Heme oxygenase-1 mediates the anti-inflammatory effect of mushroom Phellinus linteus in LPS-stimulated RAW264.7 macrophages

Journal of Ethnopharmacology 106 (2006) 364–371

Heme oxygenase-1 mediates the anti-inflammatory effect of mushroom Phellinus linteus in LPS-stimulated RAW264.7 macrophages Byung-Chul Kim a,∗ , Joung-Woo Choi a , Hye-Young Hong a , Sin-Ae Lee a , Suntaek Hong c , Eun-Hee Park b , Seong-Jin Kim c , Chang-Jin Lim a a

Division of Life Sciences, Kangwon National University, Chuncheon 200-701, Korea b College of Pharmacy, Sookmyung Women’s University, Seoul 140-742, Korea c Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD 20892, USA Received 5 December 2005; accepted 17 January 2006 Available online 20 February 2006

Abstract This work aimed to elucidate the anti-inflammatory mechanism of the n-BuOH subfraction (PL) prepared from fruiting bodies of Phellinus linteus. PL induced heme oxygenase-1 (HO-1) of the RAW264.7 macrophages in concentration- and time-dependent manner. It suppressed induction of inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO) through down-regulation of iNOS promoter activity in lipopolysaccharide (LPS)-stimulated macrophages. Zn(II) protoporphyrin IX (ZnPP), a specific inhibitor of HO-1, partly blocked suppression by PL on iNOS promoter activity and NO production, which were elevated in LPS-stimulated macrophages. LPS was able to enhance NO production via reactive oxygen species (ROS) generation, c-Jun NH2 -terminal kinase (JNK) and c-Jun induction. ZnPP prevented PL from down-regulating ROS generation and JNK activation in LPS-stimulated macrophages. Taken together, PL shows its anti-inflammatory activity via mediation of HO-1 in an in vitro inflammation model. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Phellinus linteus; Anti-inflammatory; Heme oxygenase-1; Reactive oxygen species; c-Jun NH2 -terminal kinase; Nitric oxide

1. Introduction Phellinus linteus, an orange color mushroom in the family of Hymenochaetaceae, has been used as a traditional medicine in oriental countries. In folk medicine, it is believed to possess curing effects against gastroenteric disorders, inflammation, tumors, and lymphatic diseases (Kim et al., 2004b). Some of its therapeutic effects have been pharmacologically evaluated in more detail. Phellinus linteus, as a natural antitumor product, prevents the inhibition of gap junctional intercellular communication through the inactivation of ERK1/2 and p38 MAP kinase (Cho et al., 2002). It inhibits metastasis of melanoma cells in mice via regulation of urokinase type plasminogen activator associated with tumor cell induced platelet aggregation (Lee et al., 2005a,b). Protein-bound polysaccharide, isolated ∗ Corresponding author at: Division of Life Sciences, College of Natural Sciences, Kangwon National University, 192-1 Huoja-2-Dong, Chuncheon 200701, Korea. Tel.: +82 33 250 8517; fax: +82 33 242 0459. E-mail address: [email protected] (B.-C. Kim).

0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.01.009

from Phellinus linteus, has been shown to have a direct antitumor effect through apoptosis and cell cycle blockade in human colon cancer cells (Li et al., 2004). Similarly, mycelial extract of Phellinus linteus induces apoptosis of human neuroblastoma cells via up-regulation of Bax (Choi et al., 2004a,b). Acidic polysaccharide isolated from Phellinus linteus enhances nitric oxide production, cell-mediated immunity, and anti-tumoral activity of peritoneal macrophages, which subsequently supports the antitumor action of Phellinus linteus (Kim et al., 2003, 2004a). Its antitumor activity has also assessed by antioxidant and anti-angiogenic activities of the ethanolic extract prepared from fruiting bodies of Phellinus linteus, which also contains strong anti-inflammatory and anti-nociceptive activities (Song et al., 2003; Kim et al., 2004b). Among the subfractions of the ethanolic extract, the n-butanol (n-BuOH) subfraction is most effective in anti-inflammation and anti-angiogenesis (Kim et al., 2004b). Heme oxygenases (HOs) are responsible for catalyzing the breakdown of heme into equimolar concentrations of carbon monoxide, biliverdin, and iron using molecular oxygen and

