Protective Effect of Ellagic Acid on Concanavalin A ... - ACS Publications

Sep 19, 2014 - failure induced by intravenous injection of concanavalin A. (Con A) to mice is a well-known animal model of T-cell- dependent liver inj...
2 downloads 0 Views 5MB Size
Download PDF
Article pubs.acs.org/JAFC

Protective Effect of Ellagic Acid on Concanavalin A‑Induced Hepatitis via Toll-Like Receptor and Mitogen-Activated Protein Kinase/Nuclear Factor κB Signaling Pathways Jae Hong Lee, Jong Hoon Won, Jong Min Choi, Hye Hyeon Cha, Yeo Jin Jang, Seohyeon Park, Han Gyeol Kim, Hyung Chul Kim, and Dae Kyong Kim* Department of Environmental and Health Chemistry, College of Pharmacy, Chung-Ang University, 221 Huksuk-Dong, Dongjak-Ku, Seoul 156-756, South Korea ABSTRACT: Ellagic acid (EA) is present in certain fruits and nuts, including raspberries, pomegranates, and walnuts, and has anti-inflammatory and antioxidant properties. The aims of this study were to examine the protective effect of EA on concanavalin A (Con A)-induced hepatitis and to elucidate its underlying molecular mechanisms in mice. Mice were orally administered EA at different doses before the intravenous delivery of Con A; the different experimental groups were as follows: (i) vehicle control, (ii) Con A alone without EA, (iii) EA at 50 mg/kg, (iv) EA at 100 mg/kg, and (v) EA at 200 mg/kg. We found that EA pretreatment significantly reduced the levels of plasma aminotransferase and liver necrosis in Con A-induced hepatitis. Also, EA significantly decreased the expression levels of the toll-like receptor 2 (TLR2) and TLR4 mRNA and protein in liver tissues. Further, EA decreased the phosphorylation of JNK, ERK1/2, and p38. EA-treated groups showed suppressions of nuclear factor κB (NF-κB) and IκB-α degradation levels in liver tissues. In addition, EA pretreatment decreased the expression of proinflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin 1β (IL-1β). These results suggest that EA protects against T-cell-mediated hepatitis through TLR and mitogen-activated protein kinase (MAPK)/NF-κB signaling pathways. KEYWORDS: ellagic acid, hepatitis, concanavalin A, toll-like receptors, mitogen-activated protein kinase, nuclear factor κB

■

containing adaptors, including MyD88.17 Recruitment of MyD88 leads to the activation of mitogen-activated protein kinases [MAPKs; extracellular signal-regulated kinase (ERK), cJun N-terminal kinase (JNK), and p38] and the transcription factor nuclear factor κB (NF-κB) to control the expression of genes encoding inflammatory cytokines.14,18 This activation results in the production of active proinflammatory cytokines, chemokines, interferons, and enzymes. Ellagic acid (EA) is a naturally occurring plant phenol found in certain fruits, nuts, and vegetables, including raspberries, walnuts, and pomegranates. Over the past few years, a number of studies have shown the important pharmacological properties of EA, including its antioxidant, anti-inflammatory,19−21 and anticarcinogenic22,23 activities. In addition, recent studies have shown that EA prevents liver damage in Balb/C mice, as indicated by reduced cell death and decreased levels of glutathione (GSH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) after Con A-induced fulminant liver failure.24 However, the exact mechanism of this protective effect remains unclear. In the present study, we examined the protective effect of EA in a mouse model of Con A-induced hepatitis. We evaluated the expression levels of TLR2, TLR4, and inflammatory cytokines. We suggest that the anti-inflammatory effect of EA

INTRODUCTION Liver disease is a major cause of mortality and morbidity worldwide, affecting humans of all ages. Liver disease is characterized by three major forms, namely, viral hepatitis, autoimmune hepatitis, and alcoholic liver disease. Acute liver failure induced by intravenous injection of concanavalin A (Con A) to mice is a well-known animal model of T-celldependent liver injury, which resembles virus-induced immune damage.1,2 Recent studies indicate that toll-like receptor (TLR) signaling is involved in most liver diseases and Con A-induced hepatitis.3,4 This suggests that blockade of the TLR signaling pathway may be a therapeutic strategy for the treatment of liver disease.5,6 Previous studies have shown that the TLR2 and TLR4 subtypes are particularly involved in the inflammatory mechanisms in hepatitis.7,8 Furthermore, in comparison to wild-type mice, TLR2 and TLR4 knockout mice were protected from Con A-induced hepatic injury.9 Among pattern recognition receptors (PRRs), the TLR family is well-characterized in humans and mice. TLRs are widely expressed in monocytes, mast cells, dendritic cells, macrophages, neutrophils, NK cells, B cells, and T cells in the liver.10−13 Among the TLRs, TLR2 is unique for its ability to recognize bacterial peptidoglycans,14,15 while TLR4 is a major component of the lipopolysaccharide (LPS) recognition complex.14 The liver is continuously exposed to bacterial peptidoglycans and LPS from the intestines via the portal vein.16 After ligand binding, TLRs initiate downstream signaling via recruitment of a set of intracellular toll/interleukin 1 receptor (TIR) domain© 2014 American Chemical Society

