Food and Chemical Toxicology 74 (2014) 149–155
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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x
Protective effects of maslinic acid against alcohol-induced acute liver injury in mice Sheng-lei Yan a, Hui-ting Yang b, Hsiang-lin Lee c, Mei-chin Yin b,d,* a
Division of Gastroenterology, Department of Internal Medicine, Chang Bing Show-Chwan Memorial Hospital, Changhua County, Taiwan Department of Nutrition, China Medical University, Taichung City, Taiwan Department of Surgery, Chung Shan Medical University Hospital, Taichung City, Taiwan d Department of Health and Nutrition Biotechnology, Asia University, Taichung City, Taiwan b c
A R T I C L E
I N F O
Article history: Received 28 July 2014 Accepted 25 September 2014 Available online 6 October 2014 Keywords: Alcohol Hepatotoxicity Maslinic acid ROS CYP2E1 MAPK
A B S T R A C T
Protective effects of maslinic acid (MA) at 10, 15 or 20 mg/kg body weight/day against alcohol-induced acute hepatotoxicity in mice were examined. Mice were administrated by MA for 3 weeks, and followed by alcohol treatment. Results showed that MA pre-intake at three doses resulted in its accumulation in the liver; and dose-dependently lowered cytochrome P450 2E1 activity and protein expression at 23.5– 51.2% and 21.4–62.3%, respectively (P < 0.05). MA pre-intake decreased subsequent alcohol-induced reactive oxygen species, interleukin-6, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, nitric oxide and prostaglandin E2 production; retained glutathione content; maintained catalase and glutathione peroxidase activities; and declined cyclooxygenase-2 and total nitric oxide synthase activities in the liver (P < 0.05). Furthermore, MA pre-intake suppressed 17.3–51.7% nuclear factor kappa (NF-κ)B p50, 23.5– 58.8% NF-κB p65, 25.6–62.4% p-p38 and 24.1–63.0% p-JNK expression in the liver (P < 0.05). Histological data indicated that MA intake at test doses attenuated hepatic inflammatory infiltrate. These findings support that maslinic acid is a potent preventive agent against acute alcoholic liver disease. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Alcoholic liver disease is a major cause of death from liver diseases worldwide. The deleterious effects of alcohol are mainly attributed to the massive production of reactive oxygen species (ROS) and acetaldehyde from ethanol metabolism. These ethanol metabolites deplete glutathione (GSH), and cause free radical-mediated cell apoptosis in the liver (Koch et al., 2004; Lluis et al., 2003). Moreover, ethanol and its metabolites enhance the formation of inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha (McClain et al., 2004; Naveau et al., 2005). The increased release of these inflammatory factors, partially due to the stimulation of oxidative stress (Albano, 2002), leads to cytokine imbalance and immune disorders, which further impairs
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; COX-2, cyclooxygenase-2; CRP, C-reactive protein; CYP2E1, cytochrome P450 2E1; GPX, glutathione peroxide; GSH, glutathione; GSSG, oxidized glutathione; IL, interleukin; MA, maslinic acid; MAPK, mitogen-activated protein kinase; NOS, nitric oxide synthase; MCP, monocyte chemoattractant protein; NF-κB, nuclear factor kappa B; PGE2, prostaglandin E2; ROS, reactive oxygen species; TNF, tumor necrosis factor. * Corresponding author. Department of Nutrition, China Medical University, 91, Hsueh-shih Rd., Taichung City, Taiwan. Tel.: +886 4 22053366 ext. 7510; fax: +886 4 22062891. E-mail address: [email protected] (M. Yin). http://dx.doi.org/10.1016/j.fct.2014.09.018 0278-6915/© 2014 Elsevier Ltd. All rights reserved.
hepatic functions. Thus, any agent with anti-oxidative and/or antiinflammatory abilities may potentially attenuate alcohol-induced liver injury. During ethanol metabolism, ROS could be generated through the activation of xanthine oxidase, cytochrome P450 2E1 (CYP2E1) and NADPH oxidase complex, in which CYP2E1 plays a critical role in alcohol-induced hepatic oxidative stress (Bardag-Gorce et al., 2005; Kessova and Cederbaum, 2003). In addition, alcohol promotes hepatic nitric oxide synthase (NOS) and cyclooxygenase (COX)-2 activities, and increases the formation of nitric oxide (NO) and prostaglandin E2 (PGE2), which is highly linked to necro-inflammatory injury (Chang et al., 2013; Nanji et al., 2013). Thus, inhibition on CYP2E1, NOS and COX-2 activity or expression may interrupt ethanol metabolism and improve alcoholic liver disease. It is well known that alcohol stimulates the activation of nuclear factor kappa (NFκ) B and mitogen-activated protein kinase (MAPK) pathways in the liver, which in turn accelerates the production of oxidative and inflammatory factors (Szuster-Ciesielska et al., 2013; Zima and Kalousová, 2005). Accordingly, an agent with suppressive effects upon hepatic activation of NF-κB or MAPK may contribute to diminish alcohol-induced hepatotoxicity. Maslinic acid (MA) is a pentacyclic triterpene naturally occurring in many vegetables and fruits such as basil (Ocimum basilicum), brown mustard (Brassica juncea) and olive (Olea europaea L.) (Guinda et al., 2010; Yin et al., 2012). Guillen et al. (2009) reported that dietary MA affected hepatic CYP2b9 and CYP2b13 expressions in
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apolipoprotein E-deficient mice. Lin et al. (2011) found that MA exhibited anti-angiogenic activity in human liver cancer cells. Mkhwanazi et al. (2014) indicated that MA provided hepatic antioxidative protection for diabetic rats via increasing the activity of superoxide dismutase and glutathione peroxidase (GPX). These previous studies implied that MA was a potent hepatic protective agent. So far, it remains unknown if this triterpene could protect the liver against alcohol-induced injury. The study of Huang et al. (2011) revealed that MA was able to alleviate inflammatory stress through inhibiting NF-κB pathway in lipopolysaccharide treated rat cortical astrocytes. Thus, it is possible that MA could exert antiinflammatory activity for the liver via regulating signaling pathways. The major purpose of this animal study was to examine the protective effects of MA at various doses on alcohol-induced acute hepatotoxicity. The influence of this agent upon hepatic oxidative and inflammatory factors, CYP2E1 and COX-2 activities, and protein expression of NF-κB or MAPK was evaluated in order to understand its action modes. In addition, MA content in several local vegetables and fruits was determined in order to increase the choice of MA food source. 2. Materials and methods
(Agilent Corp, Waldbronn, Germany) equipped with a C18 reversed-phase column (100 mm × 4 mm, 3 μm, Thermo Electron, Bellefonte, PA, USA) was used. MA quantification was performed using the external standard method. The limit of detection was 0.1 μg/g tissue. The relative standard deviations of precision and accuracy were less than 5%. 2.7. ROS, GSH and oxidized glutathione (GSSG) levels, catalase and GPX activities assay Liver tissue was homogenized with cold PBS containing 0.05% Tween 20 and 1 mM EDTA. After centrifuging, the supernatants were used for measurements. Sample was mixed with 25 mM 2′,7′-dichlorofluorescein diacetate. After 30 min incubation at room temperature, ROS level was determined by monitoring fluorescence change at an excitation wavelength of 488 nm and emission wavelength of 515 nm. GSH and GSSG concentrations (nmol/mg protein) in liver were measured by colorimetric GSH and GSSG assay kits (OxisResearch, Portland, OR, USA). Catalase and GPX activities (U/mg protein) were determined by catalase and GPX assay kits (Calbiochem, EMD Biosciences, Inc., San Diego, CA, USA). 2.8. Cytokine measurements Hepatic levels of IL-6 and TNF-alpha were measured by using cytoscreen immunoassay kits (BioSource International, Camarillo, CA, USA). The sensitivity of assay with the detection limit was 5 nmol/mg protein for IL-6; and 10 nmol/mg protein for TNF-alpha. The level of PGE2 was determined by a PGE2 EIA kit (Cayman Chemical Co., Ann Arbor, MI, USA). Monocyte chemoattractant protein (MCP)-1 level (pg/ mg protein) was assayed by a cytoscreen immunoassay kit (BioSource International).
