) = 32.357,
978
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
Fig. 2 – Morphological and histological evaluation of liver in mice. (A) The vehicle control mice; (B) CCl4 -treated mice; (C) CCl4 -treated mice fed with UA (25 mg/kg); (D) CCl4 -treated mice fed with UA (50 mg/kg). The black arrow indicates infiltrating leukocytes. The green arrow indicates hepatic cell necrosis. Original magnification, 20 × 10.
P < 0.01; FTBARS (3,24) = 27.131, P < 0.01; CCl4 group vs. control group]. Interestingly, treatment with low and high dose of UA in CCl4 -treated mice significantly decreased hepatic ROS (by 21% and 45%) and TBARS (by 15% and 27%), respectively (P < 0.01) (Fig. 3). As shown in Fig. 2C, the TAC level in CCl4 -treated mice was markedly decreased by 31% as compared with that of the control [F(3,24) = 22.736, P < 0.01]. However, treatment with low and high dose of UA in CCl4 -treated mice significantly increased the hepatic TAC levels by 27% and 33%, respectively (P < 0.01).
3.4. UA inhibited CCl4 -induced expression of cytochrome P450 2E1 (CYP2E1) in livers In order to determine whether UA can inhibit CCl4 -inducd the liver damage, we examined the expression of CYP2E1 in livers. As shown in Fig. 4, CCl4 treatment caused significant increase in the expression levels of CYP2E1 by 686% in mouse livers as compared with that of the control group [F(3,24) = 46.352, P < 0.01]. Interestingly, treatment with low and high dose of UA in CCl4 -treated mice significantly decreased the expression levels of CYP2E1 by 49% and 70% in the livers, respectively (P < 0.01).
3.5. UA inhibited the CCl4 -induced expression of inflammatory cytokines in livers The expression levels of IL-6, COX-2 and TNF-␣ were extensively studied in terms of their involvement in the acute
and chronic inflammation (Shin et al., 2013; Weber et al., 2003; Wang et al., 2013). As shown in Fig. 5, CCl4 treatment caused significant increase in the expression levels of TNF-␣, IL-6 and COX-2 by 772%, 672% and 920% in mouse livers as compared with those of the control group, respectively [FTNF-␣ (3,24) = 42.332, P < 0.01; FIL-6 (3,24) = 38.642, P < 0.01; FCOX-2 (3,24) = 44.263, P < 0.01, CCl4 group vs. control group]. However, treatment with low and high dose of UA in CCl4 treated mice significantly decreased the expression levels of TNF-␣ (by 49% and 81%), IL-6 (by 72% and 76%) and COX-2 (by 79% and 84%), respectively (P < 0.01).
3.6. UA-mediated protective action involves MAPK and NF-B pathway The accumulated evidence showed that the activation of MAPK and NF-B was tightly associated with inflammation. To further investigate the molecular mechanism of inflammation in mouse liver, we measured the expression levels of ERK1/2, JNK1/2, p38 and NF-B p65 (Fig. 6). The phosphorylation of JNK, p38 and ERK was increased by 639%, 504% and 841% in mice treated with CCl4 alone as compared with the control group [FJNK (3,24) = 39.412, P < 0.01; Fp38 (3,24) = 37.215, P < 0.01; FERK (3,24) = 17.384, P < 0.01, CCl4 group vs. control group]. Interestingly, treatment with low and high dose of UA in CCl4 -treated mice significantly decreased the expression levels of phosphorylated JNK (by 39% and 73%) and phosphorylated p38 (by 56% and 61%), respectively (P < 0.01) (Fig. 6C). Treatment with high dose (50 mg/(kg day)) of UA also
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
979
Fig. 4 – Western blot analysis of the expression of CYP2E1 in liver. -Actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Values are averages from seven independent experiments. Each value is expressed as mean ± S.E.M. **P < 0.01, compared with the control group; ##P < 0.01, vs. CCl4 -treated group.
of UA in CCl4 -treated mice strongly inhibited the translocation of NF-B p65 from the cytosol to the nuclear fraction by 40% and 67% (P < 0.01).