Received: Revised: Accepted: Published: 10110

July 16, 2014 September 18, 2014 September 19, 2014 September 19, 2014 dx.doi.org/10.1021/jf503188c | J. Agric. Food Chem. 2014, 62, 10110−10117

Journal of Agricultural and Food Chemistry

Article

Figure 1. Hepatoprotective effects of EA in Con A-induced liver dysfunction. Mice were pretreated with EA (50, 100, or 200 mg/kg) at both 12 and 1 h before challenge with Con A. The control mice were given distilled water. (A) Serum ALT levels were determined 8 h after Con A injection. (B) Serum AST levels were determined 8 h after Con A injection. Bar represents the mean ± SD (n = 4 mice per group). (∗) p < 0.05 versus Con A. (C) Liver sections obtained 24 h after Con A injection were assessed by H&E staining (original magnification, 40×, and 200×). described.25 EA was dissolved in 0.01 N NaOH26 at concentrations of 5, 10, or 20 mg/mL and administered by gavage at doses of 50, 100, or 200 mg/kg of body weight.27,28 The mice were randomized into five groups (n = 4/group; total = 30) as follows: (i) sham group as the vehicle control, including oral administration of the phosphatebuffered saline (PBS) and a tail vein injection with the same volume of saline, (ii) Con A administration group that received no EA treatment, (iii) EA 50 (50 mg/kg) group, (iv) EA 100 (100 mg/kg) group, and (v) EA 200 (200 mg/kg) group. Mice, deprived of food 24 h before Con A treatment with free access to water, were administered with respective doses of EA at both 12 and 1 h before challenge with Con A. Liver samples were obtained 24 h after Con A injection. Serum Transaminase Activity Assays. Blood was obtained from individual mice at 8 h after Con A injection. Serum levels of AST and ALT were used as markers of liver injury. Alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) concentrations were measured using an IDTox ALT color endpoint assay kit and an IDTox

is involved in TLR2 and TLR4 expression and MAPK/NF-κB signaling pathways.

■

MATERIALS AND METHODS

Animals. Pathogen-free male BALB/c mice (8 weeks old; weight, 23 ± 2 g) were purchased from Orient Bio, Inc. (Seoul, Korea). The animals were acclimated for 1 week before the experiments. They were allowed free access to laboratory-standard food and water on a 12 h light/12 h dark cycle in a temperature (22 ± 2 °C) and humidity (50 ± 5%) controlled environment. All procedures and animal treatments were performed in a clean animal laboratory room according to the guidelines for laboratory animal experimentation specified by ChungAng University. This work was approved by the appropriate ethics committees for the use of laboratory animals at Chung-Ang University. Animal Treatment. Con A was dissolved in pyrogen-free normal saline at a concentration of 25 mg/mL and intravenously injected at a dose of 20 mg/kg of body weight to induce hepatitis, as previously 10111

dx.doi.org/10.1021/jf503188c | J. Agric. Food Chem. 2014, 62, 10110−10117

Journal of Agricultural and Food Chemistry

Article

Cruz Biotechnology) at a dilution of 1:2000. Each filter was then washed 3 times for 10 min and incubated for 2 h at room temperature with horseradish peroxidase (HRP)-labeled secondary anti-rabbit antibody (Cell Signaling Technology) containing blocking solution. To ensure equal loading, the blots were analyzed for GAPDH expression using an anti-GAPDH antibody (Santa Cruz Biotechnology). Immunodetection was performed using Amersham ECL western blotting detection reagents (GE Healthcare, U.K.). Densitometric data were studied after normalization with the control (housekeeping gene). The signals were analyzed and quantified using ImageJ software [National Institutes of Health (NIH), Bethesda, MD]. Liver Tissue Immunofluorescence. The liver tissues were fixed in 4% paraformaldehyde, dehydrated, paraffin-embedded, and cut into 4−5 μm thick sections. After antigen retrieval, the slides were stained with primary TLR2 or TLR4 antibodies (Santa Cruz) and a fluorescently labeled secondary antibody and counterstained with 4′6-diamidino-2-phenylindole (DAPI). Confocal images were collected in fluorescence mode followed by electronic image merging. Statistical Analysis. Results are represented as the mean ± standard deviation (SD) and were analyzed statistically with analysis of variance (ANOVA), and differences between groups were determined with the Turkey method. Statistical significance was accepted when p < 0.05.

