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Aug 20, 2013 - anethole, 1), a major component of Foeniculum vulgare, and its molecular .... trauma.17 ROS may play an important role in hepatic injur...
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Protective Mechanism of Anethole on Hepatic Ischemia/Reperfusion Injury in Mice Hong-Ik Cho,† Kang-Min Kim,† Jong Hwan Kwak,† Sang Kook Lee,‡ and Sun-Mee Lee*,† †

School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, 440-746, Republic of Korea School of Pharmacy, Seoul National University, Seoul, 151-742, Republic of Korea

‡

ABSTRACT: The aim of this study was to investigate the hepatoprotective effect of anethole (transanethole, 1), a major component of Foeniculum vulgare, and its molecular mechanism during ischemia/ reperfusion (I/R). Mice were subjected to 60 min of partial hepatic ischemia followed by 1 and 6 h of reperfusion. Compound 1 (50, 100, and 200 mg/kg) or the vehicle alone (10% Tween 80-saline) was orally administered 1 h prior to ischemia. After 1 and 6 h of reperfusion, serum alanine aminotransferase, tumor necrosis factor-α, and interleukin 6 levels significantly increased, but these increases were attenuated by 1. Nuclear translocation of interferon regulatory factor (IRF)-1, release of high mobility group box (HMGB) 1 into the extracellular milieu, and the interactions between IRF-1 and histone acetyltransferase p300 increased after I/R. These increases were attenuated by 1. Compound 1 suppressed increases in toll-like receptor (TLR) 4, myeloid differentiation primary response gene 88 protein expression, phosphorylation of p38, extracellular signal-regulated kinase, c-Jun N-terminal kinase, nuclear translocation of nuclear factor kappa B, and phosphorylated c-Jun. The present findings suggest that 1 protects against hepatic I/R injury by suppression of IRF-1-mediated HMGB1 release and subsequent TLR activation.

I

nitric oxide synthase (iNOS).7 However, the source of HMGB1 acetylation and its release from the nucleus during I/R injury is unclear. Interferon regulatory factor (IRF)-1 was originally identified as a key factor in interferon expression regulating many cellular processes. IRF-1 is involved in the pathogenesis of many inflammatory disorders including I/R and hepatitis.8,9 Interestingly, a recent investigation demonstrated that activation of IRF-1 contributes to HMGB1 acetylation and release from hepatocytes during I/R injury.10 Foeniculum vulgare Mill. (Apiaceae) has been used widely as a remedy for several diseases because of its anti-inflammatory, antifungal, and hepatoregenerative activities.11 F. vulgare essential oil showed its antioxidative effects by antagonizing the reduction of cyclophosphamide-induced superoxide dismutase, catalase, and glutathione activities and attenuating increased malondialdehyde content in the mouse liver.12 Moreover, F. vulgare suppresses the inflammation response in the liver and prevents the progression of hepatic fibrosis in carbon tetrachloride-induced liver injury.13 Anethole (trans-1methoxy-4-(1-propenyl)benzene, 1), a major component of F. vulgare, has been demonstrated to have anti-inflammatory properties in lipopolysaccharide-treated RAW 264.7 cells.14 Compound 1 and its derivative, anethole ditholethione, are known to possess antioxidant effects that increase the intracellular levels of glutathione.15 Moreover, 1 blocks the inflammatory response by inhibiting nuclear factor kappa B (NF-κB), activator protein factor (AP)-1, mitogen-activated protein kinases (MAPK), and TNF-induced cellular mechanisms.16