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reducing equivalents from NADPH:cytochrome P450 reductase. Heme oxygenase-1 (HO-1), localized in the non-neural tissues, is inducible in response to oxidative stress, nitrosative stress, thiol-reactive substances, and cytokines (Ryter and Choi, 2005). Heme oxygenase-2 (HO-2) and heme oxygenase-3 (HO-3), predominantly found in neural tissues, are constitutively expressed and do not respond to transcriptional activation by xenobiotics or physical stress (Ryter and Tyrell, 2000). Induction of HO-1 is modulated by various natural and synthetic compounds in tissue culture models. Hemin-induced HO-1 expression is attenuated by flavonoids, such as apigenin known to inhibit some inducible genes (Abate et al., 2005). However, quercetin, one of other flavonoids, induces HO-1 expression leading to the prevention of oxidative stressinduced apoptosis in RAW264.7 macrophages (Chow et al., 2005). Taurine, the most abundant free amino acid in mammalian tissues, induces expression of HO-1 in both non-activated and lipopolysaccharide (LPS)-activated J774.2 macrophages (Olszanecki and Marcinkiewicz, 2004). Nicotine also enhances up-regulation of HO-1 in gingival tissues, which is dependent on the intracellular glutathione (GSH) concentration (Chang et al., 2005). Carbon monoxide, a key product of HO, is known to suppress inflammation via a mitogen-activated protein kinase (MAPK) pathway (Otterbein, 2002). The antiproliferative effect of HO-1 is also mediated primarily via the release of carbon monoxide, which inhibits vascular smooth muscle cell growth via multiple pathways (Durante, 2003). Induction of HO-1 is involved in the inhibitory mechanism of some anti-inflammatory flavonoids on LPS-induced inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in mouse macrophages (Lin et al., 2005). Opposite regulation of iNOS and HO-1 is similarly observed in response to cytokine exposure and oxidative stress, and carbon monoxide inhibits iNOS mRNA induction, in cytokine-stimulated intestinal epithelial cells (Dijkstra et al., 2004). Resveratrol, a phytoalexin found in the skin and seeds of grapes, which possesses anti-inflammatory, anticarcinogenic and antioxidant activities, augments cellular antioxidant defense capacity through upregulation of HO-1 expression via activation of NF-E2-related factor 2 (Nrf2), thereby protecting PC12 cells from oxidative stress (Chen et al., 2005). Bilirubin, a potent antioxidant, is produced from biliverdin, the other main HO product, by biliverdin reductase. Recently, it has been shown that biliverdin reductase, a serine/threonine kinase activated by free radicals, advances the role of HO-1 in cytoprotection, but also affords cytoprotection independent of heme degradation (Miralem et al., 2005). Serum levels of NO and tumor necrosis factor ␣ and hepatic iNOS expression are significantly lower in bilirubin-treated rodents versus control animals (Wang et al., 2004). Consistent with the in vivo results, bilirubin markedly inhibits iNOS expression and suppresses NO production in LPSstimulated RAW264.7 macrophage cells (Wang et al., 2004). Here in this work, it is demonstrated that the n-BuOH subfraction, which is prepared from fruiting bodies of medicinal mushroom Phellinus linteus, shows an anti-inflammatory activity through the induction of heme oxygenae-1 in an in vitro model.