AST color endpoint assay kit (ID Laboratories, Inc., London, Ontario, Canada), respectively, according to the instructions of the manufacturer, and absorbance was measured using a spectrophotometer. Histopathological Examination of the Liver. The liver tissues were excised, fixed in 10% neutral buffered formalin, and embedded in paraffin. We prepared 4 μm thick slices stained with hematoxylin and eosin (H&E) according to standard procedures. The slides were examined using light microscopy. RNA Isolation, cDNA Synthesis, and Real-Time Quantitative Polymerase Chain Reaction (qPCR) Amplification. Liver tissues were stored at −80 °C until required. RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). For real-time qPCR, 0.1 μg of total RNA was reverse-transcribed using a SuperScript III First-Strand Synthesis System for reverse transcription (RT)-PCR (Invitrogen). Real-time PCR was performed in triplicate using the MyiQ SingleColor Real-Time PCR Detection System and an iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). Relative expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels. The primer sequences used were as follows: GAPDH, forward, 5-TTC ACC ACC ATG GAG AAG GC-3 and reverse, 5-GGC ATG GAC TGT GGT CAT GA-3; TLR2, forward, 5-AAG ATG TCG TTC AAG GAG GTG CG-3 and reverse, 5-ATC CTC TGA GAT TTG ACG CTT TG-3; TLR4, forward, 5-GGT GTG AAA TTG AGA CAA TTG AAA AC-3 and reverse, 5-GTT TCC TGT CAG TAC CAA GGT TGA-3; tumor necrosis factor α (TNF-α), forward, 5-GGC AGG TCT ACT TTG GAG TC-3 and reverse, ACA TTC GAG GCT CCA GTG AA-3; interleukin 6 (IL-6), forward, 5-ACC GCT ATG AAG TTC CTC TC-3 and reverse, 5-CCT CTG TGA AGT CTC CTC TC-3; and interleukin 1β (IL-1β), forward, 5-TCA TGG GAT GAT GAT GAT AAC CTG CT-3 and reverse, 5-CCC ATA CTT TAG GAA GAC ACG GAT T-3. The thermal cycling conditions were as follows: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 10 s, annealing at 58 °C for 15 s, and extension at 72 °C for 20 s. Isolation of Cytoplasmic and Nuclear Proteins and Immunoblotting. Frozen liver tissues were weighed and homogenized in ice-cold hypotonic buffer [25 mM Tris (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 2 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.1% Triton-X 100, phosphatase inhibitor cocktail tablets, and protease inhibitor cocktail tablets]. Homogenates were incubated for 10 min on ice and centrifuged (12500g for 10 min at 4 °C). Cytoplasmic proteins were collected from the supernatants, and nuclear proteins were collected from the pellets. Cytoplasmic fractions were stored at −70 °C. Nuclear proteins were released by dropwise addition of a high-salt buffer [25 mM Tris (pH 7.5), 500 mM NaCl, 10% glycerol, 1 mM EDTA, 2 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM PMSF, 0.2% Triton-X 100, phosphatase inhibitor cocktail tablets, and protease inhibitor cocktail tablets]. Samples were incubated on ice for 30 min with smooth shaking. The nuclei-containing suspension was then centrifuged (16000g for 30 min at 4 °C). The supernatant fraction was collected as the nuclear extract. This extract was stored in aliquots at −70 °C for the subsequent NFκB assay. Protein concentrations of the homogenate were determined using the BCA protein assay with bovine serum albumin as a standard. Equal amounts of whole protein extracts (50 μg) were separated on 10% acrylamide gels using sodium dodecyl sulfate−polyacrylamide gel electrophoresis. Then, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and incubated overnight at 4 °C with specific primary antibodies, namely, rabbit polyclonal anti-TLR2 (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:500, rabbit polyclonal anti-TLR4 (Santa Cruz Biotechnology) at a dilution of 1:500, rabbit polyclonal anti-JNK and anti-p-JNK (Cell Signaling Technology, Beverly, MA) at a dilution of 1:2000, rabbit polyclonal anti-ERK and anti-p-ERK (Cell Signaling Technology) at a dilution of 1:2000, rabbit polyclonal anti-p38 and anti-p-p38 (Cell Signaling Technology) at a dilution of 1:2000, rabbit polyclonal anti-IκB-α (Santa Cruz Biotechnology) at a dilution of 1:2000, rabbit polyclonal anti-p65 (Cell Signaling Technology) at a dilution of 1:2000, and rabbit polyclonal anti-inducible nitric oxide synthase (iNOS, Santa

■

RESULTS Hepatoprotective Effect of EA on Con A-Induced Hepatitis. Acute hepatitis was induced in BALB/c mice by

