In vitro and in vivo hepatoprotective effect of ganodermanontriol against t-BHP-induced oxidative stress

Journal of Ethnopharmacology 150 (2013) 875–885

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In vitro and in vivo hepatoprotective effect of ganodermanontriol against t-BHP-induced oxidative stress Do Thi Ha a,b, Joonseok Oh c, Nguyen Minh Khoi a, Trong Tuan Dao a, Le Viet Dung a, Thi Nguyet Que Do d, Sang Myung Lee e, Tae Su Jang f, Gil-Saeng Jeong g, MinKyun Na b,n a

National Institute of Medicinal Materials (NIMM), 3B Quangtrung, Hoankiem, Hanoi, Vietnam College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea c Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677, USA d Department of Pharmacology, Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hoankiem, Hanoi, Vietnam e Korea Ginseng Corp., Central Research Institute, Daejeon 305-805, Republic of Korea f Institute of Green Bio Science and Technology, Seoul National University, Seoul 151-742, Republic of Korea g College of Pharmacy, Keimyung University, Daegu 704-701, Republic of Korea b

art ic l e i nf o

a b s t r a c t

Article history: Received 31 May 2013 Received in revised form 9 September 2013 Accepted 11 September 2013 Available online 17 October 2013

Ethnopharmacological relevance: Ganoderma lucidum (Fr.) Karst. (Ganodermataceae) is a mushroom which is used as a traditional remedy in the treatment of human diseases such as hepatitis, liver disorders, hypercholesterolemia, arthritis, bronchitis and tumorigenic diseases. This study targets the evaluation of hepatoprotective activity of ganodermanontriol, a sterol isolated from Ganoderma lucidum, and the investigation of its mechanism of action in Hepa1c1c7 and murine liver cells upon tert-butyl hydroperoxide (t-BHP)-induced inflammation. t-BHP was utilized to stimulate an anti-inflammatory reaction in the hepatic cell lines and murine hepatic tissue examined. Western blot and reverse transcription-quantitative polymerase chain reaction (RT-PCR) were used to estimate the expression of ganodermanontriol (GDT)-induced proteins, including heme oxidase-1 (HO-1) and mitogen-activated protein kinases (MAPKs) as well as the corresponding mRNA. Luciferase assays were conducted to evaluate the interaction between NF-E2-related factor-2 (Nrf-2), the antioxidant response element (ARE), and the promoter region of the HO-1 gene and subsequent gene expression. Biochemical markers for hepatotoxicity were monitored to assess whether GDT protected the cells from the t-BHP-mediated oxidative stimuli. Results: GDT induced HO-1 expression via the activation of Nrf-2 nuclear translocation and the subsequent transcription of the HO-1 gene in vitro and in vivo, which seemed to be regulated by phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and p38 signaling pathways. GDT exhibited in vitro and in vivo hepatoprotective activity as determined by the lowered levels of hepatic enzymes and malondialdehydes and the elevated glutathione levels. Conclusions: This study validates the ethnopharmacological application of Ganoderma lucidum as a treatment for hepatic disorders. GDT induced in vitro and in vivo anti-inflammatory activity in t-BHP-damaged hepatic cells through the expression of HO-1, and in which PI3K/Akt and p38 kinases are involved. Our study motivates further research in the exploration of potent hepatoprotective agents from Ganoderma lucidum. & 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Ganoderma lucidum Ganodermanontriol Heme oxygenase-1 Hepatoprotective activity Oxidative stress

1. Introduction Oxidative stress causes the pathogenesis of many disease states including atherosclerosis, carcinogenesis, ischemia-reperfusion tissue injury and inflammatory disorders (Griendling et al., 2000; Dröge, 2002). Toxic reactive oxygen species (ROS), such as the superoxide anion and hydrogen peroxide, are produced during

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cellular respiration via aerobic metabolism or are present as exogenous oxidants. ROS damage cells by oxidizing critical cellular macromolecules such as nucleic acids, proteins and membrane lipids (Griendling et al., 2000). A significant number of anti-stress protein genes are induced to protect the cells as a defense mechanism in cases where cells are subjected to proinflammatory stimuli (Oh et al., 2006). One of these stressinducible enzymes, heme oxygenase (HO, EC 1.14.99.3), regulates the oxidative degradation process of heme into bilirubin, iron and carbon monoxide (CO) (Chung et al., 2008). Three isoforms of HO have been characterized in diverse cell types (Maines, 1997); HO-2

