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Protective Effects of Lupeol against D‑Galactosamine and Lipopolysaccharide-Induced Fulminant Hepatic Failure in Mice So-Jin Kim,† Hong-Ik Cho,† Seok-Joo Kim,† Joon-Sung Kim,† Jong-Hwan Kwak,† Dong-Ung Lee,‡ Sang Kook Lee,§ and Sun-Mee Lee*,† †

School of Pharmacy, Sungkyunkwan University, Suwon, 440-746, Republic of Korea Division of Bioscience, Dongguk University, Gyeongju, 780-714, Republic of Korea § College of Pharmacy, Seoul National University, Seoul, 151-742, Republic of Korea ‡

S Supporting Information *

ABSTRACT: This study examined the hepatoprotective effects of lupeol (1, a major active triterpenoid isolated from Adenophora triphylla var. japonica) against D-galactosamine (GalN) and lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice were orally administered 1 (25, 50, and 100 mg/kg; dissolved in olive oil) 1 h before GalN (800 mg/ kg)/LPS (40 μg/kg) treatment. Treatment with GalN/LPS resulted in increased levels of serum alanine aminotransferase, tumor necrosis factor (TNF)-α, and interleukin (IL)-6, as well as increased mortality, all of which were attenuated by treatment with 1. In addition, levels of toll-like receptor (TLR)4, myeloid differentiation primary response gene 88, TIR-domain-containing adapter-inducing interferon-β (TRIF), IL-1 receptor-associated kinase (IRAK)-1, and TNF receptor associated factor 6 protein expression were increased by GalN/LPS. These increases, except TRIF, were attenuated by 1. Interestingly, 1 augmented GalN/LPS-mediated increases in the protein expression of IRAK-M, a negative regulator of TLR signaling. Following GalN/LPS treatment, nuclear translocation of nuclear factor-κB and the levels of TNF-α and IL-6 mRNA expression increased, which were attenuated by 1. Together, the present findings suggest that lupeol (1) ameliorates GalN/LPS-induced liver injury, which may be due to inhibition of IRAK-mediated TLR inflammatory signaling.

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LPS, and ligands for TLR2, -3, and -9.9 Unlike other IRAK proteins, IRAK-M is a member of the IRAK family and negatively regulates TLR signaling by interfering with the association between IRAK and its adaptor molecules including myeloid differentiation primary response gene 88 (MyD88) and tumor necrosis factor (TNF) receptor associated factor (TRAF).12−14 Sung et al. demonstrated that up-regulation of IRAK-M protein expression by procyanidin dimer B2 treatment suppresses LPS-induced maturation of macrophages and phosphorylation of mitogen-activated protein kinases.15 Furthermore, IRAK-M knockout mice exhibit an increased inflammatory response during bacterial infections.12 Adenophora triphylla has traditionally been used to treat chronic bronchitis and whooping caugh.16 A. triphylla exhibits antioxidant effects in 2,2-diphenyl-1-picrylhydrazyl/hydroxyl radical and nitrite scavenging systems in vitro16 and antiinflammatory effects in the 12-O-tetradecanoylphorbol-13acetate-induced ear edema animal model.17 In a preliminary study, the components of Adenophora triphylla (Thunb.) A.

ulminant hepatic failure (FHF) is a life-threatening clinical syndrome characterized by hepatic encephalopathy, severe coagulopathy, jaundice, and hydroperitoneum.1 There is no specific therapy for FHF other than liver transplantation, and this adverse environment is linked to high mortality in FHF patients.2 D-Galactosamine (GalN) and lipopolysaccharide (LPS)-induced hepatic failure is a widely used animal model of liver injury and is similar to acute hepatic failure in the clinic.3−5 In this model, LPS induces production of a variety of pro-inflammatory cytokines, while GalN increases sensitivity to the lethal effects of LPS.6 Furthermore, activation of the cytokine cascade in the GaIN/LPS model is considered to play a pivotal role that causes improper activation of inflammatory responses and leads to hepatocyte death.7 Toll-like receptors (TLRs) are a family of proteins that recognize pathogen- and/or damage-associated molecular patterns and activate innate immune responses.8 Among the TLR signaling-associated molecules, interleukin (IL)-1 receptor-associated kinase (IRAK) plays an important role in antipathogen responses, inflammation, and autoimmunity.9,10 The kinase domain of IRAK is essential for inflammatory signaling through nuclear factor (NF)-κB.11 Suzuki et al. demonstrated that IRAK-deficient mice do not respond to IL-1, © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 24, 2014