Figure 2. Effect of EA on the Con A-induced expression of TLR2 and TLR4 in mouse liver. Hepatic TLR2 and TLR4 protein levels were determined by immunoblotting. Densitometry was performed following normalization to the control (GADPH). Bar represents the mean ± SD (n = 4 mice per group). (∗) p < 0.05 versus Con A. (∗∗) p < 0.01 versus Con A. 10112

dx.doi.org/10.1021/jf503188c | J. Agric. Food Chem. 2014, 62, 10110−10117

Journal of Agricultural and Food Chemistry

Article

Effects of EA on the TLR2 and TLR4 Expression in Con A-Induced Hepatitis. We examined the effect of EA on the expression of TLR2 and TLR4 in Con A-induced hepatitis. We observed very low expression levels of TLR2 and TLR4 in the vehicle group. However, the Con A-treated group showed a significant increase of TLR2 and TLR4 expression levels (Figures 2 and 3). TLR2 and TLR4 were highly expressed, which suggested that both receptors were involved in the pathological sequelae of Con A-induced acute liver injury. EAtreated groups showed reduced expression levels of TLR2 and TLR4 in a dose-dependent manner (Figures 2 and 3). In immunofluorescence analysis, TLR2 and TLR4 were reduced in EA-treated groups in a dose-dependent manner (Figure 4). Effect of EA on the JNK, ERK1/2, and p38 Signaling Pathways in Con A-Induced Hepatitis. TLR signaling pathways are involved in the activation of MAPKs. These molecules play a key role in inducing gene expression, which initiates inflammatory responses. We investigated the effect of EA on the Con A-induced hepatitis-associated activation of JNK, ERK1/2, and p38. We detected JNK, ERK1/2, and p38 MAPK protein phosphorylation using western blotting in the cytosolic extracts of liver tissues using phospho-specific MAPK antibodies. Con A treatment increases phosphorylation of JNK, ERK1/2, and p38; however, the EA treatment group showed a strong inhibition of phosphorylation of JNK, ERK1/2, and p38 (panels A−C of Figure 5). These results suggested that the activation of JNK, ERK1/2, and p38 could be induced at the acute stage of hepatitis caused by Con A and treatment with EA significantly decreased the phosphorylation levels of p38, JNK, and ERK1/2. EA Inhibits NF-κB Activation and IκB-α Degradation in Con A-Induced Hepatitis. We further investigated the effect of EA on NF-κB activation and IκB degradation in a mouse model of Con A-induced hepatitis. EA pretreatment inhibits NF-κB activation and IκB-α degradation in an EA dosedependent manner (panels A and B of Figure 6). EA Pretreatment Inhibits the Expression of Proinflammatory Cytokines and iNOS in Con A-Induced Hepatitis. TNF-α, IL-6, and IL-1β have been shown to play critical roles in the progression of Con A-induced liver injury. To investigate whether EA influences the production of these cytokines, we measured the mRNA expression of TNF-α, IL-6, and IL-1β in Con A-induced hepatitis. The levels of TNF-α, IL6, and IL-1β mRNA significantly increased in the control group

Figure 3. Effect of EA treatment on the expression of (A) TLR2 and (B) TLR4 mRNAs in the liver was examined by real-time qPCR. TLR2 and TLR4 mRNA levels were normalized to GAPDH expression in each sample. Bar represents the mean ± SD (n = 4 mice per group). (∗) p < 0.05 versus Con A.

intravenous injection of Con A. Plasma levels of AST and ALT were significantly increased at 8 h after injection of Con A (Figure 1). In comparison to the vehicle-treated group, the groups receiving oral administration of EA at doses of 50, 100, or 200 mg/kg at both 12 and 1 h before Con A injection showed a significant and dose-dependent decrease in the plasma levels of AST and ALT. Histopathological examination of the liver tissue was used to determine the effects of EA on Con A-induced liver injury. Light microscopy examination showed extensive inflammatory infiltration and large areas of necrosis in the livers of Con A-treated mice. In contrast, mice pretreated with EA showed dose-dependent reduction in liver damage (Figure 1C). This histopathologic evidence of liver damage was positively correlated with increased serum levels of liver enzymes. Taken together, pretreatment with EA showed a protective effect on Con A-induced hepatitis.

Figure 4. TLR2 and TLR4 localization in murine liver by immunofluorescence microscopy after administration of Con A for 24 h (original magnification and 300×). 10113

dx.doi.org/10.1021/jf503188c | J. Agric. Food Chem. 2014, 62, 10110−10117

Journal of Agricultural and Food Chemistry

Article

Figure 6. Effect of EA on the NF-κB pathway in Con A-induced liver injury. Hepatic (A) NF-κB and (B) IκB-α protein levels determined by immunoblotting. Densitometry was performed following normalization to the control (GADPH). Bar represents the mean ± SD (n = 4 mice per group). (∗) p < 0.05 versus Con A. (∗∗) p < 0.01 versus Con A. (∗∗∗) p < 0.001 versus Con A.

the liver to Con A caused strong iNOS expression, which indicated that these proteins could be induced in the acute stage of hepatitis. Pretreatment of EA significantly decreased this upregulation of iNOS protein expression in a dosedependent manner (Figure 7D).