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and 3 are mainly detected as a constitutive form in the brains and testes of mammals (Morita et al., 1995; McCoubrey et al., 1997), while HO-1 is mostly distributed in the liver and spleen as an inducible form (Fernandez and Bonkovsky, 1999). The upregulation of HO-1 is induced by a wide range of proinflammatory stimuli such as ROS, heavy metals and nitrite (NO), and results in the catabolism of heme into the by-products, bilirubin and CO, which attenuate inflammatory reactions by scavenging ROS and NO pro-oxidants (Stocker et al., 1987; Maines, 1997; Sass et al., 2003; Ryter et al., 2006). Given previous studies that elaborate the prominent role of detoxification enzymes in the anti-inflammatory response, up-regulation of HO-1 expression is emerging as a promising therapeutic target for the treatment of inflammatory-mediated diseases (Sass et al., 2003; Tsuchihashi et al., 2003). Elevated HO-1 gene expression in response to pro-inflammatory stimuli is facilitated by several signaling cascades, such as the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), mitogen-activated protein kinase (MAPK), and NF-E2-related factor-2 (Nrf-2) signaling pathways (Kietzmann et al., 2003; Wu et al., 2006). Nrf-2 is a prominent transcription factor that enhances HO-1 gene expression via Nrf-2 and antioxidant response element (ARE) binding activity (Li et al., 2006). The transcription factor is normally sequestered in the cytoplasm as a result of binding to Keap1, an actin-binding protein (Itoh et al., 2004). In response to oxidative stress, Nrf-2 is liberated from the complex and immigrates into nuclei resulting in enhanced HO-1 gene expression, which is under control of signal transduction pathways such as MAPK and PI3K (Li et al., 2006; Oh et al., 2006). Further research verifies that the induction of HO-1 expression by natural products, including curcumin, diallyl sulfide and resveratrol, leads to anti-inflammatory activity via activation of the above-mentioned signaling pathways (Surh et al., 2008; Paine et al., 2010; Ha et al., 2013). This implies that natural products capable of inducing HO-1 expression provide a scaffold that can be used to develop potent anti-inflammatory agents for the treatment of inflammatory-mediated diseases such as vascular and liver disorders. Ganoderma lucidum (Fr.) Karst. (Ganodermataceae) is a medicinal mushroom traditionally utilized to treat various human diseases including hepatitis, liver disorders, hypercholesterolemia, arthritis, bronchitis and tumorigenic diseases (Song et al., 2004; Kuo et al., 2006; Olaku and White, 2011). Previous investigations on biologically active constituents of Ganoderma lucidum identify polysaccharides, which exhibit antiherpetic (Oh et al., 2000) and immunomodulating activity (Lin et al., 2006), p-hydroxybenzoic and cinnamic acids, which are modified to enhance antimicrobial and demelanizing activity (Heleno et al., 2013), and extracts of the fruiting bodies and cracked spores, which display in vivo and in vitro anti-breast cancer effects (Suarez-Arroyo et al., 2013). In our continuous effort to explore anti-inflammatory, natural products as prototypes capable of stimulating HO-1 expression (Ha et al., 2013), H2O, 70% EtOH and MeOH extracts from the fruiting bodies of Ganoderma lucidum, and ergosterol (1), ergosterol peroxide (2) and ganodermanontriol (GDT) (3) from the active MeOH extract are evaluated for the potential to up-regulate HO-1 expression in Hepa1c1c7 cells, a hepatic cell line commonly employed for the assessment of anti-inflammatory activity (Tan et al., 2007; Hwang et al., 2008; Kim et al., 2012a), and murine liver tissue subjected to an oxidative environment generated by tertbutyl hydroperoxide (t-BHP). Based upon the above-mentioned studies that Nrf-2 nuclear translocation is mediated via activation of PI3K/Akt and MAPK pathways, specific pharmacological inhibitors of these pathways are utilized to elucidate the pathway by which GDT mediates HO-1 related Nrf-2 nuclear translocation. GDTmediated Nrf-2 nuclear translocation seems to be regulated by

upstream signaling cascades involving PI3K/Akt and p38, which are both involved in the expression of anti-oxidant enzymes (Li et al., 2006).

2. Materials and methods 2.1. Materials α-MEM, DMEM, fetal bovine serum, sodium pyruvate, and TRIzols were purchased from Gibco BRL (Grand Island, NY). Antibodies against Akt, phospho-Akt, phospho-MAPKs and HO-1 were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against Nrf-2, Lamin B1 and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). LY294002 and SB203580 were purchased from Calbiochem (La Jolla, CA). 2.2. Cell culture and treatments Hepa1c1c7 cells (ATCC CRL-2026) were obtained from American Type Culture Collection (Manassas, VA). The cells were maintained at 37 1C in an incubator with a humidified atmosphere of 5% CO2 and cultured in α-MEM containing 10% fetal bovine serum, streptomycin (100 μg/mL) and penicillin (100 U/mL). GDT was dissolved in DMSO and the stock solutions were added directly to culture media. Control cells were treated with DMSO. The final concentration of solvent was less than 0.1% for all experiments in this study. 2.3. Preparation of samples 2.3.1. Preparation of extracts The air-dried fruiting bodies of Ganoderma lucidum (50 g) were collected in Quangnam Danang, Vietnam, authenticated by Prof. K.H. Bae and a specimen was deposited in the Department of Phytochemistry, NIMM (DP-122011). The sample was extracted with 70% EtOH, MeOH, and H2O (3  0.6 L). Each extract was passed through a No. 1 Whatman filter (Whatman Inc., Hillsboro, OR, USA) and the filtrate was evaporated to dryness under vacuum at 40 1C to obtain the 70% EtOH (2.06%; wt/wt), MeOH (2.05%; wt/wt), and H2O extracts (2.2%; wt/wt). The 70% EtOH (GLE), MeOH (GLM), and H2O (GLW) extracts were stored at  20 1C until tested. 2.3.2. Purification of the tested compounds A large scale extraction of air-dried fruiting bodies of Ganoderma lucidum (7 kg) was carried out to acquire a sufficient quantity of the bioactive GLM extract (see Section 3.1 and Fig. 2). The sample was extracted with MeOH (3  30 L) over 2 h, filtered and concentrated to afford the MeOH extract (48 g). The MeOH extract was suspended in H2O (1.5 L) and successively extracted with n-hexane (2  1.5 L) and CH2Cl2 (3  2.0 L). The CH2Cl2 extract (220 g) was dissolved in MeOH and filtered to acquire a precipitate which was crystalized in CH2Cl2 to obtain compound 1 (1.2 g). The filtrate was further fractionated using the silica gel column chromatography (60  12 cm; CH2Cl2:MeOH 200:1-1:1) and generated five fractions (GLC1–GLC5). Fraction GLC2 (20 g) was purified by the silica gel column chromatography (30  12 cm; n-hexane:EtOAc 20:11:1) to obtain compound 2 (120 mg). The fraction GLC1 (63.6 g) was chromatographed on a silica gel column (80  6 cm), eluted with CH2Cl2:MeOH (20:1) and yielded two sub-fractions (GLC1A and GLC1B). Fraction GLC1A (8.2 g) was chromatographed on a RP-18 column (80  5 cm) and eluted with MeOH:H2O (2:1) to obtain compound 3 (131 mg). The purified compounds were identified as ergosterol (1), ergosterol peroxide (2) and GDT (3) (Fig. 1) by comparison of experimental and reported spectroscopic and physicochemical data (Sato et al., 1986; Goad and Akihisa, 1997; Ishizuka et al., 1997).