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DC. var. japonica (Regel) Hara (Campanulaceae) for hepatoprotective agents were screened. Among them, lupeol (1), a major active triterpenoid constitution of A. triphylla var. japonica, was found to have hepatoprotective properties and low cytotoxicity in primary hepatocytes (data not shown). Compound 1 possesses anti-inflammatory effects by reducing the alanine/aspartate aminotransferase activity and increasing the reduced glutathione content in aflatoxin B1-induced hepatic damage in rats.18 Compound 1 also abolishes acetaminopheninduced hepatotoxicity by reducing Bax protein expression and inhibiting caspase-3 activity.19 Therefore, this study was designed to investigate the hepatoprotective effect and molecular mechanisms of lupeol (1) in a GalN/LPS-induced FHF in vivo model, particularly focusing on IRAK-mediated TLR signaling.

plasma membranes is disrupted, causing enzymes in cells to be released into the circulation, as indicated by the abnormally high serum levels of hepatospecific enzymes.20 Leist et al. showed that increased serum alanine aminotransferase (ALT) levels correlate with lethality in an experimental FHF model.21 In the control group, the serum ALT level was 57.4 ± 8.8 U/L. The serum ALT level increased by approximately 47.1-fold than that in the control group at 6 h after GalN/LPS treatment, and this was attenuated by 1 (Table 1). Histological observations of liver tissues clearly revealed the significant extensive cellular necrosis and inflammatory cell infiltration at 6 h of GalN/LPS injection. These histological changes were ameliorated by 1 (Figure 2). Compound 1 alone did not affect both serum ALT level and histological features. Taken together, these results suggest that 1 suppresses lethal liver damage induced by GalN/ LPS and thus may be useful in potential clinical applications for treating liver diseases. Based on the results of survival rate, ALT, and histological analysis, a dose of 1 at 100 mg/kg was selected as the optimal effective dose for evaluating the molecular mechanisms of 1 against GalN/LPS-induced FHF. Table 1. Effect of Lupeol (1) on Serum ALT, TNF-α, and IL6 Levels after GalN/LPS Treatment (means ± SEM, n = 6−8)a group

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control 1 GalN/LPS 1 + GalN/LPS

RESULTS AND DISCUSSION Mortality. GalN/LPS induces liver damage that closely resembles human FHF with respect to its morphological and functional features. Therefore, this animal model is used widely as an experimental liver injury model for elucidating the mechanisms of clinical FHF and evaluating the efficacy of hepatoprotective agents.3−5 In the GalN/LPS group, mice began to die 7 h after GalN/LPS treatment. The survival rate was 90% at 7 h and reached 20% at 11 h after GalN/LPS treatment. Pretreatment with 1 at 25, 50, or 100 mg/kg improved the survival rate in a dose-dependent manner (Figure 1). A dose of 1 at 100 mg/kg alone did not affect the survival rate (data not shown). Serum Aminotransferase Activity and Histological Analysis. The liver is the most vulnerable organ to GalN/LPS treatment. After GalN/LPS treatment, the permeability of

ALT (U/L) 57.4 58.1 2704.0 682.0

± ± ± ±

8.8 5.4 244.2b 96.4b,d

TNF-α (pg/mL) 42.6 41.0 250.8 109.0

± ± ± ±

1.7 1.4 5.4b 24.8d

IL-6 (pg/mL) 73.3 75.1 654.1 108.8

± ± ± ±

13.1 11.4 19.7b 24.2c

a

Compound 1 (100 mg/kg) was administered orally 1 h before GalN/ LPS treatment. Serum ALT, TNF-α, and IL-6 levels were determined at 6 h after GalN/LPS treatment. bp < 0.01 versus control group. cp < 0.05 versus GalN/LPS group. dp < 0.01 versus GalN/LPS group.