4.
Fig. 3 – Effect of UA on the oxidative stress markers in CCl4 -treated mouse liver. (A) Level of ROS; (B) Level of TBARS; (E) The total antioxidant capacity (TAC). Each value is expressed as mean ± S.E.M. (n = 7). ##P < 0.01, compared with the control group; **P < 0.01, vs. CCl4 -treated group.
decreased phosphorylated ERK as compared with the CCl4 treated group (P < 0.01), while UA (25 mg/(kg day)) did not reach the effect (P > 0.05). As shown in Fig. 6, NF-B p65 levels in nuclear fractions were significantly increased in CCl4 -treated mice as compared with vehicle controls. Accordingly, NF-B p65 levels in cytoplasma fractions were significantly reduced by 704% in CCl4 -treated mice as compared with the control group [F(3,24) = 42.432, P < 0.01]. Treatment with low and high dose
Discussion
UA is an isoflavone abundant in many kinds of plants. Recent evidences have supported the beneficial effects of UA in a variety of human diseases (Checker et al., 2012; Shanmugam et al., 2013). In this study, we showed that UA possessed the protective effects on CCl4 -induced liver injury mice. These results indicated that UA inhibited liver CCl4 -induced inflammation probably through regulating MAPK/NF-B pathway. The liver is the main site of drug and toxicant metabolism. In most cases, the metabolic process is accomplished without injury to the liver itself, whereas many inorganic or organic compounds are toxic that can cause liver injury (Lv et al., 2012). Many studies have demonstrated CCl4 can induce dysfunction and histopathologic changes in liver (Kim et al., 2011; Wang et al., 2013). The current study showed that CCl4 treatment caused the increase in serum levels of aminotransferases (ALT and AST) and histopathologic alterations, such as leukocyte infiltration and hepatic cell necrosis (Figs. 1 and 2). In this study, the results showed that UA markedly decreased the levels of aminotransferases in the CCl4 -treated group (Fig. 1) and reduced the CCl4 -induced histopathologic changes in livers (Fig. 2). These results suggest that UA could protect mouse liver against CCl4 -induced damage. CCl4 is metabolized to produce highly toxic trichloromethyl free radical (• CCl3 ) and/or trichloro methyl peroxyl (• OOCCl3 ) free radicals by cytochrome P450 enzyme and causes damage to hepatocytes within the body (Singh et al., 2008; Shim et al., 2010). Both trichloromethyl and its peroxy radical are capable of binding to proteins or lipids, or of abstracting a hydrogen atom from an unsaturated lipid, initiating lipid peroxidation
980
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
Fig. 5 – Western blot analysis of inflammatory cytokine levels in liver. -Actin was probed as an internal control in relative density analysis. The vehicle control is set as 1.0. Values are averages from seven independent experiments. Each value is expressed as mean ± S.E.M. **P < 0.01, compared with the control group; ##P < 0.01, vs. CCl4 -treated group.