■

DISCUSSION A recent study showed that the ALT and AST levels decreased after treatment with EA in Balb/C mice after Con A-induced fulminant liver failure.24 However, the exact mechanism underlying this effect remains unclear. To determine the mechanisms underlying the protective effects of EA, we examined the TLR pathway. TLR expression may be associated with the production of proinflammatory cytokines because the activation of these receptors stimulates signaling pathways, which result in activation of NF-κB and MAPK phosphorylation (Figure 8). Con A-induced hepatitis is an experimental model of immune-mediated liver disease in humans.29 Con A was originally identified as a mitogen in T lymphocytes and activates macrophages and neutrophils.30,31 The hepatitis induced by intravenous injection of Con A is mainly dependent upon T-cell activation.2 Hepatic injury caused by Con A disrupts the hepatocellular plasma membrane, and the enzymes

Figure 5. Hepatic (A) p-JNK, (B) p-ERK, and (C) p-p38 protein levels determined by immunoblotting. Densitometry was performed following normalization to the control (JNK, ERK, and p38 housekeeping genes, respectively). Bar represents the mean ± SD (n = 4 mice per group) (∗) p < 0.05 versus Con A. (∗∗) p < 0.01 versus Con A. (∗∗∗) p < 0.001 versus Con A.

(panels A−C of Figure 7). EA pretreatment decreased the expression of TNF-α, IL-6, and IL-1β in the livers of Con Atreated groups. These data suggest that EA suppresses the expression of inflammatory cytokines in Con A-induced hepatitis. iNOS is a protein that is predominantly expressed on the sites of inflammation. We measured the cytosolic iNOS expression levels in the liver using western blotting. Exposure of 10114

dx.doi.org/10.1021/jf503188c | J. Agric. Food Chem. 2014, 62, 10110−10117

Journal of Agricultural and Food Chemistry

Article

Figure 8. Hypothetic mechanism of the EA protective effect against hepatitis via TLR and MAPK/NF-κB signaling pathways. EA decreased TLR2 and TLR4 levels (Figures 2−4). EA decreased the phosphorylation of JNK, ERK1/2, and p38 (Figure 5). EA suppressed NF-κB and increased IκB-α (Figure 6). EA suppressed inflammatory cytokines (Figure 7).

activity of EA. In the previous studies, EA was administered at doses of 50, 100, or 200 mg/kg of body weight.27,28 In this study, we set the optimal dose range of the EA (0, 50, 100, or 200 mg/kg of body weight) and have shown the hepatoprotective effect of EA. Additionally, on the basis of previous studies,33−38 we chose the optimal time points of EA treatment at 12 and 1 h prior to an injection of Con A. Soyasapogenol A, which has protective effects on a Con Ainduced hepatitis model, was administered at 2 and 14 h prior to an intravenous injection of Con A. In other studies, therapeutic compounds, including allicin, Inchinko-to, and coumarin derivatives, were treated 1 h before Con A injection. Additionally, the Gal-3 inhibitor, which attenuates Con Ainduced hepatitis, was intraperitoneally administered 2 h before and immediately after Con A injection.33−38 Previous studies indicated that the pathogenesis of Con Ainduced hepatitis was mainly mediated by the release of inflammatory cytokines. TNF-α, IL-6, and IL-1β play crucial roles in the development of Con A-induced hepatitis.39,40 These studies suggested that inhibition of TNF-α, IL-6, and IL1β tended to alleviate liver injury. In the present study, we found that pretreatment with EA decreased the hepatic mRNA expression of TNF-α, IL-6, and IL-1β in Con A-induced liver injury. Therefore, we propose that EA exerts its hepatoprotective effects through the inhibition of TNF-α, IL-6, and IL-1β production. In a recent study, the TLR signaling pathway was shown to be the predominant pathway in the early phase in a model of Con A-induced hepatitis.4 The expression of TLR2 and TLR4 was increased in the liver during Con A-induced hepatitis.1 Furthermore, TLR signaling stimulates the production of type I cytokines and, thus, recruits and activates T/NK cells. Activated T/NK cells exert cytotoxic effects on hepatocytes through induction of death receptor-mediated apoptosis, which results in liver injury. Activation of TLR2 and TLR4 results in the recruitment of MyD88, an adaptor molecule, which subsequently facilitates the

Figure 7. Effect of EA on the expression of proinflammatory cytokines and iNOS protein in the liver after Con A administration. Expression of (A) TNF-α, (B) IL-6, and (C) IL-1β mRNAs in the liver were examined by real-time qPCR. TNF-α, IL-6, and IL-1β mRNA levels were normalized to GAPDH expression in each sample. (D) iNOS protein levels determined by immunoblotting. Densitometry was performed following normalization to the control (GADPH). Bar represents the mean ± SD (n = 4 mice per group). (∗) p < 0.05 versus Con A.