Serum TNF-α and IL-6 Levels. An up-regulated inflammatory response is a key mechanism and major causation of GalN/LPS-induced liver damage.22 TNF-α is a proinflammatory cytokine released by Kupffer cells and plays a critical role in modulation of the necrotic, apoptotic, and inflammatory responses in the liver.23,24 IL-6 is a typical inflammatory cytokine also produced by activated Kupffer cells in response to liver damage induced by GalN and endotoxins.24

Figure 1. Effect of lupeol (1) on mortality induced by GalN/LPS treatment. Each group consisted of 10 mice. Mice were orally administered 1 (25, 50, and 100 mg/kg) in vehicle (olive oil) 1 h before GalN (800 mg/kg)/LPS (40 μg/kg) treatment. **p < 0.01 versus GalN/LPS group. B

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increase of MyD88 protein expression, 1 did not attenuate the increase of TRIF protein expression (Figure 3). Taken

Figure 2. Effect of lupeol (1) on histological micrographs of the liver. Mice orally received 1 (100 mg/kg) in a vehicle (olive oil) 1 h before GalN (800 mg/kg)/LPS (40 μg/kg) treatment. Tissues were stained with hematoxylin and eosin (H&E) at 6 h after GalN/LPS treatment (magnification 200×). (A) control; (B) 1; (C) GalN/LPS; (D) 1 + GalN/LPS. The arrowhead indicates necrotic areas. The arrows indicate inflammatory cell infiltration. Figure 3. Effect of lupeol (1) on the levels of TLR4, MyD88, and TRIF protein expression in the liver. Mice were orally administered 1 (100 mg/kg) in a vehicle (olive oil) 1 h before GalN (800 mg/kg)/ LPS (40 μg/kg) treatment. Liver tissues were collected 6 h after GalN/LPS treatment. Densitometry was performed, and fold changes in protein expression are shown below the representative bands. Results are presented as the mean ± SEM (n = 6−8 per group). *p < 0.05, **p < 0.01 versus control group; ††p < 0.01 versus GalN/LPS group.

Because activation of the pro-inflammatory cytokine cascade plays a central role in the pathophysiology and clinical outcome of FHF, inhibition of inflammatory cytokines may be a potential target of FHF treatment. In the control group, the serum levels of TNF-α and IL-6 were 42.6 ± 1.7 and 73.3 ± 13.1 U/L, respectively. Compared with the control group, the serum TNF-α and IL-6 levels increased by approximately 5.9and 8.9-fold, respectively, at 6 h after GalN/LPS treatment, and these increases were attenuated by 1 (Table 1). Compound 1 did not affect either serum TNF-α or IL-6 levels. TLR4, MyD88, and TIR-Domain-Containing AdapterInducing Interferon-β (TRIF) Protein Expression. TLR4 is widely expressed on parenchymal and nonparenchymal cells in the liver and induces an innate immune response by recognizing a number of different microbial toxins.25 TLR4 is specific receptor for LPS and plays a crucial role in regulating inflammatory cytokine production.26 Although TLR4 plays a valuable role in the innate immune system, uncontrolled immune response following activation of TLR is noted as a fatal event in the pathophysiology of several liver diseases including steatohepatitis, fibrogenesis, and virus-infected liver.27 Indeed, Jiang et al. showed that reducing TLR4 expression led to increased survival of mice suffering from acute hepatitis by GalN/LPS treatment.28 Moreover, several therapeutic approaches controlling the TLR4 signaling pathway have been utilized to improve liver damage in experimental FHF. M62812, a TLR4 signal transduction inhibitor, prolongs survival in the GalN/LPS-induced FHF animal model.29 LPS-mediated TLR signaling consists of two pathways, namely, the MyD88- and TRIF-dependent pathways. After TLR4 activation, these adaptor molecules are recruited to stimulate downstream signaling.11 In the current study, the level of TLR4 protein expression increased by 3.2-fold compared with the control group 6 h after GalN/LPS treatment, and this increase was attenuated by 1. Furthermore, the levels of MyD88 and TRIF protein expression increased to 1.6- and 1.4-fold, respectively, compared with those in the control group 6 h after GalN/LPS treatment. However, interestingly, although 1 attenuated the