and liver damage. Reactive oxygen species (ROS) generated by metabolic intermediates of xenobiotics via induction of CYP450 families as well as activated inflammatory cells through NADPH oxidases promote the accumulation of lipidderived oxidation products that cause liver injury, resulting in cell necrosis (Weber et al., 2003). These free radicals combine with polyunsaturated fatty acids of hepatic and testicular cell membranes, cause elevation of thiobarbituric acid reactive substances (TBARS) concentration with subsequent necrosis and increase lysosomal enzymes activities (Weber et al., 2003). In the present study, we revealed CCl4 treatment induced over-production of ROS, TBARS and decreased the total antioxidant capacity (TAC) in mice, which could lead to liver oxidative damage. However, UA markedly inhibit CCl4 -induced oxidative stress in livers of mice (Fig. 3), which may be due to the powerful antioxidant and free radical scavenging activities (Xiang et al., 2012; Saravanan et al., 2006; Gayathri et al., 2009). CYP2E1 is an important hepatic P450 enzyme, which plays important role in CCl4 metabolism and ROS production (Shim et al., 2010; Ebaid et al., 2013). Our study demonstrated that CCl4 increased CYP2E1 expression in mouse liver. Interestingly, treatment with UA showed dosedependent inhibition in this elevation (Fig. 4). Consistent with this result, the experiments showed that UA decreased the CCl4 -induced production of ROS and TBARS (Fig. 3). Our finding suggests that UA could at least partly attenuate CCl4 -induced hepatotoxicity by inhibiting oxidative stress. Accumulating evidences have revealed that CCl4 and excessive ROS induced by CCl4 can stimulate circulating monocytes and tissue macrophages, which lead to the synthesis and release of a variety of proinflammatory cytokines (Wang et al., 2013; Tu et al., 2012; Shim et al., 2010). TNF-␣ and IL-1 are among the best characterized early response
proinflammatory cytokines. Many studies also demonstrated that IL-1 and TNF-␣ play a key role in the development and maintenance of inflammatory and those cytokines elevation is associated with many liver diseases (Shin et al., 2013; Weber et al., 2003; Shim et al., 2010). Cyclooxygenase-2 (COX-2) is a crucial enzyme in the biosynthesis of prostaglandins. COX-2 is inducible by a variety of pro-inflammatory stimuli. In the liver, COX-2 and prostaglandins production has been implicated in inflammation, matrix remodeling, fibrosis progress and development of hepatocellular carcinoma (Martinez et al., 2003; Ma et al., 2011). The present study showed that CCl4 treatment significantly up-regulated the expression of IL-1, TNF-␣ and COX-2 expression in the mouse liver. However, UA markedly inhibited this up-regulation (Fig. 5). These results suggested that UA could alleviate liver injury caused by CCl4 through suppressing inflammatory response. Moreover, leucocytes and macrophages respond non-specifically to foreign substances in immune system. Once tissue damage occurs, leucocytes rapidly migrate to sites of injury initiating an inflammatory response. Therefore leukocyte infiltration was considered as a marker of inflammatory response (Ma et al., 2011). As previously described, we observed that CCl4 markedly induced leukocyte infiltration in the mouse livers. Whereas, UA efficaciously suppressed the leukocyte infiltration induced by CCl4 liver injury in mice (Fig. 2). So the results of histological analysis also substantiated that UA could suppress inflammatory response induced by CCl4 in mouse livers. The MAP kinase family plays important roles in regulation of cell proliferation and cell death in response to various cellular stresses. To date, at least three major MAPK cascades have been described that involve the activation of the extracellularreceptor kinases (ERK), the c-Jun N-terminal kinases/stressactivated protein kinases (JNK/SAPK) and the p38 MAPKinases.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
981
Fig. 6 – Western blot analysis of the proteins which in association with the MAPK and NF-B pathway in liver. (A) Effect of UA on the expression of the proteins in association with the MAPK pathway in mouse liver; (B) effect of UA on the expression of the proteins in association with the NF-B pathway in mouse liver; -actin was probed as an internal control in relative density analysis. The relative density is expressed as the ratio (Phospho-p38MAPK/Total-p38MAPK, PhosphoJNK/Total-JNK and Phospho-ERK/Total-ERK). The relative density of the NF-B p65 protein bands is expressed as the ratio in cytosol and nucleus. The vehicle control is set as 1.0. Values are averages from seven independent experiments. Each value is expressed as mean ± S.E.M. ##P < 0.01, compared with the control group; **P < 0.01, vs. CCl4 -treated group.