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Fig. 1. Chemical structures of compounds (1–3) isolated from Ganoderma lucidum.

2.4. RNA extraction and RT-PCR

2.6. HO-1 activity assay

RNA was extracted by adding 1:5 (v/v) chloroform to a mixture of TRIzols and cells, and was precipitated after mixing with an equal amount of isopropanol. Extracted RNA was washed twice with 75% EtOH, dissolved in diethyl pyrocarbonate-treated H2O and stored at  70 1C. RNA was analyzed in 1% agarose gel and treated with RNase free DNase (Promega). RT-PCR was performed following the instructions provided by the manufacturer (Life Technologies). Briefly, first strand cDNA was synthesized by using the Thermoscript Reverse Transcription System (Life Technologies) with DNase-treated total RNA. Reverse transcriptase was left out of one sample as a control. PCR experiments were conducted with a Takara PCR System TP600 (Takara Bio Inc., Japan) using selective primers for HO-1 (forward, 5′-AAGGCTTTAAGCTGGTGATGG-3′; and reverse, 5′-AGCGGTGTCTGGGATGAACTA-3′). To ensure that equal amounts of cDNA were added to each PCR experiment, the β-actin gene was amplified using synthetic oligonucleotides (forward, 5′-TGTTTGAGACCTTCAACACC-3′; and reverse, 5′-CGCTCATTGCCGATAGTGAT-3′) and used as the internal standard for individual amplification reactions. PCR conditions for the amplification of HO-1 and β-actin were as follows: 25 cycles with each cycle consisting of 94 1C for 30 s, 56 1C for 30 s, and 72 1C for 45 s. PCR products were analyzed using Gel Doc 1000 (Bio-Rad). Every RT-PCR experiment was repeated at least 3 times.

HO-1 activity was determined at the end of each treatment using a method described previously (Foresti et al., 1997). Briefly, microsomes from harvested cells were added to a reaction mixture containing NADPH, glucose-6-phosphate dehydrogenase, rat liver cytosol as a source of biliverdin reductase, and the substrate hemin. The reaction was maintained in the dark at 37 1C for 1 h and terminated by the addition of chloroform (1 mL). After vigorous vortexing and centrifugation, the extracted bilirubin in the chloroform layer was measured by the difference in absorbance between 464 and 530 nm using a UV-1601 UV/vis spectrophotometer (Shimadzu, Tokyo, Japan).

2.5. Western blot analysis After treatment with GDT, cells were collected and washed with phosphate-buffered saline. The harvested cells were lysed on ice for 30 min in 200 μL lysis buffer [120 mM NaCl, 40 mM Tris (pH 8), 0.1% NP40] and centrifuged at 13,000 rpm for 30 min. Supernatants were collected from the lysates and protein concentrations were determined employing a BCA protein assay kit (Pierce, Rockford, IL). Aliquots of the lysates (30 μg of protein) were boiled for 5 min and electrophoresed on 10% SDS-polyacrylamide gels. Proteins in the gels were transferred onto nitrocellulose membranes, which were incubated with rabbit polyclonal Akt, phospho-Akt, phospho-MAPKs, Nrf-2 and HO-1 or mouse monoclonal β-actin antibodies. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive protein bands were detected using an enhanced chemiluminescence western blotting detection kit (Pierce Biotechnology, Rockford, IL).

2.7. Nuclear and cytosolic lysate preparation Nuclear extracts were prepared with a commercial kit with reference to the manufacturer's instructions (Active Motif, Carlsbad, CA). All steps were carried out on ice or at 4 1C. Protease inhibitors (10 μg/mL aprotinin and 10 μg/mL leupeptin) and reducing agents (1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) were added to each buffer prior to use. Cells were incubated in hypotonic buffer A [20 mM Hepes (pH 7.9), 1.5 mM MgCl2, 10 mM KCl] on ice for 15 min and homogenized. Nuclei were recovered by centrifugation at 3,000 rpm for 15 min, and the supernatant was reserved as the cytoplasmic extract. The nuclei were washed once using nuclei wash buffer (10 mM Hepes, pH 7.9, 0.2 mM MgCl2, 10 mM KCl) and extracted using buffer C [20 mM Hepes (pH 7.9), 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl2] for 30 min on ice. Insoluble material was removed by centrifugation at 13,000 rpm for 30 min and the supernatant was used as the nuclear extract. 2.8. Transient transfection and luciferase assay A dual-luciferase reporter assay system (Promega, Madison, WI) was utilized to determine HO-1-ARE-promoter activity. Cells were plated in 12-well plates overnight and transiently cotransfected with HO-1-ARE-promoter-luciferase construct using LipofectAMINE™ 2000 reagent (Invitrogen, Carlsbad, CA). The cells were exposed to GDT (10–40 μM) for 18 h, and luciferase activities in cell lysates were measured using an ML1000 microplate luminometer (Dynatech Laboratories, Chantilly, VA). Relative luciferase activities were calculated by normalizing HO-1-ARE-promoter