2.1. Chemicals 2.9. Nitrite assay and nitric oxide synthase (NOS) activity MA (98%) and ethanol (99%) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
Five-week-old male Balb/cA mice were obtained from the National Laboratory Animal Center (National Science Council, Taipei City, Taiwan). Mice were housed on a 12-h light–12-h dark schedule, and fed with water and mouse standard diet (PMI Nutrition International LLC, Brentwood, MO, USA). Use of the mice was reviewed and approved by the China Medical University animal care committee.
The production of NO was determined by measuring the formation of nitrite. Briefly, 100 μl supernatant was treated with nitrate reductase, NADPH and FAD, and followed by incubation for 1 h at 37 °C in the dark. After centrifuging at 6000 × g, the supernatant was mixed with Griess reagent for color development. The absorbance at 540 nm was measured and compared with a sodium nitrite standard curve. The method described in Sutherland et al. (2005) was used to assay total NOS activity (pmol/min/mg protein) by incubating 30 μl homogenate with 10 mM NADP, 10 mM L-valine, 3000 U/ml calmodulin, 5 mM tetrahydrobiopterin, 10 mM CaCl2, and a mixture of 100 μM L-arginine containing L-[3H]arginine.
2.3. Experimental design
2.10. CYP2E1 and COX-2 activities assay
MA, suspended in 1.2% methyl cellulose, was administered to mice by oral gavage. Our preliminary experiments revealed that methyl cellulose did not affect any measurement. Mice were divided into five groups: normal group (without MA intake); control group (without MA intake); MA-10 group (10 mg MA/kg BW/day); MA-15 group (15 mg MA/kg BW/day); MA-20 group (20 mg MA/kg BW/day). After 3-week supplement, control group and three MA treated groups were fasted for 12 h, and 40% (w/v) ethanol was administered at 8 ml/kg BW by gavage every 6 h for 24 h according to the method described by Yang et al. (2003). After being sacrificed, liver from each mouse was collected and weighed. Blood was also collected, and plasma was separated from erythrocyte immediately. Liver at 0.2 g was homogenized on ice in 2 ml phosphate buffer saline (PBS, pH 7.2), and the filtrate was collected. Protein concentration of liver homogenate was determined by a commercial assay kit (Pierce Biotechnology Inc., Rockford, IL, USA) with bovine serum albumin as a standard.
The activity of CYP2E1 in liver microsome was estimated by colorimetrically measuring the hydroxylation of p-nitrophenol to 4-nitrocatechol, a reaction catalyzed specifically by CYP2E1. COX-2 activity was assayed by using a kit (Cayman Chemical Co.) to monitor the variation of oxidized N,N,N′,N′-tetramethyl-p-phenylenediamine at 590 nm.
2.2. Animals
2.4. Examination of blood ethanol level Blood sample was taken from the tail vein 4 h after the last ethanol treatment, and immediately deproteinized with 6.25% trichloroacetic acid solution. Ethanol concentration was determined by using a Sigma Diagnostics Alcohol kit (Sigma Chemical Co., St. Louis, MO, USA). In the present study, the blood ethanol level was 0.69 ± 0.012% (w/v). 2.5. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and C-reactive protein (CRP) analyses Plasma activity of ALT and AST was measured by using commercial assay kits (Randox Laboratories Ltd., Crumlin, UK). Plasma CRP level (μg/ml) was measured by an ELISA kit (Anogen, ON, Canada).
2.11. Western blot analyses Liver tissue was homogenized in buffer containing 0.5% Triton X-100 and proteaseinhibitor cocktail (1:1000, Sigma-Aldrich Chemical Co.). This homogenate was further mixed with buffer (60 mM Tris–HCl, 2% SDS and 2% β-mercaptoethanol, pH 7.2), and boiled for 5 min. Sample at 40 μg protein was applied to 10% SDS-polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA) for 1 h. After blocking with a solution containing 5% nonfat milk for 1 h to prevent non-specific binding of antibody, membrane was incubated with mouse anti-CYP2E1 (1:2000), anti-iNOS, anti-COX-2, anti-NF-κB and anti-MAPK (1:1000) monoclonal antibody (Boehringer-Mannheim, Indianapolis, IN, USA) at 4 °C overnight, and followed by reacting with horseradish peroxidase-conjugated antibody for 3.5 h at room temperature. The detected bands were quantified by an image analyzer (ATTO, Tokyo, Japan), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Results were normalized to GAPDH, and given as arbitrary units (AU). 2.12. Histological analysis Partial liver tissue from each mouse was fixed in 10% phosphate-buffered formalin, and embedded in paraffin. Paraffin section at 5 μm thickness was cut and stained with hematoxylin-eosin (H&E) stain, and followed by examination under a light microscope for histological analysis.
2.6. Determination of MA content in liver 2.13. Analysis of MA content in vegetables and fruits The content of MA in liver was analyzed by an HPLC method described in Lozano-Mena et al. (2012). Liver homogenate, 100 μl, was treated with ethyl acetate for extraction. The organic layers from three-time extractions were combined and dried by nitrogen, the residue was reconstituted in 200 μl methanol/water (75:25, v/v) and centrifuged at 12,000 × g for 5 min at 4 °C. Agilent 1100 series HPLC system
The method described in Yin et al. (2012) was used to analyze the content of MA in eight fresh vegetables and seven fresh fruits. Test vegetables included bitter gourd (Momordica charantia L.), cauliflower (Brassica oleracea L.), eggplant (Solanum melongena L.), ginger (Zingiber officinale), okra (Abelmoschus esculentus L.), onion (Allium
S. Yan et al./Food and Chemical Toxicology 74 (2014) 149–155
cepa L.), snap bean (Phaseolus vulgaris) and vegetable sponge (Luffa cylindrica). Test fruits were dragon fruit (Hylocereus undatus), longan (Dimocarpus longan L.), passion fruit (Passiflora edulis), pineapple (Ananas comosus L.), pomegranate (Punica granatum L.), snaddock (Citrus grandis osbeck) and sugar apple (Anona squamosa L.). These vegetables and fruits, harvested in summer, 2014, were purchased from farms in Nanto County, Taiwan.
800
normal d
400
control
c
MA-10
300
c
MA-15
b
200
MA-20 b
3. Results
a
a
0
Table 1 Body weight (BW, g/mouse), water intake (WI, mL/mouse/day), feed intake (FI, g/mouse/day), liver weight (LW, g/mouse), and liver MA content (μg/g) in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10. Normal
Control
MA-10
MA-15
MA-20
25.1 ± 1.3a 2.2 ± 0.3a 1.8 ± 0.5a 1.34 ± 0.11a –*,a
25.6 ± 0.9a 2.0 ± 0.5a 1.6 ± 0.3a 1.29 ± 0.10a –a
24.8 ± 1.2a 2.3 ± 0.4a 1.5 ± 0.4a 1.38 ± 0.09a 0.82 ± 0.13b
24.5 ± 1.0a 2.1 ± 0.2a 1.7 ± 0.4a 1.27 ± 0.12a 1.36 ± 0.20c
25.3 ± 0.9a 2.3 ± 0.3a 1.5 ± 0.2a 1.32 ± 0.13a 2.17 ± 0.24d
* Means too low to be detected. in a row without a common letter differ, P < 0.05.
a–dMeans
Table 2 Hepatic GSH (nmol/mg protein), GSSG (nmol/mg protein) and ROS (RFU/mg protein) levels; and hepatic GPX and catalase activities (U/mg protein) in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10. Normal
Control
MA-10
MA-15
MA-20
23.1 ± 1.2e 0.27 ± 0.04a 0.24 ± 0.07a 31.3 ± 1.8e 21.1 ± 1.4d
11.2 ± 0.6a 1.13 ± 0.10d 1.63 ± 0.12e 16.8 ± 0.9a 10.9 ± 0.5a
13.3 ± 0.8b 0.98 ± 0.07d 1.31 ± 0.08d 19.5 ± 1.1b 13.1 ± 0.7b
16.9 ± 1.0c 0.71 ± 0.08c 0.96 ± 0.05c 23.0 ± 1.3c 16.9 ± 1.1c
20.4 ± 0.7d 0.51 ± 0.05b 0.62 ± 0.06b 26.8 ± 1.0d 18.0 ± 0.8c
in a row without a common letter differ, P < 0.05.