normally present in the cytosol are released into the bloodstream.32 In the present study, the ALT and AST levels in the Con A-treated mice returned to normal after pretreatment with EA, which showed the hepatoprotective 10115

dx.doi.org/10.1021/jf503188c | J. Agric. Food Chem. 2014, 62, 10110−10117

Journal of Agricultural and Food Chemistry

Article

therefore represent potential targets for inhibiting the progression of liver fibrosis. Similarly, inhibition of the phosphorylation of JNK, ERK, and p38 has protective effects in hepatitis. To our knowledge, this is the first study in which the protective effect of EA mediated by the inhibition of p38, JNK, and ERK MAPKs in hepatitis has been described. In conclusion, we showed that EA attenuates Con A-induced hepatitis. One of the possible mechanisms underlying this effect is inhibition of TLR2 and TLR4 expression, which may decrease the expression of inflammatory cytokines and, in turn, inhibit the phosphorylation of MAPK and nuclear translocation NF-κB. This may account for a reduction in TNF-α, IL-6, and IL-1β expression by inhibition of NF-κB-mediated transcriptional activation and IκB-α degradation and prevention of p38, JNK, and ERK MAPK phosphorylation. Therefore, our findings suggest that TLR2, TLR4, NF-κB, and MAPK may be the specific molecular targets of EA in its protective effects against Con A-induced hepatitis.

activation of downstream signaling pathways. Two major kinase-mediated signaling pathways are activated after TLR stimulation, the MAPK and IκB kinase (IKK) complex pathways, which transduce various upstream signals to the activation of AP-1 and NF-κB transcription factors. Both of these are essential for the expression of activation-induced inflammatory mediators, including chemokines and cytokines.17 Recent studies have shown that, in comparison to wild-type mice, TLR2 or TLR4 knockout mice are protected from Con A-induced hepatic injury.9,41 Furthermore, MyD88-knockout mice develop less severe Con A-induced hepatitis with less production of proinflammatory cytokines.42 These studies have provided evidence that TLR2 and TLR4 play an important role in Con A-induced hepatitis. Intervention via the regulation of TLR2 and TLR4 expression may therefore represent a therapeutic target for Con A-induced hepatitis. In the present study, we showed that TLR2 and TLR4 expression in liver tissues, at the mRNA and protein levels, was significantly reduced by EA pretreatment (Figures 2−4). This might be attributed, at least in part, to the amelioration of liver injury and changes in cytokine production in mice. These data are new and relevant, because the effect of EA on these receptors has not been reported thus far. We propose that EA may have general anti-inflammatory effects in diseases associated with TLR signaling. The liver is constantly exposed to bacterial peptidoglycans and LPS from the intestines via the hepatic portal vein.16 After ligand binding, the TLRs activate downstream signaling pathways via recruitment of a group of intracellular TIRdomain-containing adaptors, including MyD88.17 Recruitment of MyD88 leads to the activation of MAPKs, including ERK, JNK, and p38, and the transcription factor NF-κB, all of which regulate the expression of genes encoding the inflammatory cytokines.14,18 The transcription factor NF-κB consists of a heterodimer of p50 and p65, which is retained in the cytoplasm via the masking of a nuclear localization signal by the inhibitor IκB-α. Upon activation, IKK phosphorylates IκB-α, which promotes its translocation to the nucleus and results in the expression of various proinflammatory cytokines, including IL-2, IL-6, IL-8, IL-1β, and TNF-α.43 In addition, NF-κB activation mediates the transcription of proinflammatory genes, such as iNOS. Many studies have suggested that NF-κB may be a key regulator of inflammation in liver injury.44 Thus, inflammatory mechanisms play an important role in T-cell-mediated liver disease associated with increased activity of NF-κB. Inhibiting the activation of NF-κB exerts protective effects against liver injury; therefore, we propose that EA acts on NF-κB via the inhibition of IκB-α, which results in the prevention of NF-κB translocation into the nucleus in acute hepatitis. Furthermore, our findings show that EA can reduce the expression of the proinflammatory gene iNOS. Other important downstream signaling targets of TLRs are the MAPKs. The three major groups of MAPKs identified in mammalian cells are p38 MAPK, JNK kinase, and ERK.45 MAPKs are key modulators of several target genes that ultimately regulate innate and adaptive immunity, cell motility, and cell proliferation.46,47 Three different types of MAPKs contribute to the induction of AP-1 activity through phosphorylation of a different substrate. Activator protein (AP)-1 largely controls T-cell activation in the liver after binding of foreign antigens to the T-cell receptor, which leads to the secretion of cytokines, such as TNF-α, IL-6, and IL-1β.46,47 ERK, JNK, and p38 MAPK pathways may

■

AUTHOR INFORMATION

Corresponding Author

*Telephone: 82-2-820-5610. Fax: 82-2-3280-5610. E-mail: [email protected]. Funding