schemia/reperfusion (I/R) injury is responsible for organ damage in a variety of pathological events such as myocardial infarction, stroke, and graft failure after organ transplantation. I/R injury is a complex phenomenon involving an injurious inflammatory response that includes direct cellular damages by ischemia and delayed damages by reperfusion resulting from activation of the innate immune system.1 Innate immunity is the initial, rapid response to potentially dangerous stimuli, including pathogens, tissue injury, stress, and malignancy, and it is central to the inflammatory response.2 Many factors of innate immunity are produced and localized in the vertebrate liver, suggesting that this organ is an important site for innate immunity and systemic inflammation.3 Accumulating evidence indicates that excessive production of reactive oxygen species (ROS) during reperfusion causes cellular damage by direct attack on various cellular molecules and by indirect promotion of synthesis of proinflammatory mediators. Excessive ROS during the initial phase of reperfusion have been shown recently to act as signaling molecules inducing release of endogenous damage-associated molecular patterns (DAMPs), which are responsible for propagation of the inflammatory response4 and interact with certain pattern recognition receptors (PRRs), especially toll-like receptors (TLRs), to activate the innate immune system.5 Pardo et al. reported that the endogenous antioxidant defense in hepatic I/R injury is upregulated by DAMPs.6 High-mobility group protein box (HMGB) 1, acting as an early inflammatory mediator activating innate immune cells, has been implicated in TLR4 as a potent alarmin. Nuclear HMGB1 is rapidly acetylated and released from hepatocytes to function as a DAMP molecule in extracellular stress. HMGB1 release increases in hepatic I/R, and neutralizing antibody to HMGB1 decreases production of inflammatory mediators, including tumor necrosis factor (TNF), interleukin (IL)-6, and inducible © 2013 American Chemical Society and American Society of Pharmacognosy

Received: May 31, 2013 Published: August 20, 2013 1717

dx.doi.org/10.1021/np4004323 | J. Nat. Prod. 2013, 76, 1717−1723

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This study was conducted to investigate the effects of 1 on I/ R injury and the specific molecular mechanisms of protection, particularly on IRF-1 activation, HMGB1 release, and the subsequent TLR4-mediated inflammatory response.

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RESULTS AND DISCUSSION Serum Alanine Aminotransferase Activity (ALT) and Histological Analysis. Hepatic I/R, an exogenous antigenindependent inflammatory event, has been implicated in the pathogenesis of a variety of clinical conditions, including liver transplantation, liver resection, hypovolemic shock, and trauma.17 ROS may play an important role in hepatic injury associated with I/R and initiate lipid peroxidation, resulting in structural and functional changes to the organelles. As a result of hepatic injury, the permeability of the cell membrane is altered, causing enzymes from the cells to be released into circulation, as shown by the abnormally high level of serum hepatospecific enzymes. In the I/R group, the level of serum ALT, a marker of hepatocellular damage, increased approximately 80-fold at 1 h after reperfusion and further increased to 423-fold at 6 h after reperfusion. These increases were attenuated by pretreatment with 1 at 50, 100, and 200 mg/kg (Table 1). Compound 1 at 100 mg/kg was selected as the

Figure 1. Effect of anethole (1) on the histological changes in the liver at 6 h after reperfusion. Mice were orally administered 100 mg/kg anethole 1 h before inducing ischemia (n = 8−10). Typical images were chosen from each experimental group (original magnification 200×). (A) sham; (B) 1 + sham, normal hepatic architecture; (C) I/R, extensively damaged hepatocytes; (D) 1 + I/R, mild hepatocellular damages and inflammatory infiltration.

rapidly produced by macrophages in response to tissue damage. Increased TNF-α levels have been directly correlated with a histological evidence of hepatic necrosis and an increase in serum aminotransferase levels.19 IL-6, a typical inflammatory cytokine also produced by Kupffer cells in response to liver damage including I/R, plays a critical role in the acute phase response in the liver.20 In the I/R group, the level of serum TNF-α dramatically increased to 2.6-fold at 1 h after reperfusion and 4.5-fold at 6 h after reperfusion; both of these increases were attenuated by 1. Similar to the TNF-α level, I/R resulted in a significant increase in the serum IL-6 levels at both 1 and 6 h after reperfusion. As with TNF-α, these increases were attenuated by 1 (Table 2). TLR4 and MyD88 Protein Expression. TLRs, a family of PRRs, are central to the inflammatory response and culminate in expression of inflammatory gene products, including cytokines and chemokines.21 Myeloid differentiation primary response gene 88 (MyD88) is one of the downstream adaptor molecules of TLR4, activating MAPKs and transcription factor NF-κB.22 There is considerable evidence that I/R, specific to the liver, can induce TLR4- and MyD88-dependent inflammation and organ injury.20 Indeed, antioxidative or antiinflammatory agents, including bicyclol, N-acetylcysteine, and neutrophil elastase inhibitor sivelestat, showed protective effects through hepatic I/R by downregulation of TLR4.23−25 At 6 h after reperfusion, the levels of TLR4 and MyD88 protein expression increased to 2.2-fold and 1.7-fold of sham, respectively. Compound 1 attenuated the increases in TLR4 and MyD88 protein expression (Figure 2). MAPK Protein Expression. MAPK is a serine/threonine protein kinase subfamily playing an important role in intracellular processes responding to extracellular stimuli. Extracellular signal-related kinases (ERKs), p38 MAPK, and c-Jun amino-terminal kinases (JNKs) are members of the MAPK family. MyD88 recruitment by TLR4 results in activation of the MAPK family and leads to upregulation of proinflammatory cytokines, including iNOS, TNF-α, and IL6.26,27 In our previous study, activated MAPK contributed to