ALT
AST
CRP
120
d
100 mg/ml
MA pre-intake at three doses resulted in MA deposit in liver (Table 1, P < 0.05); but did not affect body weight, water intake, feed intake and liver weight (P > 0.05). Alcohol administration raised plasma activity of ALT and AST; and plasma CRP level (Fig. 1, P < 0.05); MA pre-treatments decreased subsequent alcohol-induced elevation of ALT and AST activities, and CRP level (P < 0.05). As shown in Fig. 2, alcohol administration increased hepatic activity and protein expression of CYP2E1. MA pre-intake dose-dependently lowered 23.5–51.2% CYP2E1 activity, and suppressed 21.4–62.3% CYP2E1 protein expression (P < 0.05). MA pre-intake significantly retained GSH content, lowered ROS and GSSG levels, and maintained catalase and GPX activities in the liver (Table 2, P < 0.05), in which dosedependent manner was presented in retaining GSH level and GPX activity, and decreasing ROS level (P < 0.05). MA pre-intake lowered alcohol-induced production of IL-6, TNFalpha, MCP-1, PGE2 and NO, and declined total NOS and COX-2 activities, in which dose-dependent manner was presented in decreasing IL-6, TNF-alpha and PGE2 level, and COX-2 activity (Table 3, P < 0.05). As shown in Fig. 3, alcohol treatment increased hepatic iNOS and COX-2 expression (P < 0.05). MA pre-intake dosedependently down-regulated 20.6–52.9% iNOS expression (P < 0.05), and at test doses declined 27.8–55.6% COX-2 expression (P < 0.05).
a–eMeans
d
500
100
GSH GSSG ROS GPX Catalase
e
600
The effect of each treatment was analyzed from 10 mice (n = 10) in each group. All data were expressed as mean ± standard deviation (SD). Statistical analysis was done using one-way analysis of variance, and post-hoc comparisons were carried out using Dunnett’s t-test. Statistical significance was considered at P < 0.05.
BW WI FI LW Liver MA
e
700
mg/ml
2.14. Statistical analysis
151
c normal
80
control
b
60
b
MA-10 MA-15
40 20
MA-20
a
0 normal
control
MA-10
MA-15
MA-20
Fig. 1. Plasma activity (U/l) of ALT and AST, and CRP level (μg/ml) in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10.
Alcohol administration up-regulated hepatic expression of NF-κB and MAPK (Fig. 4, P < 0.05). MA pre-treatments significantly suppressed 17.3–51.7% NF-κB p50, 23.5–58.8% NF-κB p65, 25.6–62.4% p-p38 and 24.1–63.0% p-JNK expression (P < 0.05), in which dosedependent manner was presented in down-regulating NF-κB p65, p-p38 and p-JNK expression (P < 0.05). As shown in Fig. 5, alcohol administration caused obvious foci of inflammatory cell infiltration in the liver, determined by H&E stain. MA intake at test doses decreased hepatic inflammatory infiltration. As shown in Table 4, MA could be detected in bitter gourd, cauliflower, ginger, okra, onion, snap bean, dragon fruit, passion fruit and pomegranate, in the range of 7–76 mg/100 g dry weight.
Table 3 Hepatic level of IL-6 (pg/mg protein), TNF-alpha (pg/mg protein), MCP-1 (pg/mg protein), PGE2 (pg/g protein) and NO (μM/mg protein); and hepatic activity of total NOS (pmol/min/mg protein) and COX-2 activity (U/mg protein) in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10. Normal IL-6 TNFalpha MCP-1 PGE2 NO Total NOS COX-2 a–eMeans
Control
MA-10
MA-15
MA-20
16 ± 4 20 ± 4a
425 ± 487 ± 26e
363 ± 401 ± 23d
275 ± 312 ± 17c
198 ± 9b 206 ± 13b
17 ± 3a 857 ± 78a 8.5 ± 0.9a 16.3 ± 1.1a
369 ± 18d 2463 ± 131e 33.4 ± 2.1d 48.2 ± 2.7d
342 ± 24d 2190 ± 106d 31.9 ± 1.6d 41.0 ± 2.0c
264 ± 20c 1808 ± 91c 24.1 ± 1.6c 34.7 ± 1.8b
157 ± 12b 1354 ± 68b 17.6 ± 1.2b 32.5 ± 1.4b
1.76 ± 0.13e
1.48 ± 0.09d
a
0.19 ± 0.04a
21e
19d
15c
1.17 ± 0.10c
in a row without a common letter differ, P < 0.05.
0.86 ± 0.08b
152
a.
S. Yan et al./Food and Chemical Toxicology 74 (2014) 149–155
NF-κB p50
nmol/min/mg
2.5
e
2
d
NF-κB p65
c
1.5
b
1
p38
a
0.5
p-p38
0 normal
control
MA-10
MA-15
MA-20
JNK
b. CYP2E1
p-JNK GAPDH
ERK normal
control
3.5
MA-10
MA-20
GAPDH
3
normal
d
2
c
1.5 1 0.5
p-ERK
e
2.5 AU
MA-15
Fig. 2. Hepatic CYP2E1 activity (a) and protein expression (b) in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10. a–eMeans among bars without a common letter differ, P < 0.05.
d c
2
b
1.5 1
c
d
c
2.5
d
e
d
3
MA-20
AU
MA-15
MA-20
e
3.5
0 MA-10
MA-15
e
4
a
control
MA-10
4.5
b
normal
control
b
a
c b
a
b b
a
a
0.5 0 NF-kB p50 normal
NF-kB p65 control
MA-10
p-p38 MA-15
p-JNK MA-20
Fig. 4. Hepatic protein expression of NF-κB and MAPK in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10. a–eMeans among bars without a common letter differ, P < 0.05.
iNOS COX-2 GAPDH
4. Discussion normal
control
MA-10
MA-15
MA-20
4.5 d
4
e
3.5
d
AU
3 2.5
c
normal control
c
2 1.5
c
b
b
MA-10 MA-15
a
MA-20
1
a
0.5 0 iNOS
COX-2
Fig. 3. Hepatic protein expression of iNOS and COX-2 in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10. a–eMeans among bars without a common letter differ, P < 0.05.
In our present study, MA pre-intake at three doses, 10–20 mg/ kg BW/day, increased its bioavailability in the liver, and mitigated subsequent alcohol-induced hepatic oxidative and inflammatory injury via retaining GSH content and GPX activity, decreasing ROS, NO and inflammatory cytokines production, and suppressing CYP2E1, COX-2, iNOS, NF-κB and MAPK expression. These data indicated that dietary MA could be absorbed and used for liver anti-oxidative and anti-inflammatory protection. Furthermore, our histological data supported that MA intake improved alcohol caused lobular inflammation and ballooning dgeneration. Therefore, MA was a potent preventive agent against alcoholic liver disease. Oxidative stress is a crucial etiologic factor responsible for acute alcoholic liver injury, especially when liver has less antioxidant protection to cope with the ROS generation (Wu and Cederbaum, 2009; Zhou et al., 2003). Moreover, it is reported that GSH restoration could improve alcohol-induced hepatotoxicity through lowering oxidative stress (Fernandez-Checa and Kaplowitz, 2005; Powell et al., 2010). The results from our present study supports the claim that oxidative damage was an important contributor to acute alcoholic liver injury. Furthermore, we found that MA supplement resulted
S. Yan et al./Food and Chemical Toxicology 74 (2014) 149–155
153
Fig. 5. Effects of MA upon hepatic inflammation, determined by H&E stain, in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Magnification: 200×.
in its accumulation in the liver, and spared GSH content, maintained catalase and GPX activities, as well as decreased alcoholinduced ROS and GSSG formation in the liver. These findings revealed that MA was able to exert hepatic anti-oxidative protection via both enzymatic and non-enzymatic actions. In addition, CYP2E1 is a microsomal alcohol-oxidizing system. It is documented that alcohol administration enhances CYP2E1 activity, which in turn facilitates ROS overproduction through catalytic reaction from alcohol to acetaldehyde (Bardag-Gorce et al., 2005; Zhou et al., 2014). In our present study, MA intake markedly declined subsequent alcoholinduced increase in CYP2E1 activity and protein expression, which consequently decreased the generation of oxidative factors such as ROS. Thus, the anti-oxidative action from this triterpene was partially due to its suppressive effect on hepatic CYP2E1. Mandrekar et al. (2011) and Cohen et al. (2011) reported that alcohol evoked the release of inflammatory cytokines such as IL-6, TNF-alpha and MCP-1 in the liver, which intensified hepatic inflammation and cell apoptosis. We found that MA intakeeffectively lowered
Table 4 Content (mg/100 g dry weight) of MA in eight vegetables and seven fruits harvested in summer, 2013. Data are mean ± SD (n = 8). MA Bitter gourd Cauliflower Eggplant Ginger Okra Onion Snap bean Vegetable sponge Dragon fruit Longan Passion fruit Pineapple Pomegranate Snaddock Sugar apple * Means too low to be detected.