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2. Materials and methods 2.1. Cell culture and DNA constructs RAW264.7, a mouse macrophage cell line, obtained from American Type Culture Collection (Manassas, VA), was cultured in Dulbecco’s modified Eagle’s medium supplemented with 2 mM l-glutamine, 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 ␮g/ml streptomycin. The mammalian cells were maintained at 37 ◦ C in a humidified air/CO2 (19:1) atmosphere. A murine iNOS promoterluciferase construct was kindly provided by Dr. YoungMyeong Kim (Kangwon National University, Chuncheon, Korea). GAL4-transactivating system (PathDetect) was purchased from Stratagene (La Jolla, CA). The PathDetect system consists of the reporter vector pFR-Luc (5X GAL4 binding element) and the GAL4 fusion vector pFA2-c-Jun (1-223). 2.2. Preparation of the n-BuOH subfraction (PL) of Phellinus linteus The n-BuOH subfraction was prepared from fruiting bodies of Phellinus linteus as previously described (Kim et al., 2004b). Fruiting bodies of Phellinus linteus, ground under liquid nitrogen, were extracted with 70% ethanol at room temperature to generate the ethanolic extract (yield, 6.3%). The ethanolic extract, suspended in water, was successively extracted with equal volumes of n-hexane and ethyl acetate. The residual aqueous fraction was then extracted with equal volume of n-BuOH to give the n-BuOH subfraction (PL; yield, 41.5%). 2.3. Preparation of cytosolic extracts Cytosolic extracts from the mouse cell line were prepared as described previously (Lee et al., 2002). Briefly, grown cells were disrupted in animal cell lysis buffer (50 mM HEPES, 10% sucrose, 0.1% Triton X-100, and 1 mM PMSF, pH 7.5) and centrifuged at 12,000 × g for 15 min. The protein concentration was determined according to the method of Bradford, using bovine serum albumin as a standard (Bradford, 1976). 2.4. Transfection and luciferase assay The mammalian cells were transfected using FuGENE 6 (Roche, Mannheim, Germany). To control for variations in transfection efficiency, all clones were co-transfected with 0.2 ␮g of CMV-␤-GAL, a eukaryotic expression vector in which Escherichia coli ␤-galactosidase (Lac Z) structural gene is under the transcriptional control of the CMV promoter. Cytosolic extracts were prepared 24 h after transfection by using a luciferase cell lysis buffer (Promega, Madison, WI). Luciferase reporter activity was assessed on a luminometer with a luciferase assay system (Promega, Madison, WI) as suggested by the manufacturer.

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2.5. Measurement of intracellular ROS For measurement of intracellular ROS, the redox-sensitive fluorescent probe 2 ,7 -dihydrodichlorofluorescein diacetate (DCFH-DA) was used as previously described (Royall and Ischiropoulos, 1993). Cells were incubated with 5 ␮M DCFHDA for 30 min at 37 ◦ C. The harvested cells were immediately analyzed by a flow cytometry. DCFH-DA is nonfluorescent and can readily diffuse across cell membranes and hydrolyzed by intracellular esterases to non-fluorescent 2 ,7 dihydrodichlorofluorescein (DCFH), which is converted to the highly fluorescent 2 ,7 -dichlorofluorescein (DCF) after reacting with intracellular ROS. 2.6. Nitrite assay The nitrite level is generally accepted as indicative of NO production. Accumulated nitrite (NO2 − ) in cell culture supernatants was determined using a colorimetric assay based on the Griess reaction (Sherman et al., 1993). The supernatants (100 ␮l) were reacted with a 100 ␮l Griess reagent (6 mg/ml) at room temperature for 10 min, and then NO2 − concentration was determined by measuring absorbance at 540 nm. A standard curve was constructed using known concentrations of sodium nitrite. 2.7. Immunoblot analysis Cytosolic extracts were obtained in 1% Triton X-100 lysis buffer (50 mM Tris–Cl, pH 8.0, 150 mM sodium chloride, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM ␤-glycerophosphate, 1 ␮g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Western blotting was performed using anti-HO-1 (H105; Santa Cruz Biotechnology, Santa Cruz, CA), anti-iNOS (54; Transduction Laboratories, Lexington, KY), anti-phospho-JNK (G9; Cell Signaling Technology, Beverly, MA), and anti-␤-actin (AC-15; Sigma, St. Louis, MO) antibodies. Protein samples were heated at 95 ◦ C for 5 min and analyzed by SDS-PAGE. Immunoblot signals were developed by Super Signal Ultra chemiluminescence detection reagents (Pierce Biotechnology, Rockford, IL). 2.8. Statistical analysis All data presented are expressed as mean ± S.D., and representative of three or more independent experiments. Statistical analysis was assessed by Student’s t-test for paired data. Results were considered significant at p < 0.05. 3. Results 3.1. Induction of HO-1 expression Treatment with PL at 0.1, 0.25 and 0.5 mg/ml for 24 h enhanced HO-1 expression in RAW264.7 macrophages in a concentration-dependent manner (Fig. 1A). When the mammalian cells were incubated with 0.5 mg/ml PL for varying time periods, the HO-1 expression was time-dependently increased in