Table 1. Effect of Anethole (1) on Serum ALT Activity during Hepatic I/Ra ALT group sham 1 + sham I/R 1 + I/R

dose (mg/kg) 100 50 100 200

1h

6h

28.3 ± 3.98 34.4 ± 4.79 2322.0 ± 234.9** 1939.5 ± 304.3** 1335.6 ± 114.2*,+ 1478.6 ± 145.4*

26.1 ± 6.03 30.9 ± 3.68 11044.2 ± 839.9** 7113.1 ± 1642.6** 5115.6 ± 413.2*,++ 4652.8 ± 296.4*,++

a

Liver damage was assessed at 1 and 6 h after reperfusion by measurement of circulating serum ALT activity. Mice were orally administered anethole 1 h before inducing ischemia (n = 8−10). The values are represented as mean ± SEM. *, ** denote significant differences (p < 0.05, p < 0.01) versus sham group; +, ++ denote significant differences (p < 0.05, p < 0.01) versus I/R group.

optimal effective dose based on serum ALT activity for evaluating the molecular mechanisms of 1 against I/R-induced hepatic injury. As shown in Figure 1, normal lobular architecture and cell structure were observed in the sham group. However, liver sections obtained from the I/R group showed multiple and extensive areas of portal inflammation and hepatocyte necrosis. These histologic damages were ameliorated by pretreatment with 1, indicating a hepatoprotective effect of this compound. Serum TNF-α and IL-6 Levels. The liver is a major inflammatory organ, and inflammatory processes contribute to a number of pathological events. During reperfusion, activated Kupffer cells, the resident macrophages of the liver, produce a number of signaling molecules that promote inflammatory reactions.18 TNF-α, a pleiotropic proinflammatory cytokine, is 1718

dx.doi.org/10.1021/np4004323 | J. Nat. Prod. 2013, 76, 1717−1723

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Table 2. Effect of Anethole (1) on Serum TNF-α and IL-6 during Hepatic I/Ra TNF-α

IL-6

group

1h

6h

1h

6h

sham 1 + sham I/R 1 + I/R

49.1 ± 2.38 49.0 ± 1.02 128.6 ± 20.6** 77.5 ± 6.14+

47.3 ± 1.34 51.2 ± 3.07 221.5 ± 19.8** 116.5 ± 3.32**,++

61.7 ± 2.40 63.4 ± 2.58 305.6 ± 35.2** 148.4 ± 19.0*,++

62.9 ± 2.13 65.7 ± 1.86 1271.8 ± 166.6** 607.7 ± 40.2*,++

a

The levels of serum TNF-α and IL-6 were determined at 1 and 6 h after reperfusion. Mice were orally administered 100 mg/kg anethole 1 h before inducing ischemia (n = 8−10). The values are represented as mean ± SEM. *, ** denote significant differences (p < 0.05, p < 0.01) versus sham group; +, ++ denote significant differences (p < 0.05, p < 0.01) versus I/R group.