25 ± 3 43 ± 5 –* 14 ± 4 76 ± 7 10 ± 3 41 ± 43 – 7±2 – 26 ± 4 – 30 ± 6 – –
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hepatic IL-6, TNF-alpha and MCP-1 production, which contributed to diminished hepatic inflammatory stress. On the other hand, NO derived from iNOS and PGE2 synthesized by COX-2 play pivotal roles in the pathogenesis of acute and chronic hepatic inflammation (Baldwin, 1996). Besides acting as inflammatory enhancers, excessive NO impairs hepatic insulin signaling (Martyn et al., 2008), and excessive PGE2 increases hepatic triglyceride accumulation via interacting with prostanoid receptors of hepatocytes (Enomoto et al., 2000). Thus, the agent with the capability to decrease NO and PGE2 production in the liver not only mitigates hepatic inflammatory injury but also avoids the occurrence of fatty liver or diabetes. Our present study found that the dietary MA intake declined hepatic activity and/ or protein expression of total NOS, iNOS and COX-2, which in turn limited the production of NO and PGE2. Obviously, MA could exert anti-inflammatory action through regulating NOS and COX-2. These results also implied that MA might retard the development of other hepatic complications such as steatosis. In addition, the lower CRP level, and ALT and AST activities in plasma from MA treated mice also proved that hepatic inflammatory injury had been ameliorated. Oxidation and inflammation are closely interrelated in alcohol-induced liver injury because inflammatory cytokines favor ROS generation, and mitochondrial GSH depletion enhances liver sensitization to alcohol through TNF-alpha mediated hepatocellular death (Fernandez-Checa and Kaplowitz, 2005; Hoek and Pastorino, 2002). We found that MA pre-intake markedly reduced hepatic production of both oxidative and inflammatory factors in alcohol treated mice. Apparently, this triterpene could protect the liver through multiple actions. NF-κB and MAPK are important signaling pathways responsible for the formation of oxidative and inflammatory reactants. It is documented that alcohol could activate these pathways, and promote the expression of inflammatory mediators such as iNOS and COX-2, which in turn accelerate the production of down-stream factors including NO, TNF-alpha and PGE2 in the liver (Beier and McClain, 2010; Tipoe et al., 2008). Our western blot data revealed that MA intake downregulated hepatic protein expression of NF-κB p50, NF-κB p65, p-p38 and p-JNK in alcohol treated mice. Since both NF-κB and MAPK pathways had been declined, the lower hepatic expression and activity of iNOS and COX-2 could be explained. Consequently, it is also reasonable to observe lower hepatic levels of ROS, NO, PGE2 and TNFalpha in MA treated mice. These findings indicated that hepatic protective effects from dietary MA could be ascribed to this triterpene suppressed NF-κB and MAPK pathways, which in turn decreased the production of down-stream oxidative and inflammatory factors, and finally alleviated alcohol evoked hepatotoxicity. MA is a triterpene occurring in many plant foods or botanicals used as food supplement ingredients (Caligiani et al., 2013). Our previous study reported that MA was present in gynara (Gynura bicolor DC), basil (O. basilicum), brown mustard (B. juncea) and Chinese mahogany (Toona sinensis) (Yin et al., 2012). Our present study further found that this compound could be detected in six vegetables and three fruits. Thus, consumers could obtain this compound from eating these foods. Liu et al. (2007) reported that MA intake at 10 and 30 mg/ kg BW/day for 2 weeks improved glycemic control in diabetic mice. Furthermore, it is reported that a single oral administration of MA at 1000 mg/kg to mice did not cause any signs of morbidity or mortality; and daily MA oral administration at 50 mg/kg for 28 days did not induce any sign of toxicity in mice (Sánchez-González et al., 2013). These previous studies suggested that MA was a natural and safe nutraceutical. Thus, using this agent or foods rich in this agent for liver protection against alcohol seems feasible. In conclusion, maslinic acid pre-intake led to its deposit in liver, and markedly decreased subsequent alcohol elicited acute hepatic oxidative and inflammatory stress via suppressing CYP2E1, NF-κB and MAPK pathways. These results support that this triterpene is a potent preventive agent against acute alcoholic liver disease.
Conflict of interest 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. Acknowledgement This study was partially supported by a grant from Ministry of Science and Technology, Taiwan (NSC 102-2313-B-039-002-MY3). References Albano, E., 2002. Free radical mechanisms in immune reactions associated with alcoholic liver disease. Free Radic. Biol. Med. 32, 110–114. Baldwin, A.S., Jr., 1996. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu. Rev. Immunol. 14, 649–683. Bardag-Gorce, F., Li, J., French, B.A., French, S.W., 2005. The effect of ethanol-induced CYP2E1 on proteasome activity: the role of 4-hydroxynonenal. Exp. Mol. Pathol. 78, 109–115. Beier, J.I., McClain, C.J., 2010. Mechanisms and cell signaling in alcoholic liver disease. Biol. Chem. 391, 1249–1264. Caligiani, A., Malavasi, G., Palla, G., Marseglia, A., Tognolini, M., Bruni, R., 2013. A simple GC-MS method for the screening of betulinic, corosolic, maslinic, oleanolic and ursolic acid contents in commercial botanicals used as food supplement ingredients. Food Chem. 136, 735–741. Chang, Y.Y., Lin, Y.L., Yang, D.J., Liu, C.W., Hsu, C.L., Tzang, B.S., et al., 2013. Hepatoprotection of noni juice against chronic alcohol consumption: lipid homeostasis, antioxidation, alcohol clearance, and anti-inflammation. J. Agric. Food Chem. 61, 11016–11024. Cohen, J.I., Chen, X., Nagy, L.E., 2011. Redox signaling and the innate immune system in alcoholic liver disease. Antioxid. Redox Signal. 15, 523–534. Enomoto, N., Ikejima, K., Yamashina, S., Enomoto, A., Nishiura, T., Nishimura, T., et al., 2000. Kupffer cell-derived prostaglandin E(2) is involved in alcohol-induced fat accumulation in rat liver. Am. J. Physiol. Gastrointest. Liver Physiol. 279, 100–106. Fernandez-Checa, J.C., Kaplowitz, N., 2005. Hepatic mitochondrial glutathione: transport and role in disease and toxicity. Toxicol. Appl. Pharmacol. 