together, although both MyD88- and TRIF-dependent pathways of the TLR system were activated in GalN/LPS-induced FHF, 1 appeared to suppress inflammatory cytokines only through inhibition of the MyD88 pathway, but not the TRIFdependent pathway. IRAK-1, TRAF6, and IRAK-M Protein Expression. IRAK, a serine/threonine kinase, binds to MyD88 through a Nterminal death domain interaction after TLR stimulation, where it continuously associates with TRAF6. LPS-induced lipid peroxidation in plasma coincides with decreased activities of antioxidant enzymes, but not in IRAK-1-deficient mice.30 Importantly, this mechanism of TLR signaling leads to activation of NF-κB.31 Different from other IRAK proteins, IRAK-M negatively regulates TLR signaling by preventing both dissociation of IRAK from MyD88 and sequential binding of IRAK with TRAF6.12 Previous studies demonstrated that IRAK-M-deficient mice exhibit increased NF-κB activation and elevated expression of inflammatory cytokines upon stimulation with TLR ligands.12,32 Furthermore, a recent study performed by Wang et al. reported that, in the absence of IRAK-M, alcoholic fatty liver model mice develop worse liver injury including altered inflammation and increased gut permeability.33 These findings indicate that IRAK-M negatively regulates TLR-signaling pathways. In this study, the levels of IRAK-1 and TRAF6 protein expression were increased significantly by 1.9and 5.0-fold, respectively, compared with the control group at 6 h after GalN/LPS treatment, and increases in expression of these proteins were attenuated by 1. Interestingly, the level of IRAK-M protein expression markedly increased by 1.7-fold compared with the control group at 6 h after GalN/LPS C

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treatment, which was augmented by 1 (Figure 4). Given the inhibitory role of IRAK-M on MyD88 activation, these results

Figure 5. Effect of lupeol (1) on nuclear translocation of NF-κB in liver. Mice were orally administered 1 (100 mg/kg) in a vehicle (olive oil) 1 h before GalN (800 mg/kg)/LPS (40 μg/kg) treatment. The liver tissues were collected 6 h after GalN/LPS treatment. Densitometry was performed, and fold changes in protein expression are shown below the representative bands. Results are presented as the mean ± SEM (n = 6−8 per group). **p < 0.01 versus control group; † p < 0.05, ††p < 0.01 versus GalN/LPS group.

Figure 4. Effect of lupeol (1) on the levels of IRAK-1, TRAF-6, and IRAK-M protein expression in liver. Mice were orally administered 1 (100 mg/kg) in a vehicle (olive oil) 1 h before GalN (800 mg/kg)/ LPS (40 μg/kg) treatment. The liver tissues were collected 6 h after GalN/LPS treatment. Densitometry was performed, and fold changes in protein expression are shown below the representative bands. Results are presented as the mean ± SEM (n = 6−8 per group). **p < 0.01 versus control group; †p < 0.05 versus GalN/LPS group.