transcription. IκB-α inhibits the transcription ability of the NFκB family by forming a complex with NF-κB and blocking nuclear translocation from the cytosol. Previous studies demonstrated that activation of NF-κB is involved in TLR4mediated liver I/R injury, and increased IκB-α attenuated this injury.29,30 In our study, the nuclear level of NF-κB increased to 5.2 times the sham value in I/R mice. In contrast, the cytosolic level of IκB-α in the I/R group decreased to about 60% of the level seen in the sham group. These changes were attenuated by compound 1. AP-1, as a transcription factor like NF-κB, regulates a number of cellular responses, including inflammation. c-Jun is one member of the AP-1 subfamily.31 In this study, the nuclear protein level of phospho-c-Jun in the I/R group increased to 4.1 times the sham value. The increase in phospho-c-Jun was attenuated by compound 1 (Figure 4). IRF-1 and HMGB1 Translocations and Interaction between IRF-1 and Histone Acetyltransferase (HAT) p300. The IRF family is a group of regulatory proteins involved in a number of cellular process including cell proliferation and gene expression.32 IRF-1, the first identified member of the IRF family, was originally identified due to its ability to enhance the expression of the type 1 interferon gene and other interferon inducible genes.33 However, several studies have illustrated that IRF-1 is involved in responses to viral infections, innate and adaptive immunity, and pathogenesis of inflammatory disorders including I/R and hepatitis.8,34 Nuclear factor HMGB1 is a proinflammatory mediator, which is rapidly acetylated and released into the serum from activated macrophages during cell injury and necrosis.35 Extracellular HMGB1 acts as a DAMP molecule to TLR4 and exerts its proinflammatory effects by upregulating NF-κB, JNK, p38 kinases, oxygen free radical, and neutrophil recruitment during I/R injury.28,36 Furthermore, HMGB1 might contribute to the underlying mechanism for I/R injury as downstream of TLR4. Activation of TLR4 signaling in I/R injury promotes IRF-1-mediated HMGB1 release and causes hepatocellular damage immediately.37 Specifically, HAT is a complex of enzymes acetylating conserved lysine amino acids by transferring an acetyl group and has been related to HMGB1 acetylation and release from the nucleus; Gcn5, p300, and cAMP-response element-binding protein-binding protein are some examples of HAT complexes.38−40 HMGB1 increases and functions as an early mediator of organ damage and inflammation in hepatic I/R.7 Nuclear translocation of IRF-1 is responsible for the acetylation and release of HMGB1.41 In detail, IRF-1 closely interacts with the HAT complex and enhances its acetylation activity on HMGB1 to release it from the nucleus.10 In this study, the I/R group showed a marked 2.4-fold increase in nuclear translocation of IRF-1 over that of the sham group, and compound 1 treatment attenuated this increase in nuclear translocation (Figure 5). I/R caused the

Figure 2. Effect of anethole (1) on TLR4 (A) and MyD88 (B) protein expression during hepatic I/R. Mice were orally administered 100 mg/ kg anethole 1 h before inducing ischemia (n = 8−10). The protein expression was measured by Western blot analysis at 6 h after reperfusion. [The values are represented as mean ± SEM. *, ** denote significant differences (p < 0.05, p < 0.01) versus sham group; ++ denotes significant difference (p < 0.01) versus I/R group.]

the inflammatory response in I/R, and MAPK inhibition showed improvement in survival, indicating that MAPK plays a pivotal role in hepatic I/R injury.28 In the I/R group, phosphorylated (phospho)-p38, ERK, and JNK increased to 4.8 times, 4.1 times, and 5.3 times the sham group, respectively. These increases were attenuated by 1 (Figure 3). NF-κB, Inhibitor of Kappa B (IκB)-α and Phospho-cJun Protein Expression. NF-κB is a protein complex that regulates transcription of various gene encoding proinflammatory mediators. In the presence of extracellular stimuli, NF-κB in the cytoplasm translocates to the nucleus and induces gene 1719

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Figure 3. Effect of anethole (1) on MAPK phosphorylation during hepatic I/R. Mice were orally administered 100 mg/kg anethole 1 h before inducing ischemia (n = 8−10). Total and phospho-p38, ERK, and JNK level in the liver was measured by Western blot analysis at 6 h after reperfusion. [The values are represented as mean ± SEM. *, ** denote significant differences (p < 0.05, p < 0.01) versus sham group; +, ++ denote significant differences (p < 0.05, p < 0.01) versus I/R group.]