204, 263–273. Guillen, N., Acín, S., Surra, J.C., Arnal, C., Godino, J., García-Granados, A., et al., 2009. Apolipoprotein E determines the hepatic transcriptional profile of dietary maslinic acid in mice. J. Nutr. Biochem. 20, 882–893. Guinda, A., Rada, M., Delgado, T., Gutiérrez-Adánez, P., Castellano, J.M., 2010. Pentacyclic triterpenoids from olive fruit and leaf. J. Agric. Food Chem. 58, 9685–9691. Hoek, J.B., Pastorino, J.G., 2002. Ethanol, oxidative stress, and cytokine-induced liver cell injury. Alcohol 27, 63–68. Huang, L., Guan, T., Qian, Y., Huang, M., Tang, X., Li, Y., et al., 2011. Anti-inflammatory effects of maslinic acid, a natural triterpene, in cultured cortical astrocytes via suppression of nuclear factor-kappa B. Eur. J. Pharmacol. 672, 169–174. Kessova, I., Cederbaum, A.I., 2003. CYP2E1: biochemistry, toxicology, regulation and function in ethanol-induced liver injury. Curr. Mol. Med. 3, 509–518. Koch, O.R., Pani, G., Borrello, S., Colavitti, R., Cravero, A., Farre, S., et al., 2004. Oxidative stress and antioxidant defenses in ethanol-induced cell injury. Mol. Aspects Med. 25, 191–198. Lin, C.C., Huang, C.Y., Mong, M.C., Chan, C.Y., Yin, M.C., 2011. Antiangiogenic potential of three triterpenic acids in human liver cancer cells. J. Agric. Food Chem. 59, 755–762. Liu, J., Sun, H., Duan, W., Mu, D., Zhang, L., 2007. Maslinic acid reduces blood glucose in KK-Ay mice. Biol. Pharm. Bull. 30, 2075–2078. Lluis, J.M., Colell, A., Garcia-Ruiz, C., Kaplowitz, N., Fernandez-Checa, J.C., 2003. Acetaldehyde impairs mitochondrial glutathione transport in HepG2 cells through endoplasmic reticulum stress. Gastroenterology 124, 708–724. Lozano-Mena, G., Juan, M.E., García-Granados, A., Planas, J.M., 2012. Determination of maslinic acid, a pentacyclic triterpene from olives, in rat plasma by highperformance liquid chromatography. J. Agric. Food Chem. 60, 10220–10225. Mandrekar, P., Ambade, A., Lim, A., Szabo, G., Catalano, D., 2011. An essential role for monocyte chemoattractant protein-1 in alcoholic liver injury: regulation of proinflammatory cytokines and hepatic steatosis in mice. Hepatology 54, 2185–2197. Martyn, J.A., Kaneki, M., Yasuhara, S., 2008. Obesity-induced insulin resistance and hyperglycemia: etiologic factors and molecular mechanisms. Anesthesiology 109, 137–148. McClain, C.J., Song, Z., Barve, S.S., Hill, D.B., Deaciuc, I., 2004. Recent advances in alcoholic liver disease. IV. Dysregulated cytokine metabolism in alcoholic liver disease. Am. J. Physiol. Gastrointest. Liver Physiol. 287, 497–502. Mkhwanazi, B.N., Serumula, M.R., Myburg, R.B., van Heerden, F.R., Musabayane, C.T., 2014. Antioxidant effects of maslinic acid in livers, hearts and kidneys of streptozotocin-induced diabetic rats: effects on kidney function. Ren. Fail. 36, 419–431.
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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x
Protective effects of maslinic acid against alcohol-induced acute liver injury in mice Sheng-lei Yan a, Hui-ting Yang b, Hsiang-lin Lee c, Mei-chin Yin b,d,* a
Division of Gastroenterology, Department of Internal Medicine, Chang Bing Show-Chwan Memorial Hospital, Changhua County, Taiwan Department of Nutrition, China Medical University, Taichung City, Taiwan Department of Surgery, Chung Shan Medical University Hospital, Taichung City, Taiwan d Department of Health and Nutrition Biotechnology, Asia University, Taichung City, Taiwan b c
A R T I C L E
I N F O
Article history: Received 28 July 2014 Accepted 25 September 2014 Available online 6 October 2014 Keywords: Alcohol Hepatotoxicity Maslinic acid ROS CYP2E1 MAPK
A B S T R A C T
Protective effects of maslinic acid (MA) at 10, 15 or 20 mg/kg body weight/day against alcohol-induced acute hepatotoxicity in mice were examined. Mice were administrated by MA for 3 weeks, and followed by alcohol treatment. Results showed that MA pre-intake at three doses resulted in its accumulation in the liver; and dose-dependently lowered cytochrome P450 2E1 activity and protein expression at 23.5– 51.2% and 21.4–62.3%, respectively (P < 0.05). MA pre-intake decreased subsequent alcohol-induced reactive oxygen species, interleukin-6, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, nitric oxide and prostaglandin E2 production; retained glutathione content; maintained catalase and glutathione peroxidase activities; and declined cyclooxygenase-2 and total nitric oxide synthase activities in the liver (P < 0.05). Furthermore, MA pre-intake suppressed 17.3–51.7% nuclear factor kappa (NF-κ)B p50, 23.5– 58.8% NF-κB p65, 25.6–62.4% p-p38 and 24.1–63.0% p-JNK expression in the liver (P < 0.05). Histological data indicated that MA intake at test doses attenuated hepatic inflammatory infiltrate. These findings support that maslinic acid is a potent preventive agent against acute alcoholic liver disease. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Alcoholic liver disease is a major cause of death from liver diseases worldwide. The deleterious effects of alcohol are mainly attributed to the massive production of reactive oxygen species (ROS) and acetaldehyde from ethanol metabolism. These ethanol metabolites deplete glutathione (GSH), and cause free radical-mediated cell apoptosis in the liver (Koch et al., 2004; Lluis et al., 2003). Moreover, ethanol and its metabolites enhance the formation of inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha (McClain et al., 2004; Naveau et al., 2005). The increased release of these inflammatory factors, partially due to the stimulation of oxidative stress (Albano, 2002), leads to cytokine imbalance and immune disorders, which further impairs
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; COX-2, cyclooxygenase-2; CRP, C-reactive protein; CYP2E1, cytochrome P450 2E1; GPX, glutathione peroxide; GSH, glutathione; GSSG, oxidized glutathione; IL, interleukin; MA, maslinic acid; MAPK, mitogen-activated protein kinase; NOS, nitric oxide synthase; MCP, monocyte chemoattractant protein; NF-κB, nuclear factor kappa B; PGE2, prostaglandin E2; ROS, reactive oxygen species; TNF, tumor necrosis factor. * Corresponding author. Department of Nutrition, China Medical University, 91, Hsueh-shih Rd., Taichung City, Taiwan. Tel.: +886 4 22053366 ext. 7510; fax: +886 4 22062891. E-mail address: [email protected] (M. Yin). http://dx.doi.org/10.1016/j.fct.2014.09.018 0278-6915/© 2014 Elsevier Ltd. All rights reserved.