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luciferase activity to Renilla luciferase activity using the pRL-SV plasmid (Promega, Madison, WI). 2.9. In vitro determination of hepatic alanine transaminase (ALT) and aspartate transaminase (AST) production The levels of ALT and AST in culture media were measured using commercially available assay kits (Randox Laboratories, Antrim, UK). Hepa1c1c7 cells were incubated in the presence or absence of various concentrations of GDT for 1 h and cells were treated with t-BHP for 24 h. After incubation, cultures were centrifuged and the supernatant was removed. Total ALT and AST were measured at 340 nm using a Biochrom EZ Read 400 ELISA microplate reader. 2.10. In vitro determination of lipid peroxidation and glutathione (GSH)

0.01 mM phenylmethoxysulfonyl fluoride in a Potter–Elvehjem homogenizer. Hepatic microsomal fractions were obtained by differential centrifugation as described previously (Lee et al., 2001), and hepatic GSH and MDA levels were assessed colorimetrically using Ellman's reagent (Lee et al., 2004). 2.12. Measurement of cell viability Cells were plated in 96-well plates and cell viability was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay. After incubation, cells were treated with MTT solution at up to 1 mg/mL for 1 h. The dark blue formazan crystals that formed in intact cells were dissolved with DMSO and the absorbance at 570 nm was measured with a UV-1601 spectrophotometer (Shimadzu, Tokyo, Japan). 2.13. Statistical analysis

t-BHP-altered hepatic GSH and lipid peroxidation levels were assessed in Hepa1c1c7 cells. Briefly, 1  107 cells/mL were seeded in 6-well culture plates. Cells were pretreated with different concentrations of GDT for 1 h. Toxicity was induced by the addition of t-BHP to culture media for 24 h. Cells were lysed with 100 μL of the lysis buffer. Lysates were centrifuged at 16,000g for 15 min at 4 1C and the supernatant was mixed with 100 μL trichloroacetic acid to induce precipitation and then was further centrifuged at 16,000g for 5 min. Cellular GSH levels were measured in non-protein cell lysates by using a commercially available GSH assay kit (Oxis International, Foster City, CA, USA). Lipid peroxidation was assessed by the formation of malondialdehyde (MDA: the lipid peroxidation product) in cultured cell lysates using a lipid peroxidation assay kit according to the supplier's instructions (Oxford Biomedical Research, Oxford, MI, USA). 2.11. Animals and experimental protocols Male ICR mice (25–30 g) were obtained from the Korean Food and Drug Administration (Seoul, Korea) and allowed access to Purinas rodent chow and tap water. The animals were maintained in a controlled environment at 21 1C and 50% relative humidity with a 12 h dark/light cycle. The animal protocol used in this study was reviewed by the Chungnam National University on the ethical procedures and scientific care and was approved. Five experimental groups (I–V) of six mice each were randomly assigned for in vivo experiments (see Section 3.5 and Fig. 8). Group I, shown on the first lane in Fig. 8A and B, and indicated “control” in Fig. 8C–F, represents a negative control; group II, shown on the second lane in Fig. 8A and B, and denoted “þ ” in Fig. 8C–F, is an experimental group treated only with the indicated concentration of t-BHP; group III, IV and V are experimental groups treated with 2 mmol/ kg of t-BHP upon pretreatment of GDT 250, 500 and 1000 mg/kg, respectively. GDT in saline was administered intraperitoneally (i.p.) at the indicated concentrations once daily for three consecutive days. Three hours after the final administration, the mice were treated with t-BHP (2 mmol/kg, i.p., 100:1 dissolved in saline). Twenty-four hours after t-BHP treatment, mice were anesthetized with CO2, blood was removed by cardiac puncture to evaluate serum ALT and AST activities, and mice were euthanized by cervical dislocation. Hepatotoxicity was assessed using mice liver tissues by measuring the serum activities of ALT, AST, GSH and MDA. Serum ALT and AST activities were assessed employing spectrophotometric diagnostic kits (Sigma Co., St. Louis, MO, USA). To measure hepatic GSH and MDA levels, livers were quickly harvested, weighed and perfused with ice-cold 0.15 M KCl, and then homogenized with 4% (w/v) of 10 mM Tris–HCl (pH 7.4) containing 0.15 M KCl, 0.1 mM EDTA, 1.0 mM dithiothreitol and