Figure 4. Effect of anethole (1) on nuclear NF-κB, phospho-c-Jun, and cytosolic IκB-α protein level during hepatic I/R. Mice were orally administered 100 mg/kg anethole 1 h before inducing ischemia (n = 8−10). NF-κB and phospho-c-Jun were measured on the nuclear extracts from liver by Western blot analysis at 6 h after reperfusion. IκB-α was measured on the cytosolic extracts. [The values are represented as mean ± SEM. ** denotes significant difference (p < 0.01) versus sham group; ++ denotes significant difference (p < 0.01) versus I/R group.]

increase to 8.9-fold compared with the sham group, and compound 1 suppressed the release of HMGB1 to the circulatory system (Figure 6B). To determine whether HMGB1 acetylation and release were dependent on the interaction between HAT p300 and IRF-1, liver samples were subjected to immunoprecipitation using p300 antibody and immunoblot analysis using IRF-1 antibody. The level of IRF-1 interacting with HAT p300 significantly increased after I/R injury, which was attenuated by 1 (Figure 7). In summary, our findings suggest that 1 protects the liver against I/R injury, which might be due to the inhibition of IRF1-mediated HMGB1 release and TLR signaling. Thus, compound 1 may provide a new pharmacological intervention strategy for organ I/R injuries.

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Figure 5. Effect of anethole (1) on nuclear IRF-1 protein level during hepatic I/R. Mice were orally administered 100 mg/kg anethole 1 h before inducing ischemia (n = 8−10). Western blot analysis for IRF-1 was performed on the nuclear extracts from liver at 6 h after reperfusion. [The values are represented as mean ± SEM. ** denotes significant difference (p < 0.01) versus sham group; + denotes significant difference (p < 0.05) versus I/R group.]

EXPERIMENTAL SECTION

General Experimental Procedures. NMR experiments were performed on a Varian Unity INOVA 500 spectrometer with the usual pulse sequences. The GC/EIMS spectrum was obtained on a HewlettPackard HP 6890 series GC system with a Hewlett-Packard 5973 mass selective detector. Column chromatography was carried out on silica gel 60 (230−400 mesh; Merck, Darmstadt, Germany) and LiChroprep RP-18 (40−63 μm; Merck). TLC was performed on precoated silica gel 60 F254 plates (20 × 20 cm, 0.25 mm; Merck) and RP-18 F254s plates (20 × 20 cm, 0.25 mm; Merck). Plant Material. The fruits of Foeniculum vulgare were purchased in February 2010 from a traditional medicine market, Kyung-dong yakryong-si, in Seoul, Korea, and identified by Prof. Je-Hyun Lee, College of Oriental Medicine, Dongguk University. A voucher specimen was

level of HMGB1 translocation from nucleus to cytosol to increase dramatically to 3.0 times that of the sham value. Compound 1 ameliorated the increase in HMGB1 translocation (Figure 6A). Moreover, HMGB1 release into the extracellular milieu in the I/R group showed a significant 1720