hepatic functions. Thus, any agent with anti-oxidative and/or antiinflammatory abilities may potentially attenuate alcohol-induced liver injury. During ethanol metabolism, ROS could be generated through the activation of xanthine oxidase, cytochrome P450 2E1 (CYP2E1) and NADPH oxidase complex, in which CYP2E1 plays a critical role in alcohol-induced hepatic oxidative stress (Bardag-Gorce et al., 2005; Kessova and Cederbaum, 2003). In addition, alcohol promotes hepatic nitric oxide synthase (NOS) and cyclooxygenase (COX)-2 activities, and increases the formation of nitric oxide (NO) and prostaglandin E2 (PGE2), which is highly linked to necro-inflammatory injury (Chang et al., 2013; Nanji et al., 2013). Thus, inhibition on CYP2E1, NOS and COX-2 activity or expression may interrupt ethanol metabolism and improve alcoholic liver disease. It is well known that alcohol stimulates the activation of nuclear factor kappa (NFκ) B and mitogen-activated protein kinase (MAPK) pathways in the liver, which in turn accelerates the production of oxidative and inflammatory factors (Szuster-Ciesielska et al., 2013; Zima and Kalousová, 2005). Accordingly, an agent with suppressive effects upon hepatic activation of NF-κB or MAPK may contribute to diminish alcohol-induced hepatotoxicity. Maslinic acid (MA) is a pentacyclic triterpene naturally occurring in many vegetables and fruits such as basil (Ocimum basilicum), brown mustard (Brassica juncea) and olive (Olea europaea L.) (Guinda et al., 2010; Yin et al., 2012). Guillen et al. (2009) reported that dietary MA affected hepatic CYP2b9 and CYP2b13 expressions in
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apolipoprotein E-deficient mice. Lin et al. (2011) found that MA exhibited anti-angiogenic activity in human liver cancer cells. Mkhwanazi et al. (2014) indicated that MA provided hepatic antioxidative protection for diabetic rats via increasing the activity of superoxide dismutase and glutathione peroxidase (GPX). These previous studies implied that MA was a potent hepatic protective agent. So far, it remains unknown if this triterpene could protect the liver against alcohol-induced injury. The study of Huang et al. (2011) revealed that MA was able to alleviate inflammatory stress through inhibiting NF-κB pathway in lipopolysaccharide treated rat cortical astrocytes. Thus, it is possible that MA could exert antiinflammatory activity for the liver via regulating signaling pathways. The major purpose of this animal study was to examine the protective effects of MA at various doses on alcohol-induced acute hepatotoxicity. The influence of this agent upon hepatic oxidative and inflammatory factors, CYP2E1 and COX-2 activities, and protein expression of NF-κB or MAPK was evaluated in order to understand its action modes. In addition, MA content in several local vegetables and fruits was determined in order to increase the choice of MA food source. 2. Materials and methods
(Agilent Corp, Waldbronn, Germany) equipped with a C18 reversed-phase column (100 mm × 4 mm, 3 μm, Thermo Electron, Bellefonte, PA, USA) was used. MA quantification was performed using the external standard method. The limit of detection was 0.1 μg/g tissue. The relative standard deviations of precision and accuracy were less than 5%. 2.7. ROS, GSH and oxidized glutathione (GSSG) levels, catalase and GPX activities assay Liver tissue was homogenized with cold PBS containing 0.05% Tween 20 and 1 mM EDTA. After centrifuging, the supernatants were used for measurements. Sample was mixed with 25 mM 2′,7′-dichlorofluorescein diacetate. After 30 min incubation at room temperature, ROS level was determined by monitoring fluorescence change at an excitation wavelength of 488 nm and emission wavelength of 515 nm. GSH and GSSG concentrations (nmol/mg protein) in liver were measured by colorimetric GSH and GSSG assay kits (OxisResearch, Portland, OR, USA). Catalase and GPX activities (U/mg protein) were determined by catalase and GPX assay kits (Calbiochem, EMD Biosciences, Inc., San Diego, CA, USA). 2.8. Cytokine measurements Hepatic levels of IL-6 and TNF-alpha were measured by using cytoscreen immunoassay kits (BioSource International, Camarillo, CA, USA). The sensitivity of assay with the detection limit was 5 nmol/mg protein for IL-6; and 10 nmol/mg protein for TNF-alpha. The level of PGE2 was determined by a PGE2 EIA kit (Cayman Chemical Co., Ann Arbor, MI, USA). Monocyte chemoattractant protein (MCP)-1 level (pg/ mg protein) was assayed by a cytoscreen immunoassay kit (BioSource International).
2.1. Chemicals 2.9. Nitrite assay and nitric oxide synthase (NOS) activity MA (98%) and ethanol (99%) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
Five-week-old male Balb/cA mice were obtained from the National Laboratory Animal Center (National Science Council, Taipei City, Taiwan). Mice were housed on a 12-h light–12-h dark schedule, and fed with water and mouse standard diet (PMI Nutrition International LLC, Brentwood, MO, USA). Use of the mice was reviewed and approved by the China Medical University animal care committee.
The production of NO was determined by measuring the formation of nitrite. Briefly, 100 μl supernatant was treated with nitrate reductase, NADPH and FAD, and followed by incubation for 1 h at 37 °C in the dark. After centrifuging at 6000 × g, the supernatant was mixed with Griess reagent for color development. The absorbance at 540 nm was measured and compared with a sodium nitrite standard curve. The method described in Sutherland et al. (2005) was used to assay total NOS activity (pmol/min/mg protein) by incubating 30 μl homogenate with 10 mM NADP, 10 mM L-valine, 3000 U/ml calmodulin, 5 mM tetrahydrobiopterin, 10 mM CaCl2, and a mixture of 100 μM L-arginine containing L-[3H]arginine.
2.3. Experimental design
2.10. CYP2E1 and COX-2 activities assay
MA, suspended in 1.2% methyl cellulose, was administered to mice by oral gavage. Our preliminary experiments revealed that methyl cellulose did not affect any measurement. Mice were divided into five groups: normal group (without MA intake); control group (without MA intake); MA-10 group (10 mg MA/kg BW/day); MA-15 group (15 mg MA/kg BW/day); MA-20 group (20 mg MA/kg BW/day). After 3-week supplement, control group and three MA treated groups were fasted for 12 h, and 40% (w/v) ethanol was administered at 8 ml/kg BW by gavage every 6 h for 24 h according to the method described by Yang et al. (2003). After being sacrificed, liver from each mouse was collected and weighed. Blood was also collected, and plasma was separated from erythrocyte immediately. Liver at 0.2 g was homogenized on ice in 2 ml phosphate buffer saline (PBS, pH 7.2), and the filtrate was collected. Protein concentration of liver homogenate was determined by a commercial assay kit (Pierce Biotechnology Inc., Rockford, IL, USA) with bovine serum albumin as a standard.
The activity of CYP2E1 in liver microsome was estimated by colorimetrically measuring the hydroxylation of p-nitrophenol to 4-nitrocatechol, a reaction catalyzed specifically by CYP2E1. COX-2 activity was assayed by using a kit (Cayman Chemical Co.) to monitor the variation of oxidized N,N,N′,N′-tetramethyl-p-phenylenediamine at 590 nm.
2.2. Animals
2.4. Examination of blood ethanol level Blood sample was taken from the tail vein 4 h after the last ethanol treatment, and immediately deproteinized with 6.25% trichloroacetic acid solution. Ethanol concentration was determined by using a Sigma Diagnostics Alcohol kit (Sigma Chemical Co., St. Louis, MO, USA). In the present study, the blood ethanol level was 0.69 ± 0.012% (w/v). 2.5. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and C-reactive protein (CRP) analyses Plasma activity of ALT and AST was measured by using commercial assay kits (Randox Laboratories Ltd., Crumlin, UK). Plasma CRP level (μg/ml) was measured by an ELISA kit (Anogen, ON, Canada).
2.11. Western blot analyses Liver tissue was homogenized in buffer containing 0.5% Triton X-100 and proteaseinhibitor cocktail (1:1000, Sigma-Aldrich Chemical Co.). This homogenate was further mixed with buffer (60 mM Tris–HCl, 2% SDS and 2% β-mercaptoethanol, pH 7.2), and boiled for 5 min. Sample at 40 μg protein was applied to 10% SDS-polyacrylamide gel electrophoresis, and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA) for 1 h. After blocking with a solution containing 5% nonfat milk for 1 h to prevent non-specific binding of antibody, membrane was incubated with mouse anti-CYP2E1 (1:2000), anti-iNOS, anti-COX-2, anti-NF-κB and anti-MAPK (1:1000) monoclonal antibody (Boehringer-Mannheim, Indianapolis, IN, USA) at 4 °C overnight, and followed by reacting with horseradish peroxidase-conjugated antibody for 3.5 h at room temperature. The detected bands were quantified by an image analyzer (ATTO, Tokyo, Japan), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Results were normalized to GAPDH, and given as arbitrary units (AU). 2.12. Histological analysis Partial liver tissue from each mouse was fixed in 10% phosphate-buffered formalin, and embedded in paraffin. Paraffin section at 5 μm thickness was cut and stained with hematoxylin-eosin (H&E) stain, and followed by examination under a light microscope for histological analysis.