All experiments were repeated at least 3 times. Mean 7standard deviation (SD) were calculated for each group and Dunnett's t-test was used to calculate statistical significance. Differences were considered statistically significant when p o0.01. 3. Results 3.1. Effects of extracts and purified compounds on HO-1 and Nrf-2 expression Nrf-2 plays a vital role in the escalation of HO-1 expression in response to various oxidative stimuli and the up-regulation of HO-1 is known to elicit hepatoprotective activity (Abraham and Kappas, 2008) (Surh et al., 2008; Paine et al., 2010). Based on these findings, Hepa1c1c7 cells were treated with 40 μM of H2O (GLW), 70% EtOH (GLE), and MeOH extracts (GLM) to evaluate the potential to increase Nrf-2 and HO-1 expression. Based on RTPCR and western blot analysis, GLE and GLM induced the expression of Nrf-2 and HO-1 (Fig. 2A and B). GLM showed the most significant effect on up-regulation of Nrf-2 and HO-1 expression and further purification of the GLM extract was conducted. Three compounds, ergosterol (1), ergosterol peroxide (2) and GDT (3), were purified using various chromatographic techniques and identified from the active extract (See Section 2.3.2; Fig. 1). Hepa1c1c7 cells were treated with 20 μM of the purified compounds to assess the potential to up-regulate Nrf-2 and HO-1 expression. According to RT-PCR and western blot analysis (Fig. 2C and D), GDT (3) exhibited the most potent stimulating effects on HO-1 and Nrf-2 expression, thus, it was employed in further studies examining upstream signaling pathways involved in the induction of HO-1 expression. Furthermore, the proportional level of expression of HO-1 and Nrf-2 induced by each compound is in agreement with previous studies highlighting the predominant role of Nrf-2 on HO-1 expression (Surh et al., 2008; Paine et al., 2010). 3.2. Non-cytotoxic GDT stimulated HO-1 expression via activation of Nrf-2 nuclear translocation and transcription in Hepa1c1c7 cells Prior to a mechanistic study of GDT, the compound was evaluated for cytotoxic activity against hepatic cells using an MTT assay and was observed to be non-cytotoxic at concentrations lower than 100 μM (Fig. 3A). Consequently, the ability of GDT to protect cells from t-BHP-induced oxidative damage was examined. Hepatic cell viability, after a reduction to 40% of that of the control upon t-BHP-induced oxidative stress, recovered to 70% of that of

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Fig. 2. The effect of GLW, GLE, GLM and three isolated compounds (1–3) on the expression of HO-1 mRNA and protein and Nrf2. HO-1 and Nrf2 expression were analyzed by RT-PCR and western blot analyses. Effects of the three extracts from Ganoderma lucidum on HO-1 mRNA (top) and protein expression (bottom) (A) and on Nrf-2 protein expression (B) in Hepa1c1c7 cells. Cells were exposed to 20 μg/mL of each extract for 6 h. Effects of compounds (1–3) on HO-1 mRNA (top) and protein expression (bottom) (C) and on Nrf-2 protein expression (D) in Hepa1c1c7 cells. Cells were exposed to 20 μM of each compound for 6 h.

Fig. 3. Cell viability was assessed by an MTT assay. Each bar represents the mean 7SD calculated from three independent experiments (significant as compared to control, n p o 0.01). (A) Effect of GDT on cell viability. Hepa1c1c7 cells were seeded in a 96-well plate and incubated in various concentrations of GDT (2–200 μM) for 24 h. (B) Cell viability of Hepa1c1c7 cells pretreated with GDT (2–100 μM) for 24 h, and incubated with t-BHP (500 μM) for an additional 24 h. #Significantly different from t-BHP-treated cells, p o 0.01. The “þ ” column represents treatment with t-BHP (500 mM) without GDT pretreatment.

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Fig. 4. Effect of GDT pretreatment on HO-1 activity, mRNA and protein expression in Hepa1c1c7 cells. (A) Effect of GDT on HO-1 activity was assessed (see Section 2.6). n Significantly different from untreated cells (p o 0.01). Hepatic cells were exposed to various concentrations of GDT (10–40 μM) for 6 h (B) or 40 μM GDT for the indicated times (C). Total RNA was extracted and HO-1 mRNA was analyzed by RT-PCR (top of B and C) and resultant HO-1 protein expression was evaluated using western blot analysis (bottom of B and C). β-actin band is shown to confirm integrity and equal loading of RNA.

the control as the concentration of GDT increased to 40 μM (Fig. 3B). This suggests that GDT protects hepatic cells from proinflammatory stimuli generated by t-BHP. To examine if the observed GDT-mediated hepatoprotective activity was related to the activation of HO-1 expression, Hepa1c1c7 cells were exposed to GDT at the indicated concentrations for 24 h or at 40 μM for the indicated times (Fig. 4B and C) and nuclear extracts were subjected to RT-PCR. HO-1 activity increased with GDT pretreatment of 2–200 μM and peaked at 40 μM with a 5.5-fold increase compared to that of the control. A slight decrease from the maximum GDTinduced activity was observed at concentrations higher than 40 μM (Fig. 4A, top). The enzyme activity increased from 3 h to 24 h and reached its maximum after 6 h of exposure (7-fold over 0 h of exposure) (Fig. 4A, bottom) and slightly diminished from the maximum by 24 h of exposure. A similar dose–response and timedependent HO-1 activity following treatment with another compound was observed in a previous study (Kim et al., 2012b). As shown in Fig. 4B and C, the indicated concentrations and times of GDT pretreatment enhanced HO-1 mRNA and consequent protein expression in a dose- and time-dependent manner, which implies that GDT-promotes HO-1 protein expression. According to previous studies, HO-1 induction is dependent on nuclear translocation of the transcription factor Nrf-2 (Li et al., 2006; Surh et al., 2008). Upon activation by oxidative stress, Nrf-2 translocates into the nucleus and binds to ARE in the promoter region of a wide array of target genes and facilitates the expression of genes encoding anti-oxidant enzymes including HO-1 (Li et al., 2006). Based on these studies, we further explored the effect of GDT on HO-1 expression via the enhancement of nuclear translocation of Nrf-2 and the increase in the interaction between Nrf-2 and ARE in the promoter region of the HO-1 gene. Hepatic cells were treated with GDT at concentrations of 0, 10, 20 and 40 μM for 6 h and a western blot of the Nrf-2 in nuclear extracts