This research was supported by the Chung-Ang University Excellent Student Scholarship and the Public Welfare and Safety Research Program through the National Research Foundation of Korea (NRF), as funded by the Ministry of Science, ICT and Future Planning (NRF-2010-0020844). Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Ajuebor, M. N.; Carey, J. A.; Swain, M. G. CCR5 in T cellmediated liver diseases: What’s going on? J. Immunol. 2006, 177 (4), 2039−2045. (2) Tiegs, G.; Hentschel, J.; Wendel, A. A T cell-dependent experimental liver injury in mice inducible by concanavalin A. J. Clin. Invest. 1992, 90 (1), 196−203. (3) Ojiro, K.; et al. MyD88-dependent pathway accelerates the liver damage of concanavalin A-induced hepatitis. Biochem. Biophys. Res. Commun. 2010, 399 (4), 744−749. (4) Cao, Q. Y.; et al. A microarray analysis of early activated pathways in concanavalin A-induced hepatitis. J. Zhejiang Univ., Sci., B 2010, 11 (5), 366−377. (5) Tu, C. T.; et al. Curcumin attenuates concanavalin A-induced liver injury in mice by inhibition of toll-like receptor (TLR)2, TLR4 and TLR9 expression. Int. Immunopharmacol. 2012, 12 (1), 151−157. (6) Chen, Y.; Sun, R. Toll-like receptors in acute liver injury and regeneration. Int. Immunopharmacol. 2011, 11 (10), 1433−1441. (7) Soares, J. B.; et al. Increased hepatic expression of TLR2 and TLR4 in the hepatic inflammation-fibrosis-carcinoma sequence. Innate Immun. 2012, 18 (5), 700−708. (8) Berzsenyi, M. D.; et al. Hepatic TLR2 and TLR4 expression correlates with hepatic inflammation and TNF-α in HCV and HCV/ HIV infection. J. Viral Hepatitis 2011, 18 (12), 852−860. (9) Xu, J.; et al. Extracellular histones are mediators of death through TLR2 and TLR4 in mouse fatal liver injury. J. Immunol. 2011, 187 (5), 2626−2631. (10) Bourke, E.; et al. The toll-like receptor repertoire of human B lymphocytes: Inducible and selective expression of TLR9 and TLR10 in normal and transformed cells. Blood 2003, 102 (3), 956−963. (11) Bosisio, D.; et al. Stimulation of toll-like receptor 4 expression in human mononuclear phagocytes by interferon-γ: A molecular basis for 10116

dx.doi.org/10.1021/jf503188c | J. Agric. Food Chem. 2014, 62, 10110−10117

Journal of Agricultural and Food Chemistry

Article

priming and synergism with bacterial lipopolysaccharide. Blood 2002, 99 (9), 3427−3431. (12) Kadowaki, N.; et al. Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J. Exp. Med. 2001, 194 (6), 863−869. (13) Muzio, M.; et al. Differential expression and regulation of tolllike receptors (TLR) in human leukocytes: Selective expression of TLR3 in dendritic cells. J. Immunol. 2000, 164 (11), 5998−6004. (14) Schwabe, R. F.; Seki, E.; Brenner, D. A. Toll-like receptor signaling in the liver. Gastroenterology 2006, 130 (6), 1886−1900. (15) Akira, S.; Uematsu, S.; Takeuchi, O. Pathogen recognition and innate immunity. Cell 2006, 124 (4), 783−801. (16) Seki, E.; Brenner, D. A. Toll-like receptors and adaptor molecules in liver disease: Update. Hepatology 2008, 48 (1), 322−335. (17) Akira, S.; Takeda, K. Toll-like receptor signalling. Nat. Rev. Immunol. 2004, 4 (7), 499−511. (18) Antosz, H.; Choroszynska, D. Negative regulation of toll-like receptor signalling. Postepy Hig. Med. Dosw. 2013, 67, 339−350. (19) Suzuki, N.; et al. Ellagic acid inhibits pancreatic fibrosis in male Wistar Bonn/Kobori rats. Dig. Dis. Sci. 2009, 54 (4), 802−810. (20) Chao, P. C.; Hsu, C. C.; Yin, M. C. Anti-inflammatory and anticoagulatory activities of caffeic acid and ellagic acid in cardiac tissue of diabetic mice. Nutr. Metab. 2009, 6, 33. (21) Rogerio, A. P.; et al. Anti-inflammatory effects of Lafoensia pacari and ellagic acid in a murine model of asthma. Eur. J. Pharmacol. 2008, 580 (1−2), 262−270. (22) Bell, C.; Hawthorne, S. Ellagic acid, pomegranate and prostate cancerA mini review. J. Pharm. Pharmacol. 2008, 60 (2), 139−144. (23) Falsaperla, M.; et al. Support ellagic acid therapy in patients with hormone refractory prostate cancer (HRPC) on standard chemotherapy using vinorelbine and estramustine phosphate. Eur. Urol. 2005, 47 (4), 449−454. (24) Hwang, J. M.; et al. Ellagic acid protects hepatocytes from damage by inhibiting mitochondrial production of reactive oxygen species. Biomed. Pharmacother. 2010, 64 (4), 264−270. (25) Bozza, M.; et al. Interleukin-11 reduces T-cell-dependent experimental liver injury in mice. Hepatology 1999, 30 (6), 1441− 1447. (26) Atessahin, A.; et al. Modulatory effects of lycopene and ellagic acid on reproductive dysfunction induced by polychlorinated biphenyl (Aroclor 1254) in male rats. Basic Clin. Pharmacol. Toxicol. 2010, 106 (6), 479−489. (27) Girish, C.; Pradhan, S. C. Hepatoprotective activities of picroliv, curcumin, and ellagic acid compared to silymarin on carbontetrachloride-induced liver toxicity in mice. J. Pharmacol. Pharmacother. 2012, 3 (2), 149−155. (28) Singh, K.; Jaggi, A. S.; Singh, N. Exploring the ameliorative potential of Punica granatum in dextran sulfate sodium induced ulcerative colitis in mice. Phytother. Res. 2009, 23 (11), 1565−1574. (29) Kaneko, Y.; et al. Augmentation of Vα14 NKT cell-mediated cytotoxicity by interleukin 4 in an autocrine mechanism resulting in the development of concanavalin A-induced hepatitis. J. Exp. Med. 2000, 191 (1), 105−114. (30) Cameron, A. R.; Nelson, J.; Forman, H. J. Depolarization and increased conductance precede superoxide release by concanavalin Astimulated rat alveolar macrophages. Proc. Natl. Acad. Sci. U. S. A. 1983, 80 (12), 3726−3728. (31) Boldt, D. H.; MacDermott, R. P.; Jorolan, E. P. Interaction of plant lectins with purified human lymphocyte populations: Binding characteristics and kinetics of proliferation. J. Immunol. 1975, 114 (5), 1532−1536. (32) Rajesh, M. G.; Latha, M. S. Preliminary evaluation of the antihepatotoxic activity of Kamilari, a polyherbal formulation. J. Ethnopharmacol. 2004, 91 (1), 99−104. (33) Volarevic, V.; et al. Galectin-3 deficiency prevents concanavalin A-induced hepatitis in mice. Hepatology 2012, 55 (6), 1954−1964. (34) Itoh, A.; et al. Hepatoprotective effect of syringic acid and vanillic acid on concanavalin A-induced liver injury. Biol. Pharm. Bull. 2009, 32 (7), 1215−1219.