The ERK cascade is mostly responsive to mitogenic and differentiation stimuli, where as the JNK and p38 MAPK pathways are preferentially activated by pro-inflammatory cytokines and extracellular stress (Ikeda et al., 2005). One type of stress that induces potential activation of MAPK pathways is the oxidative stress caused by ROS. ROS activated the MAPK signaling pathways, which further activates several inflammatory cytokines (Ikeda et al., 2005). Our results also showed that CCl4 exposure induced activation of MAPKs in mouse liver (Fig. 6). In vivo and in vitro studies have demonstrated that UA can protect cell against inflammatory responses by MAPK signaling pathway (Ikeda et al., 2007; Wu et al., 2011; Checker et al., 2012). In the present study, our data showed that UA inhibited CCl4 -induced JNK, ERK and p38 phosphorylation (particular JNK and p38) and verified the proinflammatory nature of the MAPK pathway in mouse liver (Fig. 6). It demonstrates, further, an additional anti-inflammatory property of UA. Based on these results, we ensure that MAPK pathways have important roles in inflammatory processes in
CCl4 -stimulated mouse liver. UA may exert its anti-inflammatory effect on rat liver by inhibiting the phosphorylation of MAPKs. NF-B is a nuclear transcription factor that regulates expression of a large number of genes that are critical for the regulation of apoptosis, viral replication, tumorigenesis, inflammation, and various autoimmune diseases (Ma et al., 2011). In liver, NF-B is activated by a variety of cytokines and stimuli. CCl4 can activate numerous intracellular signaling pathways (such as MAPK, TGF-1), which may converge on NFB (Ma et al., 2011; Wang et al., 2013). Excessive ROS induced by CCl4 intermediates can also function as signaling messengers to NF-B, and ultimately lead to increased cytokine production, such as COX-2, IL-6 and TNF-␣ (Li et al., 2013). Previous studies had demonstrated that UA can protect cell against inflammatory responses by inhibiting NF-B activity (Checker et al., 2012; Xiang et al., 2012). In the present study, we found that the translocation of activated NF-B to the nucleus was markedly increased in mouse liver under CCl4 treatment.
982
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
However, this NF-B activation was inhibited by UA, implying that inhibition of NF-B activation was tightly involved in the anti-inflammatory action of UA (Fig. 6). So the results in vitro also substantiated that UA could suppress inflammatory response through by MAPK and NFB pathway. In conclusion, this study demonstrates for the first time that UA has potent protective effects against CCl4 -induced inflammation through inhibiting TNF-␣, IL-1 and COX-2 protein expression, at least in part, by suppressing the MAPKs and NF-B activation. Here we demonstrated that UA administration attenuated CCl4 -induced hepatic dysfunction and histopathologic changes. UA attenuated CCl4 -induced oxidative damage by inhibiting ROS generation in liver. UA seems to be potent hepatoprotective drug and its use in maintaining a healthy liver and preventing toxic liver damage deserves consideration and further examination.
Conflict of interest statement The authors declare that there are no conflicts of interest.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgments This work is supported from Startup Project of Doctor scientific research by Sichuan University of Science and Engineering (2013RC14).
references
Checker, R., Sandur, S.K., Sharma, D., Patwardhan, R.S., Jayakumar, S., Kohli, V., Sethi, G., Aggarwal, B.B., Sainis, K.B., 2012. Potent anti-inflammatory activity of ursolic acid, a triterpenoid antioxidant, is mediated through suppression of NF-kB, AP-1 and NF-AT. Plos One 7, e31318. Ebaid, H., Bashandy, S.A., Alhazza, I.M., Rady, A., EI-Shehry, S., 2013. Folic acid and melatonin ameliorate carbon tetrachloride-induced hepatic injury, oxidative stress and inflammation in rats. Nutr. Metab. 10, 20–31. Gayathri, R., Priya, D.K., Gunassekaran, G.R., Sakthisekaran, D., 2009. Ursolic acid attenuates oxidative stress-mediated hepatocellular carcinoma induction by diethylnitrosamine in male Wistar rats. Asian Pac. J. Cancer Prev. 10, 933–938. Hamdy, N., El-Demerdash, E., 2012. New therapeutic aspect for carvedilol: anti fibrotic effects of carvedilol in chronic carbon tetrachloride-induced liver damage. Toxicol. Appl. Pharmacol. 261, 292–299. Iida, C., Fujii, K., Koga, E., Washino, Y., Kitamura, Y., Ichi, I., Abe, K., Matsura, T., Kojo, S., 2009. Effect of ␣-tocopherol on carbon tetrachloride intoxication in the rat liver. Arch. Toxicol. 83, 477–483. Ikeda, Y., Murakami, A., Ohigashi, H., 2005. Ursolic acid promotes the release of macrophag emigration inhibitory factor via ERK2 activation in resting mouse macrophages. Biochem. Pharmacol. 70, 1497–1505. Ikeda, Y., Murakami, A., Fujimura, Y., Tachibana, H., Yamada, K., Masuda, D., Hirano, K., Yamashita, S., Ohigashi, H., 2007.