was conducted. GDT enriched nuclear Nrf-2 expression in a dosedependent manner (Fig. 5A). To examine whether GDT promoted the interaction between Nrf-2 and the ARE binding site in the HO1 promoter region, Hepa1c1c7 cells were transfected with luciferase expression vectors containing the wild-type promoter ARE sequence and the ARE activity was examined (Fig. 5B). The observed ARE activity was augmented as the concentration of GDT was increased and treatment with 40 μM GDT significantly elevated the ARE promoter activity by 3-fold in the examined cells compared to the control (Fig. 5B). These data indicated that GDT induced the Nrf-2 nuclear translocation and transactivation of HO-1 expression. 3.3. GDT diminishes the levels of hepatic enzymes in t-BHP-induced Hepa1c1c7 cells To examine whether GDT-induced HO-1 expression exerted a hepatoprotective effect, Hepa1c1c7 cells were exposed to a t-BHPinduced hepatotoxic environment. AST and ALT were employed as biochemical markers of early acute hepatic damage. Both AST and ALT increased in cells after a 24 h exposure to 500 μM t-BHP indicating that the pro-inflammatory stimulus resulted in hepatotoxicity in Hepa1c1c7 cells (Fig. 6A and B). Pretreatment with GDT prior to t-BHP-induced hepatotoxicity diminished the elevated ALT and AST levels in a dose-dependent manner (Fig. 6A and B), and upon pretreatment with 40 μM of GDT the levels were restored to those near the control. To examine whether GDT prevents t-BHPinduced oxidative damage on cellular macromolecules in Hepa1c1c7 cells, MDA, a biochemical marker of membrane lipid peroxidation, was measured upon treatment of the indicated concentrations of GDT (Fig. 6C). The exposure of hepatic cells to t-BHP for 6 h escalated the level of cell-associated MDA in Hepa1c1c7 cells, and GDT reduced MDA production in a dose-dependent manner (Fig. 6C). GSH levels

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Fig. 5. Effect of GDT on Nrf-2 translocation into the nucleus and transcriptional activity in Hepa1c1c7 cells. (A) Effect of GDT on nuclear translocation of Nrf2. The cells were treated with GDT (10–40 μM) for 6 h and nuclear extracts were subjected to western blotting. (B) Effect of GDT on ARE promoter activity. Cells were transfected with the HO1-ARE reporter plasmid and treated with GDT (10–40 μM). After 18 h, the cells were harvested and luciferase activities were determined to estimate the transactivation of HO-1 expression. nSignificantly different from untreated cells (p o0.01).

Fig. 6. Effects of GDT on ALT, AST, MDA and GSH levels in Hepa1c1c7 cell lysates. Cell were treated with t-BHP for 24 h upon pretreatment of the indicated concentrations of GDT for an hour. Hepatic ALT (A) and AST (B) were measured in the culture supernatant. Hepatic MDA (C) and GSH (D) cellular levels were assessed using Ellman's reagent. Values represent the mean 7 SD of three experiments. nSignificantly different from untreated cells, #significantly different from GDT-treated cells (po 0.01). The “þ ” column represents treatment with t-BHP (500 mm) without GDT pretreatment.

were examined in Hepa1c1c7 cells with t-BHP-induced oxidative damage, as the GSH system is known to be involved in alleviating oxidative stress (Applegate et al., 1991). As shown in Fig. 6D, the exposure of the hepatic cells to t-BHP significantly depleted GSH. GDT pretreatment (10 μM) restored the reduced GSH level to that of the unstressed cells (control), and pretreatment with 40 μM of GDT resulted in a 1.4 fold increase in GSH levels compared to the control (Fig. 6D). The observed levels of known hepatotoxicity biomarkers support that GDT promotes hepatoprotective activity in a t-BHPinduced hepatotoxic model. 3.4. GDT-induced HO-1 and Nrf-2 expression through the PI3K/Akt and p38 pathway A network of signaling cascades is involved in the up-regulation of HO-1 gene expression resulting in anti-inflammatory activity.

Recent reports demonstrate that the PI3K/Akt and MAPK pathways are involved in the induction of HO-1 in response to oxidative stimuli (Park et al., 2011; Kim et al., 2012b). To investigate which pathways were involved in the GDT-mediated HO-1 expression, Hepa1c1c7 cells were treated with GDT (40 mM) for the indicated times and phosphorylation of Akt kinase, related to the PI3K/Akt pathway, and that of p38, ERK 1/2 and JNK kinases, associated with MAPK pathways, were measured by the western blot (Fig. 7A). Phosphorylation of Akt and p38 were enhanced in GDT-treated Hepa1c1c7 cells while phosphorylation of ERK 1/2 and JNK were not affected (Fig. 7A). LY294002 and SB203580, pharmacologic inhibitors of PI3K/Akt and p38 pathways, respectively, were used to address the role of each pathway in GDTinduced HO-1 expression. Hepatic cells were pre-incubated in 10 μM of each inhibitor for 30 min and then treated with 40 μM of GDT for 24 h. Based on the observation of a reduction in HO-1