2.6. Determination of MA content in liver 2.13. Analysis of MA content in vegetables and fruits The content of MA in liver was analyzed by an HPLC method described in Lozano-Mena et al. (2012). Liver homogenate, 100 μl, was treated with ethyl acetate for extraction. The organic layers from three-time extractions were combined and dried by nitrogen, the residue was reconstituted in 200 μl methanol/water (75:25, v/v) and centrifuged at 12,000 × g for 5 min at 4 °C. Agilent 1100 series HPLC system
The method described in Yin et al. (2012) was used to analyze the content of MA in eight fresh vegetables and seven fresh fruits. Test vegetables included bitter gourd (Momordica charantia L.), cauliflower (Brassica oleracea L.), eggplant (Solanum melongena L.), ginger (Zingiber officinale), okra (Abelmoschus esculentus L.), onion (Allium
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cepa L.), snap bean (Phaseolus vulgaris) and vegetable sponge (Luffa cylindrica). Test fruits were dragon fruit (Hylocereus undatus), longan (Dimocarpus longan L.), passion fruit (Passiflora edulis), pineapple (Ananas comosus L.), pomegranate (Punica granatum L.), snaddock (Citrus grandis osbeck) and sugar apple (Anona squamosa L.). These vegetables and fruits, harvested in summer, 2014, were purchased from farms in Nanto County, Taiwan.
800
normal d
400
control
c
MA-10
300
c
MA-15
b
200
MA-20 b
3. Results
a
a
0
Table 1 Body weight (BW, g/mouse), water intake (WI, mL/mouse/day), feed intake (FI, g/mouse/day), liver weight (LW, g/mouse), and liver MA content (μg/g) in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10. Normal
Control
MA-10
MA-15
MA-20
25.1 ± 1.3a 2.2 ± 0.3a 1.8 ± 0.5a 1.34 ± 0.11a –*,a
25.6 ± 0.9a 2.0 ± 0.5a 1.6 ± 0.3a 1.29 ± 0.10a –a
24.8 ± 1.2a 2.3 ± 0.4a 1.5 ± 0.4a 1.38 ± 0.09a 0.82 ± 0.13b
24.5 ± 1.0a 2.1 ± 0.2a 1.7 ± 0.4a 1.27 ± 0.12a 1.36 ± 0.20c
25.3 ± 0.9a 2.3 ± 0.3a 1.5 ± 0.2a 1.32 ± 0.13a 2.17 ± 0.24d
* Means too low to be detected. in a row without a common letter differ, P < 0.05.
a–dMeans
Table 2 Hepatic GSH (nmol/mg protein), GSSG (nmol/mg protein) and ROS (RFU/mg protein) levels; and hepatic GPX and catalase activities (U/mg protein) in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10. Normal
Control
MA-10
MA-15
MA-20
23.1 ± 1.2e 0.27 ± 0.04a 0.24 ± 0.07a 31.3 ± 1.8e 21.1 ± 1.4d
11.2 ± 0.6a 1.13 ± 0.10d 1.63 ± 0.12e 16.8 ± 0.9a 10.9 ± 0.5a
13.3 ± 0.8b 0.98 ± 0.07d 1.31 ± 0.08d 19.5 ± 1.1b 13.1 ± 0.7b
16.9 ± 1.0c 0.71 ± 0.08c 0.96 ± 0.05c 23.0 ± 1.3c 16.9 ± 1.1c
20.4 ± 0.7d 0.51 ± 0.05b 0.62 ± 0.06b 26.8 ± 1.0d 18.0 ± 0.8c
in a row without a common letter differ, P < 0.05.
ALT
AST
CRP
120
d
100 mg/ml
MA pre-intake at three doses resulted in MA deposit in liver (Table 1, P < 0.05); but did not affect body weight, water intake, feed intake and liver weight (P > 0.05). Alcohol administration raised plasma activity of ALT and AST; and plasma CRP level (Fig. 1, P < 0.05); MA pre-treatments decreased subsequent alcohol-induced elevation of ALT and AST activities, and CRP level (P < 0.05). As shown in Fig. 2, alcohol administration increased hepatic activity and protein expression of CYP2E1. MA pre-intake dose-dependently lowered 23.5–51.2% CYP2E1 activity, and suppressed 21.4–62.3% CYP2E1 protein expression (P < 0.05). MA pre-intake significantly retained GSH content, lowered ROS and GSSG levels, and maintained catalase and GPX activities in the liver (Table 2, P < 0.05), in which dosedependent manner was presented in retaining GSH level and GPX activity, and decreasing ROS level (P < 0.05). MA pre-intake lowered alcohol-induced production of IL-6, TNFalpha, MCP-1, PGE2 and NO, and declined total NOS and COX-2 activities, in which dose-dependent manner was presented in decreasing IL-6, TNF-alpha and PGE2 level, and COX-2 activity (Table 3, P < 0.05). As shown in Fig. 3, alcohol treatment increased hepatic iNOS and COX-2 expression (P < 0.05). MA pre-intake dosedependently down-regulated 20.6–52.9% iNOS expression (P < 0.05), and at test doses declined 27.8–55.6% COX-2 expression (P < 0.05).
a–eMeans
d
500
100
GSH GSSG ROS GPX Catalase
e
600
The effect of each treatment was analyzed from 10 mice (n = 10) in each group. All data were expressed as mean ± standard deviation (SD). Statistical analysis was done using one-way analysis of variance, and post-hoc comparisons were carried out using Dunnett’s t-test. Statistical significance was considered at P < 0.05.
BW WI FI LW Liver MA
e
700
mg/ml
2.14. Statistical analysis
151
c normal
80
control
b
60
b
MA-10 MA-15
40 20
MA-20
a
0 normal
control
MA-10
MA-15
MA-20
Fig. 1. Plasma activity (U/l) of ALT and AST, and CRP level (μg/ml) in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10.
Alcohol administration up-regulated hepatic expression of NF-κB and MAPK (Fig. 4, P < 0.05). MA pre-treatments significantly suppressed 17.3–51.7% NF-κB p50, 23.5–58.8% NF-κB p65, 25.6–62.4% p-p38 and 24.1–63.0% p-JNK expression (P < 0.05), in which dosedependent manner was presented in down-regulating NF-κB p65, p-p38 and p-JNK expression (P < 0.05). As shown in Fig. 5, alcohol administration caused obvious foci of inflammatory cell infiltration in the liver, determined by H&E stain. MA intake at test doses decreased hepatic inflammatory infiltration. As shown in Table 4, MA could be detected in bitter gourd, cauliflower, ginger, okra, onion, snap bean, dragon fruit, passion fruit and pomegranate, in the range of 7–76 mg/100 g dry weight.
Table 3 Hepatic level of IL-6 (pg/mg protein), TNF-alpha (pg/mg protein), MCP-1 (pg/mg protein), PGE2 (pg/g protein) and NO (μM/mg protein); and hepatic activity of total NOS (pmol/min/mg protein) and COX-2 activity (U/mg protein) in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10. Normal IL-6 TNFalpha MCP-1 PGE2 NO Total NOS COX-2 a–eMeans
Control
MA-10
MA-15
MA-20
16 ± 4 20 ± 4a
425 ± 487 ± 26e
363 ± 401 ± 23d
275 ± 312 ± 17c
198 ± 9b 206 ± 13b
17 ± 3a 857 ± 78a 8.5 ± 0.9a 16.3 ± 1.1a
369 ± 18d 2463 ± 131e 33.4 ± 2.1d 48.2 ± 2.7d
342 ± 24d 2190 ± 106d 31.9 ± 1.6d 41.0 ± 2.0c
264 ± 20c 1808 ± 91c 24.1 ± 1.6c 34.7 ± 1.8b
157 ± 12b 1354 ± 68b 17.6 ± 1.2b 32.5 ± 1.4b
1.76 ± 0.13e
1.48 ± 0.09d
a
0.19 ± 0.04a
21e
19d
15c
1.17 ± 0.10c
in a row without a common letter differ, P < 0.05.
0.86 ± 0.08b
152
a.