are consistent with the inhibitory effect of 1 on MyD88 protein expression. In addition, these findings may have important implications for the use of anti-TLR4 protein signaling, especially focusing on IRAK as a potential target for therapeutic intervention in FHF. Nuclear Translocation of NF-κB. NF-κB has been implicated in the regulation of a number of genes that encode pro-inflammatory cytokines, chemokines, and adhesion molecules.34,35 In its dormant form, NF-κB is sequestered in the cytoplasm in an inactive form by inhibitory (I)κB. After activation by upstream signals, IκB is phosphorylated, degraded, and then detached from NF-κB. Activated NF-κB then translocates to the nucleus, where it induces transcriptional up-regulation.36 Heyninck et al. suggested that NF-κB is a key regulator of liver damage in a GalN/LPS- and GalN/TNF-αinduced hepatotoxicity model.37 In the present study, the level of nuclear NF-κB protein expression was significantly increased by 2.3-fold compared with the control group at 6 h after GalN/ LPS treatment, and this effect was attenuated by 1. Likewise, levels of cytosolic IκB protein expression were significantly decreased by 2.8-fold compared with the control group at 6 h after GalN/LPS treatment, which was attenuated by 1 (Figure 5). Taken together, these results indicate that 1 may inhibit the release of inflammatory cytokines by suppressing transactivation of NF-κB. TNF-α and IL-6 mRNA Expression. Nuclear translocation of NF-κB results in increased transcription of genes encoding inflammatory cytokines. As shown in Figure 6, the levels of TNF-α and IL-6 mRNA expression were significantly increased by 235.8- and 473.9-fold, respectively, compared with the

Figure 6. Effect of lupeol (1) on the levels of TNF-α and IL-6 mRNA expression in mouse livers. Mice were orally administered 1 (100 mg/ kg) in a vehicle (olive oil) 1 h before GalN (800 mg/kg)/LPS (40 μg/ kg) treatment. The liver tissues were collected 6 h after GalN/LPS treatment. Results are presented as the mean ± SEM (n = 6−8 per group). **p < 0.01 versus control group; †p < 0.05, ††p < 0.01 versus GalN/LPS group.

control group at 6 h after GalN/LPS treatment. Consistent with results above, administration of 1 attenuated the GalN/ LPS-induced increased levels of TNF-α and IL-6 mRNA expression. In summary, the present findings suggest that lupeol (1) possesses the hepatoprotective properties consisting of improvement of survival rate and alleviation of liver injury induced by GalN/LPS. GalN/LPS-induced liver injury was attenuated by 1 through suppression of the IRAK-mediated TLR4 signaling pathway, which may lead to the inhibition of expression of inflammatory cytokines. Taken together, these results provide evidence that 1 may be a novel therapeutic agent for the treatment of hepatic failure. D