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Figure 7. Effect of anethole (1) on interaction between IRF-1 and HAT p300 during hepatic I/R. Mice were orally administered 100 mg/ kg anethole 1 h before inducing ischemia (n = 8−10). Immunoprecipitation with p300 antibody was performed on 100 μg of whole liver tissue lysate. The precipitates were subjected to immunoblot analysis using IRF-1 antibody. [The values are represented as mean ± SEM. *, ** denote significant differences (p < 0.05, p < 0.01) versus sham group; ++ denotes significant difference (p < 0.01) versus I/R group.] and GC/EIMS) consistent with literature values.42 This substance was determined to be 98.9% pure by HPLC. Animal Treatment. Male, 6-week-old ICR mice (24−26 g, Orient Bio, Inc., Gapyeong, Korea) were kept in a temperature- and humiditycontrolled room (25 ± 1 °C and 55 ± 5%, respectively) under a 12 h light−dark cycle. All experiments were approved by the Animal Care Committee of Sungkyunkwan University School of Pharmacy (SUSP12-18) and performed in accordance with the guidelines of the National Institutes of Health (NIH publication No. 86-23, revised 1985). Mice were fasted for 18 h before the experiments but allowed free access to tap water. Following intraperitoneal administration of ketamine (55 mg/kg) and xylazine (7 mg/kg), body temperature was maintained at 37 °C using heating pads. After a midline laparotomy, partial hepatic ischemia was induced by occluding the blood supply to the left and median lobes of the liver with a microserrefine clip (Fine Science Tools Inc., Vancouver, BC, Canada). After 60 min of ischemia, the clamp was removed to allow reperfusion. Sham-operated animals underwent the same procedure but without vessel occlusion. At 1 and 6 h after reperfusion, blood and liver tissues were collected and stored at −75 °C for later analysis; part of the left lobe was used for histological analysis. Experimental Treatment. Compound 1 (50, 100, and 200 mg/ kg/10 mL) or 10% Tween 80-saline (vehicle) was orally administered 1 h before inducing ischemia. The dose and timing of 1 treatment were selected based on previous reports43 and on preliminary studies. Mice were randomly assigned to one of the following six groups (each group, n = 8−10): (1) vehicle-treated sham (sham), (2) 1 100 mg/kgtreated sham (1 + sham), (3) vehicle-treated I/R (I/R), (4) 1 at 50 mg/kg-treated I/R (1 50 mg/kg + I/R), (5) 1 at 100 mg/kg-treated I/ R (1 100 mg/kg + I/R), (6) 1 at 200 mg/kg-treated I/R (1 200 mg/kg + I/R). Based on ALT activity, a dose of 100 mg/kg was chosen for further biochemical studies. Serum ALT Activity. Serum ALT activity was measured by the standard spectrophotometric procedure using a ChemiLab ALT assay kit (IVDLab Co., Ltd., Uiwang, Korea). Serum TNF-α and IL-6 Levels. Circulating levels of TNF-α and IL-6 were quantified at 1 and 6 h after reperfusion using commercial mouse enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, San Diego, CA, USA), according to the manufacturer’s instructions. Histological Analysis. Liver specimens for histopathological analysis were obtained 6 h after reperfusion. Samples were fixed in

Figure 6. Effect of anethole (1) on cytosolic and nuclear HMGB1 protein level (A) and serum HMGB1 release (B) during hepatic I/R. Mice were orally administered 100 mg/kg anethole 1 h before inducing ischemia (n = 8−10). Western blot analysis for nuclear and cytosolic HMGB1 was performed on the nuclear and cytosolic extracts from liver at 6 h after reperfusion, respectively. [The values are represented as mean ± SEM. ** denotes significant difference (p < 0.01) versus sham group; ++ denotes significant difference (p < 0.01) versus I/R group.]

deposited in the Laboratory of Pharmacognosy, School of Pharmacy, Sungkyunkwan University (skku-10-002). Extraction and Isolation. The dried fruits of F. vulgare (5.0 kg) were extracted with MeOH twice at room temperature. After maceration, total filtrate was concentrated to obtain a MeOH extract (450.9 g). Then, the MeOH extract (420 g) was suspended in distilled water (1.8 L) and successively partitioned with n-hexane to yield a hexane fraction (221.4 g). A portion (30.0 g) of the hexane fraction was subjected to silica gel column chromatography (stepwise elution with hexane−CH2Cl2, 2:1, 1:1, CH2Cl2, hexane−CH2Cl2−MeOH, 10:10:0.2, 10:10:0.5, 10:10:2, and CH2Cl2−MeOH, 1:1) to give 10 subfractions (H-1 to H-10). Subfraction H-1 was rechromatographed over a silica gel column (hexane−EtOAc, 30:1) to afford five further subfractions (H-1-1−H-1-5). Among these subfractions, H-1-2 was applied to a silica gel column with hexane−CH2Cl2 (3:1) for elution and also on a RP-C18 column with 97% MeOH−H2O as eluents to obtain compound 1 (1.7 g). This compound was obtained as a colorless oil and exhibited spectroscopic data (1H NMR, 13C NMR, 1721