Aggregated ursolic acid, a natural triterpenoid, induces IL-1 release from murine peritoneal macrophages: role of CD36. J. Immunol. 178, 4854–4864. Kim, H.Y., Park, J., Lee, K.H., Lee, D.U., Kwak, J.H., Kim, Y.S., Lee, S.M., 2011. Ferulic acid protects against carbon tetrachlorideinduced liver injury in mice. Toxicology 282, 104–111. Laskin, D.L., Laskin, J.D., 2001. Role of macrophages and inflammatory mediators in chemically induced toxicity. Toxicology 160, 111–118. Li, R., Xu, L., Liang, T., Li, Y., Zhang, S., Duan, X., 2013. Puerarin mediates hepatoprotection against CCl4 -induced hepatic fibrosis rats through attenuation of inflammation response and amelioration of metabolic function. Food Chem. Toxicol. 52, 69–75. Liu, J., Liu, Y., Mao, Q., Klaassen, C.D., 1994. The effects of 10 triterpenoid compounds on experimental liver injury in mice. Fundam. Appl. Toxicol. 22, 34–40. Liu, C.M., Ma, J.Q., Sun, Y.Z., 2012. Puerarin protects rat kidney from lead-induced apoptosis by modulating the PI3K/Akt/eNOS pathway. Toxicol. Appl. Pharmacol. 258, 330–342. Liu, C.M., Zheng, G.H., Ming, Q.L., Chao, C., Sun, J.M., 2013a. Sesamin protects mouse liver against nickel-induced oxidative DNA damage and apoptosis by the PI3K-Akt pathway. J. Agric. Food Chem. 61, 1146–1154. Liu, C.M., Zheng, G.H., Ming, Q.L., Sun, J.M., Cheng, C., 2013b. Protective effect of quercetin on lead-induced oxidative stress and endoplasmic reticulum stress in rat liver via the IRE1/JNK and PI3K/Akt pathway. Free Radic. Res. 47, 192–201. Lv, Y.Y., Jin, Y., Han, G.Z., Liu, Y.X., Wu, T., Liu, P., Zhou, Q., Liu, K.X., Sun, H.J., 2012. Ursolic acid suppresses IL-6 induced C-reactive protein expression in HepG2 and protects HUVECs from injury induced by CRP. Eur. J. Pharm. Sci. 45, 190–194. Ma, J.Q., Liu, C.M., Qin, Z.H., Jiang, J.H., Sun, Y.Z., 2011. Ganoderma applanatum terpenes protect mouse liver against benzo(␣)pyren-induced oxidative stress and inflammation. Environ. Toxicol. Pharmacol. 31, 460–468. Martin-Aragon, S., de las Heras, B., Sanchez-Reus, M.I., Benedi, J., 2001. Pharmacological modification of endogenous antioxidant enzymes by ursolic acid on tetrachloride-induced liver damage in rats and primary cultures of rat hepatocytes. Exp. Toxicol. Pathol. 53, 199–206. Martinez, O.N., Ricote, G.C., Monzon, C.G., 2003. Role of cyclooxygenase-2 in the pathogenesis of chronic liver diseases. Med. Clin. 121, 743–748. Onkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Saravanan, R., Viswanathan, P., Pugalendi, K.V., 2006. Protective effect of ursolic acid on ethanol-mediated experimental liver damage in rats. Life Sci. 78, 713–718. Shanmugam, M.K., Dai, X., Kumar, A.P., Tan, B.K.H., Sethi, G., Bishayee, A., 2013. Ursolic acid in cancer prevention and treatment: molecular targets, pharmacokinetics and clinical studies. Biochem. Pharmacol. 