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thickness of 5 mm and fixed immediately in 10% neutral-buffered formalin. Samples were then embedded with paraffin, sliced into 5 μm sections, and stained with H&E for a blinded histological assessment. All liver samples (n = 6−8) were evaluated by a single pathologist in nonconsecutive, randomly chosen fields at 200× magnification (Olympus BX51/Olympus DP71, Olympus, Tokyo, Japan). Serum TNF-α and IL-6 Levels. Serum TNF-α and IL-6 levels were quantified 6 h after GalN/LPS treatment using commercial mouse enzyme-linked immunosorbent assay kits (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. Preparation of Protein Extracts. Fresh liver tissue was isolated and homogenized in PRO-PREP (iNtRON Biotechnology Co., Ltd., Seongnam, Korea) for total protein samples and in NE-PER (Pierce Biotechnology, Inc., Rockford, IL, USA) for extraction of nuclear and cytosolic protein samples, according to the manufacturer’s instructions. Protein concentrations were determined using the bicinchoninic acid protein assay kit (Pierce Biotechnology). Western Blot. Total protein extracts (16−20 μg) were used to determine the content of TLR4, MyD88, TRIF, IRAK-1, TRAF6, and IRAK-M. Cytosolic protein (16 μg) was used to determine the content of IκB, while nuclear protein (12 μg) was used to determine the relative abundance of the NF-κB/p65 subunit. Protein samples were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) using a Semi-Dry Trans-Blot Cell (Bio-Rad Laboratories, Hercules, CA, USA). After transfer, the membranes were washed with 0.1% Tween-20 in Tris-buffered saline (TBS/T) and blocked for 1 h at room temperature with 5% (w/v) skim milk powder in TBS/T. Blots were then incubated overnight at 4 °C with the primary antibodies. After overnight incubation, the blots were incubated with appropriate secondary antibodies and detected using an ECL detection system (iNtRON Biotechnology Co.), according to the manufacturer’s instructions. Visualized immunoreactive bands were evaluated densitometrically with ImageQuant TL software (Amersham Biosciences/GE Healthcare, Piscataway, NJ, USA). Primary antibodies against mouse TLR4, MyD88, TRIF, TRAF6, IRAK-M, NF-κB/p65, IκB-α (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:1000), and IRAK-1 (Abcam, Cambridge, UK; 1:1000) were used, and the signals were standardized against that of βactin (Sigma-Aldrich Chemical Co.; 1:5000) for whole and cytosolic proteins or lamin B1 (Abcam; 1:5000) for nuclear proteins. Total RNA Extraction and Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Total RNA was extracted from the tissue using RNAiso Plus (Takara Bio, Inc., Shiga, Japan). Next, cDNA was synthesized using a reverse transcription reaction (EcoDry cDNA Synthesis Premix; Takara Bio) and amplified using real-time RT-PCR with a thermocycler (Lightcycler Nano; Roche Applied Science, Mannheim, Germany) and SYBR green detection system (Roche Applied Science). Gene-specific primers are listed in Table S1, Supporting Information. Real-time PCR was carried out with an initial denaturation step at 94 °C for 5 min. The amplification cycling conditions were as follows: 45 cycles of 30 s at 94 °C, 30 s at 65 °C, and 30 s at 72 °C for TNF-α; 32 cycles of 30 s at 94 °C, 45 s at 58 °C, and 30 s at 72 °C for IL-6; and 45 cycles of 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C for β-actin. The relative mRNA levels were normalized to the expression level of the β-actin and reported relative to the average of all Δcycle threshold (Ct) values in each sample using the Ct method. All samples were assayed in duplicate to ensure amplification integrity. Statistical Analysis. Survival data were analyzed by Kaplan−Meier curves and log-rank test. All other data were analyzed by one-way analysis of variance. Differences between groups were considered significant at p < 0.05, and the appropriate Bonferroni correction was made for multiple comparisons. All results are presented as the mean ± SEM.