S. Yan et al./Food and Chemical Toxicology 74 (2014) 149–155
NF-κB p50
nmol/min/mg
2.5
e
2
d
NF-κB p65
c
1.5
b
1
p38
a
0.5
p-p38
0 normal
control
MA-10
MA-15
MA-20
JNK
b. CYP2E1
p-JNK GAPDH
ERK normal
control
3.5
MA-10
MA-20
GAPDH
3
normal
d
2
c
1.5 1 0.5
p-ERK
e
2.5 AU
MA-15
Fig. 2. Hepatic CYP2E1 activity (a) and protein expression (b) in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10. a–eMeans among bars without a common letter differ, P < 0.05.
d c
2
b
1.5 1
c
d
c
2.5
d
e
d
3
MA-20
AU
MA-15
MA-20
e
3.5
0 MA-10
MA-15
e
4
a
control
MA-10
4.5
b
normal
control
b
a
c b
a
b b
a
a
0.5 0 NF-kB p50 normal
NF-kB p65 control
MA-10
p-p38 MA-15
p-JNK MA-20
Fig. 4. Hepatic protein expression of NF-κB and MAPK in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10. a–eMeans among bars without a common letter differ, P < 0.05.
iNOS COX-2 GAPDH
4. Discussion normal
control
MA-10
MA-15
MA-20
4.5 d
4
e
3.5
d
AU
3 2.5
c
normal control
c
2 1.5
c
b
b
MA-10 MA-15
a
MA-20
1
a
0.5 0 iNOS
COX-2
Fig. 3. Hepatic protein expression of iNOS and COX-2 in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Values are mean ± SD, n = 10. a–eMeans among bars without a common letter differ, P < 0.05.
In our present study, MA pre-intake at three doses, 10–20 mg/ kg BW/day, increased its bioavailability in the liver, and mitigated subsequent alcohol-induced hepatic oxidative and inflammatory injury via retaining GSH content and GPX activity, decreasing ROS, NO and inflammatory cytokines production, and suppressing CYP2E1, COX-2, iNOS, NF-κB and MAPK expression. These data indicated that dietary MA could be absorbed and used for liver anti-oxidative and anti-inflammatory protection. Furthermore, our histological data supported that MA intake improved alcohol caused lobular inflammation and ballooning dgeneration. Therefore, MA was a potent preventive agent against alcoholic liver disease. Oxidative stress is a crucial etiologic factor responsible for acute alcoholic liver injury, especially when liver has less antioxidant protection to cope with the ROS generation (Wu and Cederbaum, 2009; Zhou et al., 2003). Moreover, it is reported that GSH restoration could improve alcohol-induced hepatotoxicity through lowering oxidative stress (Fernandez-Checa and Kaplowitz, 2005; Powell et al., 2010). The results from our present study supports the claim that oxidative damage was an important contributor to acute alcoholic liver injury. Furthermore, we found that MA supplement resulted
S. Yan et al./Food and Chemical Toxicology 74 (2014) 149–155
153
Fig. 5. Effects of MA upon hepatic inflammation, determined by H&E stain, in mice treated with MA at 0 (control), or 10, 15, 20 mg/kg BW/day for 3 weeks, and followed by alcohol treatment. Normal groups had neither MA nor alcohol treatments. Magnification: 200×.
in its accumulation in the liver, and spared GSH content, maintained catalase and GPX activities, as well as decreased alcoholinduced ROS and GSSG formation in the liver. These findings revealed that MA was able to exert hepatic anti-oxidative protection via both enzymatic and non-enzymatic actions. In addition, CYP2E1 is a microsomal alcohol-oxidizing system. It is documented that alcohol administration enhances CYP2E1 activity, which in turn facilitates ROS overproduction through catalytic reaction from alcohol to acetaldehyde (Bardag-Gorce et al., 2005; Zhou et al., 2014). In our present study, MA intake markedly declined subsequent alcoholinduced increase in CYP2E1 activity and protein expression, which consequently decreased the generation of oxidative factors such as ROS. Thus, the anti-oxidative action from this triterpene was partially due to its suppressive effect on hepatic CYP2E1. Mandrekar et al. (2011) and Cohen et al. (2011) reported that alcohol evoked the release of inflammatory cytokines such as IL-6, TNF-alpha and MCP-1 in the liver, which intensified hepatic inflammation and cell apoptosis. We found that MA intakeeffectively lowered
Table 4 Content (mg/100 g dry weight) of MA in eight vegetables and seven fruits harvested in summer, 2013. Data are mean ± SD (n = 8). MA Bitter gourd Cauliflower Eggplant Ginger Okra Onion Snap bean Vegetable sponge Dragon fruit Longan Passion fruit Pineapple Pomegranate Snaddock Sugar apple * Means too low to be detected.
25 ± 3 43 ± 5 –* 14 ± 4 76 ± 7 10 ± 3 41 ± 43 – 7±2 – 26 ± 4 – 30 ± 6 – –
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hepatic IL-6, TNF-alpha and MCP-1 production, which contributed to diminished hepatic inflammatory stress. On the other hand, NO derived from iNOS and PGE2 synthesized by COX-2 play pivotal roles in the pathogenesis of acute and chronic hepatic inflammation (Baldwin, 1996). Besides acting as inflammatory enhancers, excessive NO impairs hepatic insulin signaling (Martyn et al., 2008), and excessive PGE2 increases hepatic triglyceride accumulation via interacting with prostanoid receptors of hepatocytes (Enomoto et al., 2000). Thus, the agent with the capability to decrease NO and PGE2 production in the liver not only mitigates hepatic inflammatory injury but also avoids the occurrence of fatty liver or diabetes. Our present study found that the dietary MA intake declined hepatic activity and/ or protein expression of total NOS, iNOS and COX-2, which in turn limited the production of NO and PGE2. Obviously, MA could exert anti-inflammatory action through regulating NOS and COX-2. These results also implied that MA might retard the development of other hepatic complications such as steatosis. In addition, the lower CRP level, and ALT and AST activities in plasma from MA treated mice also proved that hepatic inflammatory injury had been ameliorated. Oxidation and inflammation are closely interrelated in alcohol-induced liver injury because inflammatory cytokines favor ROS generation, and mitochondrial GSH depletion enhances liver sensitization to alcohol through TNF-alpha mediated hepatocellular death (Fernandez-Checa and Kaplowitz, 2005; Hoek and Pastorino, 2002). We found that MA pre-intake markedly reduced hepatic production of both oxidative and inflammatory factors in alcohol treated mice. Apparently, this triterpene could protect the liver through multiple actions. NF-κB and MAPK are important signaling pathways responsible for the formation of oxidative and inflammatory reactants. It is documented that alcohol could activate these pathways, and promote the expression of inflammatory mediators such as iNOS and COX-2, which in turn accelerate the production of down-stream factors including NO, TNF-alpha and PGE2 in the liver (Beier and McClain, 2010; Tipoe et al., 2008). Our western blot data revealed that MA intake downregulated hepatic protein expression of NF-κB p50, NF-κB p65, p-p38 and p-JNK in alcohol treated mice. Since both NF-κB and MAPK pathways had been declined, the lower hepatic expression and activity of iNOS and COX-2 could be explained. Consequently, it is also reasonable to observe lower hepatic levels of ROS, NO, PGE2 and TNFalpha in MA treated mice. These findings indicated that hepatic protective effects from dietary MA could be ascribed to this triterpene suppressed NF-κB and MAPK pathways, which in turn decreased the production of down-stream oxidative and inflammatory factors, and finally alleviated alcohol evoked hepatotoxicity. MA is a triterpene occurring in many plant foods or botanicals used as food supplement ingredients (Caligiani et al., 2013). Our previous study reported that MA was present in gynara (Gynura bicolor DC), basil (O. basilicum), brown mustard (B. juncea) and Chinese mahogany (Toona sinensis) (Yin et al., 2012). Our present study further found that this compound could be detected in six vegetables and three fruits. Thus, consumers could obtain this compound from eating these foods. Liu et al. (2007) reported that MA intake at 10 and 30 mg/ kg BW/day for 2 weeks improved glycemic control in diabetic mice. Furthermore, it is reported that a single oral administration of MA at 1000 mg/kg to mice did not cause any signs of morbidity or mortality; and daily MA oral administration at 50 mg/kg for 28 days did not induce any sign of toxicity in mice (Sánchez-González et al., 2013). These previous studies suggested that MA was a natural and safe nutraceutical. Thus, using this agent or foods rich in this agent for liver protection against alcohol seems feasible. In conclusion, maslinic acid pre-intake led to its deposit in liver, and markedly decreased subsequent alcohol elicited acute hepatic oxidative and inflammatory stress via suppressing CYP2E1, NF-κB and MAPK pathways. These results support that this triterpene is a potent preventive agent against acute alcoholic liver disease.
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