85, 1579–1587. Shim, J.Y., Kim, M.H., Kim, H.D., Ahn, J.Y., Yun, Y.S., Song, J.Y., 2010. Protective action of the immunomodulator ginsan against carbon tetrachloride-induced liver injury via control of oxidative stress and the in flammatory response. Toxicol. Appl. Pharmacol. 242, 318–325. Shin, D.S., Kim, K.W., Chung, H.Y., Yoon, S., Moon, J.O., 2013. Effect of sinapic acid against carbon tetrachloride-induced acute hepatic injury in rats. Arch. Pharm. Res. 36, 626–633. Shinomol, G.K., Muralidhara, 2007. Differential induction of oxidative impairments in brain regions of male mice following subchronic consumption of Khesari dhal (Lathyrus sativus) and detoxified Khesari dhal. Neurotoxicology 28, 798–806. Singh, N., Kamath, V., Narasimhamurthy, K., Rajini, P.S., 2008. Protective effect of potato peel extract against carbon
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 7 ( 2 0 1 4 ) 975–983
tetrachloride-induced liver injury in rats. Environ. Toxicol. Pharmacol. 26, 241–246. Tacke, F., Luedde, T., Trautwein, C., 2009. Inflammatory pathways in liver homeostasis and liver injury. Clin. Rev. Allergy Immunol. 36, 4–12. Tseng, C.K., Lin, C.K., Chang, H.W., Wu, Y.H., Yen, F.L., Chang, F.R., Chen, W.C., Yeh, C.C., Lee, J.C., 2014. Aqueous extract of Gracilaria tenuistipitata suppresses LPS-Induced NF-kB and MAPK Activation in RAW 264.7 and rat peritoneal macrophages and exerts hepatoprotective effects on carbon tetrachloride treated rat. PLoS One 9, e86557. Tu, C.T., Yao, Q.Y., Xu, B.L., Wang, J.Y., Zhou, C.H., Zhang, S.C., 2012. Protective effects of curcumin against hepatic fibrosis induced by carbon tetrachloride: modulation of high-mobility group box 1. Toll-like receptor 4 and 2 expression. Food Chem. Toxicol. 50, 3343–3351.
983
Wang, J.Q., Chen, X., Zhang, C., Tao, L., Zhang, Z.H., Liu, X.Q., Xu, Y.B., Wang, H., Li, J., Xu, D.X., 2013. Phenylbutyric acid protects against carbon tetrachloride-induced hepatic fibrogenesis in mice. Toxicol. Appl. Pharmacol. 266, 307–316. Weber, L.W., Boll, M., Stampfl, A., 2003. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit. Rev. Toxicol. 33, 105–136. Wu, H.Y., Chang, C.I., Lin, B.W., Yu, F.L., Lin, P.Y., Hsu, J.L., Yen, C.H., Liao, M.H., Shih, W.L., 2011. Suppression of hepatitis B virus X protein-mediated tumorigenic effects by ursolic acid. J. Agric. Food Chem. 59, 1713–1722. Xiang, M., Wang, J., Zhang, Y., Ling, J., Xu, X., 2012. Attenuation of aortic injury by ursolic acid through RAGE-Nox-NFB pathway in streptozocin-induced diabetic rats. Arch. Pharm. Res. 35, 877–886.