EXPERIMENTAL SECTION

General Experimental Procedures. NMR experiments were performed with Varian Unity INOVA 500 and Bruker AVANCE III 700 spectrometers with the usual pulse sequences. The GC/EIMS data were obtained on a Hewlett-Packard HP 6890 series GC system with a Hewlett-Packard 5973 mass selective detector (Agilent Technologies Inc., Santa Clara, CA, USA). Column chromatography was carried out on silica gel 60 (230−400 mesh; Merck, Darmstadt, Germany) and Cosmosil 75C18-PREP (Nacalai Tesque, Kyoto, Japan). Plant Material. The roots of A. triphylla var. japonica were collected in September 2011 at Jeongseon County, Gangwon-do, Korea, and identified by Prof. Je-Hyun Lee, College of Oriental Medicine, Dongguk University. A voucher specimen was deposited in the Laboratory of Pharmacognosy, School of Pharmacy, Sungkyunkwan University (SKKU-Ph-11-01). Extraction and Isolation. The dried roots of A. triphylla (4.67 kg) were cut into small pieces and extracted with MeOH three times at room temperature. After maceration, total filtrate was concentrated, and the residue (427 g) was suspended in distilled water (2.4 L). The resulting solution was consecutively partitioned with CH2Cl2, EtOAc, and n-BuOH to yield CH2Cl2 (42.8 g), EtOAc (1.0 g), n-BuOH (16.5 g), and H2O (357.0 g) fractions. The CH2Cl2 fraction was fractionated on a silica gel column using a stepwise elution with hexane−CH2Cl2 (5:1, 1:1), CH2Cl2, and CH2Cl2−MeOH (10:1, 3:1) to give nine subfractions (D-1 to D-9). Subfraction D-7 was rechromatographed over a silica gel column using hexane−EtOAc (10:1) as an eluent to afford four further subfractions (D-7-1 to D-7-4). Compound 1 (522 mg) was obtained with a silica gel column (hexane−CH2Cl2, 1:1) and RP-C18 column chromatography (MeOH only) of subfraction D-7-3. This compound was identified as lupeol by comparing its spectroscopic data (1H NMR, 13C NMR, and GC/EIMS) with literature-reported values.38 Compound 1 (lupeol) was determined to be 96.2% pure by HPLC analysis. Animals and Treatment Regimens. Male ICR mice (23−25 g) were supplied from Orient Bio Inc. (Sungnam, Korea). Mice were housed in cages located in temperature- and humidity-controlled rooms (25 ± 1 °C and 55 ± 5%, respectively) with a 12 h light−dark cycle and received water and food ad libitum. All experiments were approved by the Animal Care Committee of Sungkyunkwan University (SUSP13-14) and performed in accordance with National Institutes of Health guidelines (NIH publication No. 86-23, revised 1985). For procedures, mice were fasted but given tap water ad libitum. Eighteen hours after starvation, the mice were intraperitoneally treated with vehicle (phosphate-buffered saline; PBS) or GalN (800 mg/kg; SigmaAldrich Chemical Co., St Louis, Mo, USA)/LPS (40 μg/kg; Escherichia coli O11:B4; Sigma-Aldrich Chemical Co.). One hour before GalN/ LPS treatment, mice were orally administered a vehicle (olive oil) and 1 (25, 50, and 100 mg/kg). Animals were randomly divided into six groups with 6−8 mice per group for biochemical study and 10 mice for survival analysis: (a) vehicle-treated control (control), (b) 1 (100 mg/ kg)-treated control (1), (c) vehicle-treated GalN/LPS (GalN/LPS), (d−f) 1 (25, 50, and 100 mg/kg)-treated GalN/LPS (1 + GalN/LPS). With mice under anesthetization with ketamine/xylazine, the blood was collected 6 h after GalN/LPS treatment from the inferior vena cava, and serum was separated by centrifugation (10000g, 10 min). The liver sample was obtained 6 h after GalN/LPS treatment. Liver tissue was embedded in 10% neutral-buffered formalin for histological assay, and the remainder was kept at −75 °C for further study. The dose and timing of 1 administration were selected based on a previous report and results of a preliminary study (data not shown).18,39 Mortality and Serum ALT Activity. Mice were monitored up to 24 h after GalN/LPS treatment for the survival test. Six hours after GalN/LPS treatment, serum ALT activity was determined by standard spectrophotometric procedures using the ChemiLab ALT assay kit (IVDLab Co., Uiwang, Korea). Histological Analysis. Six hours after GalN/LPS treatment, the liver was perfused with cold saline via hepatic portal vein, and liver tissues were removed from a portion of the left lobe. After washing off any blood or debris with ice-cold PBS, liver tissue was sectioned at a E

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ASSOCIATED CONTENT

S Supporting Information *

Gene-specific primers list. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Grant (12172MFDS989) from Ministry of Food and Drug Safety in 2012 (“Studies on the Identification of Efficacy of Biologically Active Components from Oriental Herbal Medicines”). So-Jin Kim (NRF2013H1A2A1034472) and Hong-Ik Cho (NRF2012H1A2A1016419) received ‘Global Ph.D. Fellowship Program’ support from the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (MEST).

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