(35) Bruck, R.; et al. Allicin, the active component of garlic, prevents immune-mediated, concanavalin A-induced hepatic injury in mice. Liver Int. 2005, 25 (3), 613−621. (36) Okamoto, T.; et al. Inhibition of concanavalin A-induced mice hepatitis by coumarin derivatives. Jpn. J. Pharmacol. 2001, 85 (1), 95− 97. (37) Yamashiki, M.; et al. Effects of the Japanese herbal medicine ‘Inchinko-to’ (TJ-135) on concanavalin A-induced hepatitis in mice. Clin. Sci. 2000, 99 (5), 421−431. (38) Kuzuhara, H.; et al. Protective effects of soyasapogenol A on liver injury mediated by immune response in a concanavalin A-induced hepatitis model. Eur. J. Pharmacol. 2000, 391 (1−2), 175−181. (39) Sass, G.; et al. Cytokine expression in three mouse models of experimental hepatitis. Cytokine 2002, 19 (3), 115−120. (40) Kesherwani, V.; Sodhi, A. Quantitative role of p42/44 and p38 in the production and regulation of cytokines TNF-α, IL-1β and IL-12 by murine peritoneal macrophages in vitro by concanavalin A. Cytokine 2007, 37 (1), 62−70. (41) Gong, Q. A.; et al. High-mobility group box 1 exacerbates concanavalin A-induced hepatic injury in mice. J. Mol. Med. 2010, 88 (12), 1289−1298. (42) Ojiro, K.; et al. MyD88-dependent pathway accelerates the liver damage of concanavalin A-induced hepatitis. Biochem. Biophys. Res. Commun. 2010, 399 (4), 744−749. (43) Ghosh, S.; Hayden, M. S. New regulators of NF-κB in inflammation. Nat. Rev. Immunol. 2008, 8 (11), 837−848. (44) He, G.; Karin, M. NF-κB and STAT3Key players in liver inflammation and cancer. Cell Res. 2011, 21 (1), 159−168. (45) De Keulenaer, G. W.; et al. Convergence of redox-sensitive and mitogen-activated protein kinase signaling pathways in tumor necrosis factor-α-mediated monocyte chemoattractant protein-1 induction in vascular smooth muscle cells. Arterioscler., Thromb., Vasc. Biol. 2000, 20 (2), 385−391. (46) Wang, X. J.; et al. B7-H4 treatment of T cells inhibits ERK, JNK, p38, and AKT activation. PLoS One 2012, DOI: 10.1371/journal.pone.0028232. (47) Plotnikov, A.; et al. The MAPK cascades: Signaling components, nuclear roles and mechanisms of nuclear translocation. Biochim. Biophys. Acta, Mol. Cell Res. 2011, 1813 (9), 1619−1633.

10117

dx.doi.org/10.1021/jf503188c | J. Agric. Food Chem. 2014, 62, 10110−10117