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Fig. 7. Induction of HO-1 and activation of Nrf-2 by GDT via the phosphorylation of Akt and p38 in Hepa1c1c7. (A) Effect of GDT treatment on phosphorylation of Akt and MAPK-related kinases. Hepa1c1c7 cells were treated with 40 μM GDT for 5–45 min and immunoblotted with the specific antibodies. (B) Effect of PI3K (LY) and p38 (SB) pathway inhibitors on GDT-induced HO-1 expression. Cells were pre-incubated with 10 μM LY294002 (LY) or 10 μM SB203580 (SB) for 30 min and then incubated with 40 μM GDT for 24 h. Whole of the cell lysates were subjected to western blotting analyses with anti-HO-1. (C) Effect of PI3K and p38 inhibitors on GDT-induced Nrf-2 translocation. Cells were pre-incubated with 10 μM LY294002 or 10 μM SB203580 for 30 min and then incubated with 40 μM GDT for 3 h. Nuclear extracts were subjected to western blotting with anti-Nrf-2. Anti-lamin B1 antibodies were used for a loading control. (D) Effect of PI3K and p38 inhibitors on GDT-induced HO-1-ARE-luciferase activity. Cells were transfected with the HO-1-ARE-luciferase plasmid construct. After overnight incubation, cells were treated with 40 μM GDT for 18 h in the presence or absence of LY294002 or SB203580 and cell lysates were mixed with the luciferase substrate. nSignificantly different from untreated cells, #significantly different from GDTtreated cells (p o0.01).

expression only when Hepa1c1c7 cells were treated with both GDT and each respective inhibitor, it is established that inhibition of the PI3K/Akt and p38 pathways results in attenuation of GDTinduced HO-1 expression (Fig. 7B). To determine whether the activation of the PI3K/Akt and p38 pathways were involved in the process by which GDT activated Nrf-2 nuclear translocation and transactivity, the hepatic cells were pre-incubated with the specific inhibitors and treated with GDT. The inhibitors (10 μM) of PI3K/Akt and p38 pathways hampered the GDT-induced Nrf-2 nuclear accumulation and enhancement of ARE activity (Fig. 7C and D). As seen with HO-1 expression, Nrf-2 accumulation was attenuated only upon co-treatment with GDT and each inhibitor (Fig. 7C). The observed ARE activity increased 4.5-fold compared to the control upon pretreatment of GDT (40 μM) and this level diminished by half with co-treatment of GDT and each inhibitor (Fig. 7D). These results suggest that GDT-induced Nrf-2 expression occurs via activation of PI3K/Akt and p38 signaling pathways, and the increased Nrf-2–ARE binding interaction results in the up-regulation of HO-1 expression. 3.5. In vivo hepatoprotective effects of GDT via enhancement of HO-1 and Nrf-2 expression In vivo experiments assessing liver tissue harvested from GDTtreated mice were conducted to confirm the observed in vitro GDTinduced hepatoprotective effect; in addition, the enhancement of HO-1 and Nrf-2 expression was examined. Mice were treated with the indicated GDT concentrations followed by t-BHP, which was utilized to induce a hepatotoxic environment, and HO-1 expression was measured employing western blot analysis of harvested

cell lysates. GDT treatment enhanced HO-1 expression in a dosedependent manner (Fig. 8A). Accumulation of Nrf-2 was observed at a GDT pretreatment concentration of 250 mg/kg and was maintained up to 1000 mg/kg indicating that the enhanced HO-1 gene expression was associated with nuclear translocation of Nrf-2 (Fig. 8A). These in vivo results confirmed the in vitro observation of GDT-induced HO-1 expression via the activation of Nrf-2 nuclear translocation in Hepa1c1c7 cells. To further address the upstream signaling cascades related to GDT-induced Nrf-2 and consequent HO-1 expression, the activation of PI3K/Akt and MAPK pathways in the murine liver cells were examined in mice treated with tBHP. As shown in Fig. 8B, the phosphorylation of Akt and p38 was enhanced upon GDT pretreatment at concentrations higher than 250 mg/kg but that of ERK 1/2 and JNK was not affected by GDT pretreatment. This implied that GDT enhanced Nrf-2 translocation via PI3K/Akt and p38 signaling pathways, and hence increased HO-1 expression in vivo, which is consistent with the in vitro results. Serum hepatic enzyme levels of MDA and GSH were measured to investigate whether GDT protected murine hepatic cells from t-BHP-mediated oxidative stress. In t-BHP-treated mice without GDT pretreatment, acute hepatic damage by the stressor was explicit as manifested by the increased serum ALT, AST, and MDA as well as the decreased GSH levels (Fig. 8C–F). GDT treatment prior to t-BHP-induced oxidative damage reduced the ALT, AST, and MDA levels in a dose-dependent manner, and 1000 mg/kg of the pretreatment diminished the hepatic enzyme and membrane lipid peroxidation levels by 3.2-, 3.2- and 2.2-fold, respectively, as compared to mice without GDT treatment (Fig. 8C–E). GDT pretreatment in mice at 250 mg/kg restored the depleted GSH level to