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10% neutral-buffered formalin, embedded in paraffin, sliced into 5 μm sections, and stained with hematoxylin and eosin, followed by blinded histological assessment. Histological changes were evaluated in randomly chosen histological fields at 200× magnification. Isolation of Cytosolic and Nuclear Proteins. Cytosolic and nuclear proteins were isolated as described previously.44 Fresh liver tissue was isolated and homogenized in PRO-PREP (iNtRON Biotechnology Inc., Seongnam, Korea) for whole protein samples and in NE-PER (Pierce Biotechnology Inc., Rockford, IL, USA) for nuclear and cytosolic protein samples, according to the manufacturer’s instructions. Protein concentration was determined using a BCA Protein Assay kit (Pierce Biotechnology Inc.). Serum Preparation for HMGB1 Analysis. Serum samples were filtered and concentrated through Centricon YM-100 and YM-10 (Millipore, Billerica, MA, USA) with fixed-angle (35°), 7500g for 15 min, 4 °C. The concentrated samples were then subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). Western Blot Immunoassay. To determine the content of TLR4, MyD88, phospho-p38, p38, phospho-ERK, ERK, phospho-JNK, JNK, IκB-α, NF-κB, phospho-c-Jun, IRF-1, HAT p300, and HMGB1 protein expression, 16 μg of soluble protein from each whole liver homogenate, cytosolic fraction, nuclear fraction, and concentrated serum were used. Protein samples were loaded per lane on 6−12.5% polyacrylamide gels, separated by SDS/PAGE, and transferred to nitrocellulose membranes using the Semi-Dry Trans-Blot Cell (BioRad Laboratories, Hercules, CA, USA). After transfer, membranes were washed with 0.1% Tween 20 in 1× Tris-buffered saline (TBS/T) and blocked for 1 h at room temperature with 5% (w/v) skim milk powder or 5% (w/v) bovine serum albumin powder in TBS/T. Blots were then incubated overnight at 4 °C with primary antibodies. After washing five times for 7 min each in TBS/T, the membranes were incubated with appropriate secondary antibodies for 1 h at room temperature, followed by detection using an ECL detection system (iNtRON Biotechnology Inc.), according to the manufacturer’s instructions. The intensity of the immunoreactive bands was determined using TotalLab TL 120 software (Nonlinear Dynamics Ltd., Newcastle, UK). The following primary antibodies were used: TLR4, MyD88, IκB-α, p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:1000), phospho-p38, p38, phospho-ERK, ERK, phosphoJNK, JNK (Cell Signaling Technology, Beverly, MA, USA; 1:1000), NF-κB, HMGB1 (Abcam, Cambridge, MA, USA; 1:1000), phospho-cJun (Santa Cruz Biotechnology; 1:500), IRF-1 (Santa Cruz Biotechnology; 1:2000). All signals were normalized to those of βactin (Abcam; 1:5000) for whole lysate and cytosolic fraction and lamin B1 (Abcam; 1:2000) for the nuclear fraction. Immunoprecipitation. Whole liver tissues (100 μg) were homogenized with ice-cold radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, pH 7.4) containing a protease and phosphatase inhibitor cocktail set (Calbiochem, La Jolla, CA, USA). Aliquots of 500 μg of protein diluted to 1 mg/mL in RIPA buffer were precleared with protein A/G agarose beads (Santa Cruz Biotechnology) for 30 min and then incubated overnight at 4 °C with anti-p300 (Santa Cruz Biotechnology) with a constant rotation of the sample. Fresh protein A/G agarose beads were added to the supernatant, and the samples were incubated for a further 4 h at 4 °C with constant rotation. Immune complexes were washed three times in RIPA buffer for 30 s and boiled for 10 min. The samples were then analyzed by Western blotting as described above. Statistical Analysis. All results are presented as means ± standard error of the mean (SEM). The overall significance of results was analyzed by two-way ANOVA. Differences between compared groups were considered statistically significant at p < 0.05 with the appropriate Bonferroni correction for multiple comparisons. Survival data were analyzed by Kaplan−Meier curves and the log-rank test.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-31-290-7712. Fax: +82-31-292-8800. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the Korea Food and Drug Administration (Studies on the Identification of Efficacy of Biologically Active Components from Oriental Herbal Medicines).

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