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Fig. 8. Effect of GDT pretreatment on t-BHP (2 mmol/kg) induced hepatotoxicity in liver tissue harvested from GDT-treated mice. Five groups (I–V) of six mice each were employed for the in vivo experiment. Group I (the first lane in Fig. 8A and B, “control” in Fig. 8C–F) is a negative control and group II (the second lane in Fig. 8A and B, “ þ” in Fig. 8C–F) is an experimental group only treated with the indicated concentration of t-BHP. Group III, IV and V are experimental groups treated with 2 mmol/kg of t-BHP upon pretreatment of GDT 250, 500 and 1000 mg/kg, respectively. (A) Effects of GDT on HO-1 and Nrf-2 protein expression levels in t-BHP-treated liver lysates from the mice with GDT pretreatment. Cytosolic lysates were prepared and subjected to western blot to analyze HO-1 protein expression level. Nrf-2 protein expression was monitored in nuclear fraction employing western blot analysis. (B) Effects of GDT on Akt and MAPK activation in GDT-treated mice liver cells with t-BHP-induced hepatotoxicity. Cytosolic lysates were prepared and subjected to western blot to measure the phosphorylated forms of Akt, p38, JNK and ERK 1/2 kinases expression levels. Effects of GDT on serum ALT, AST, MDA and GSH levels in mouse liver tissues in the response to t-BHP-induced oxidative stimuli. The liver cells were pretreated with the indicated concentrations of GDT as described in Section 2.11. Serum ALT (C) and AST (D) were measured and hepatocellular MDA (E) and GSH (F) levels were assessed from the harvested mice liver lysates. Values represent the mean 7 SD of three experiments. nSignificantly different from untreated cells, #significantly different from GDT-treated cells (p o 0.01).

that of the negative control and GSH continued to accumulate in a dose-dependent manner (Fig. 8F).

4. Discussion and conclusions In spite of numerous attempts to elucidate the mechanisms of ethnopharmacological properties of Ganoderma lucidum, few studies have been conducted to address the anti-inflammatory properties of this medicinal mushroom. In this study, three compounds, ergosterol (1), ergosterol peroxide (2) and GDT (3) were obtained from the active MeOH extract and further utilized in the investigation of the mechanism by which Ganoderma lucidum alleviates oxidant-induced cellular damage in Hepa1c1c7 and mice liver cells. t-BHP was used to induce liver toxicity because this stressor was reported to generate harmful free radical intermediates which subsequently react with DNA, proteins, and lipids leading to hepatotoxicity (Grunberger et al., 1988). The stimulation of the phase II enzyme systems, such as HO-1, glutathione S-transferase, and glutamylcysteine synthetase, plays a significant role in the removal of generated ROS before ROS can interact with and damage cellular macromolecules (Rushmore and Tony Kong, 2002). Anti-inflammatory agents are able to either

remove ROS directly or activate the phase II enzyme systems ultimately resulting in ROS scavenging. Our study suggested that GDT exerted anti-inflammatory biological activity in t-BHPinduced hepatotoxicity present in cells and mice via the activation of HO-1 enzyme expression. This expression was regulated by Nrf-2 activation and upstream PI3K/Akt and MAPK signaling pathways as shown by phosphorylation of Akt and p38. Even though there have been several studies attempting to link the activation of MAPK pathways with transcription factors that bind to ARE regions and induce antioxidant enzymes, the mechanism by which activated MAPK pathway influences Nrf-2–ARE-dependent transcriptional activation is still elusive. Oxidized low-density lipoproteins induced nuclear translocation of Nrf-2 via activation of p38, JNK and ERK 1/2 leading to HO-1 expression in human vascular smooth muscle cells (Anwar et al., 2005). On the other hand, the transcriptional activation of the detoxifying enzyme human NADPH:quinone oxidoreductase was mediated by PI3K without the involvement of ERK (1/2) in the IMR-32 human neuroblastoma cells damaged by tert-butylhydroquinone (Lee et al., 2001). A similar activation pattern to the latter example was observed when HO-1 expression was induced in PC12 cells upon treatment with 3-morpholinosydnonimine (Li et al., 2006). In our study, the inhibition of either the PI3K/Akt or the p38

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pathway abrogated the phosphorylation of Akt and p38 thus inhibiting the GDT-inducible enhancement of Nrf-2–ARE binding transcriptional activity. In line with our observation, a mechanistic study by Hseu et al. (2012) found that the PI3K/Akt and p38 pathways were involved in the up-regulation of HO-1 expression through the induction of Nrf-2–ARE nuclear translocation and transcription activity in human oral carcinoma (HSC-3) cells treated with flavokawain B. In light of these findings, it is plausible that different upstream signaling kinases participate in facilitating the activation of Nrf-2 depending on the examined cell types and the nature of the treated protective compounds. The increased serum levels of ALT, AST and MDA in Hepa1c1c7 and mouse liver cells treated with t-BHP suggests that the stressor induces hepatic structural damage because these hepatic enzymes are generally localized in the cytoplasm and are released into circulation in response to cellular damage. GDT treatment decreased ALT, AST and MDA implying that GDT protected the hepatic cells from acute hepatic and membrane damage induced by t-BHP. The mechanism by which GDT treatment affected levels of the liver enzymes and MDA could be at least partially correlated to its potential to increase HO-1 expression considering that the in vitro and in vivo levels of the liver enzymes and MDA were reduced in correlation with increased GDT dose and subsequent expression of HO-1. This is supported by the similar observation that CO, a product of HO-1 catabolism in the HO-1 induced-C57BL/ 6J mouse model, was capable of lowering levels of liver enzymes to prevent hepatic damage and hepatic failure (Zuckerbraun et al., 2003). Furthermore, our study showed a GDT-induced restoration and subsequent accumulation of GSH serum levels depleted as a result of t-BHP treatment in hepatic cells and mice. The accumulated GSH levels upon GDT-induced HO-1 expression suggested that oxidative stimuli were alleviated, exemplifying HO-1 expression and its influence on GSH levels in response to a natural product-stimulated hepatoprotective reaction. In conclusion, this study supports the ethnopharmacological application of Ganoderma lucidum in the alleviation of inflammatory hepatic disorders and motivates additional research for the exploration of a potent hepatoprotective compound from this medicinal mushroom.

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