Fig. 1. Induction by PL of HO-1 expression in RAW264.7 cells. (A) The mammalian cells were treated with 0.1, 0.25 and 0.5 mg/ml PL for 24 h. (B) The cells were treated with 0.5 mg/ml PL for 6, 12 and 24 h. (C) The cells were treated with 0.5 mg/ml PL for 24 h in the absence or presence of 100 ng/ml cycloheximide (CHX), a protein synthesis inhibitor.

RAW264.7 cells (Fig. 1B). To confirm whether the induction of HO-1 expression by PL arises from increased protein synthesis or not, cycloheximide (CHX), a protein synthesis inhibitor, was added into the cells together with PL. As shown in Fig. 1C, CHX markedly inhibited enhanced expression of HO-1 by PL, indicating that PL increased the production of HO-1 on an expression level. Taken together, PL induces expression of the HO-1 protein in non-stimulated RAW264.7 macrophages. 3.2. Mediation of HO-1 in the suppression of LPS-stimulated NO production and iNOS expression in macrophages Lipopolysaccharide (LPS) is a bacterial endotoxin, which promotes the secretion of pro-inflammtory cytokines and related molecules, including inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-␣ in many cell types. Stimulation of the transfected RAW264.7 cells with 1 ␮g/ml LPS for 24 h caused a massive increase in iNOS promoter activity (Fig. 2A). Subsequently, expression of iNOS and nitrite content were significantly elevated in the macrophages after the treatment with LPS (Fig. 3A). Pretreatment with PL at 0.01, 0.1, 0.5 and 1 mg/ml concentration-dependently diminished LPS-stimulated iNOS promoter activity in the macrophages (Fig. 2A), and iNOS expression and NO production were significantly decreased in LPS-stimulated RAW264.7 cells after the pretreatment with PL (Fig. 3A). These results indicate that PL is able to suppress LPS-stimulated iNOS promoter activity, iNOS expression and NO production in a sequential manner. Pretreatment with Zn(II) protoporphyrin IX (ZnPP), a HO-1 inhibitor, at 0.1, 1 and 5 ␮M for 1 h gave rise to suppression in the inhibition by PL of LPSstimulated iNOS promoter activity in the macrophages (Fig. 2B). This was accompanied by a significant suppression in the inhi-

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Fig. 2. HO-1 mediates the inhibition of lipopolysaccharide (LPS)-stimulated iNOS promoter activity in RAW264.7 cells, transiently transfected with murine iNOS promoter, following prolonged treatment with PL. (A) The transfected cells were pretreated with varying amounts of PL for 1 h, and stimulated with 1 ␮g/ml LPS for 24 h. ** p < 0.01; * p < 0.05 vs. LPS alone. (B) The transfected cells were pretreated with 1 mg/ml PL for 1 h, in the absence or presence of varying amounts of Zn(II) protoporphyrin IX (ZnPP), and stimulated with 1 ␮g/ml LPS for 24 h. * p < 0.01.

bition of LPS-stimulated iNOS expression and NO production in the same macrophages after the treatment with 5 ␮M ZnPP (Fig. 3B). Collectively, PL suppresses NO production in LPSstimulated RAW264.7 macrophages through the mediation of HO-1. 3.3. Involvement of ROS and JNK in LPS-stimulated NO production in macrophages Since ROS is known to contribute to inflammatory reactions, changes in ROS level were examined in LPS-stimulated RAW264.7 cells. As shown in Fig. 4A, ROS level increased in the macrophages up to 30 min after the treatment with 1 ␮g/ml LPS, and gradually decreased afterwards. The ROS level nearly dropped to the pretreated level of ROS 12 h after the treatment with LPS (Fig. 4A). This result indicates that LPS transiently enhances ROS level in the macrophages. In order to figure out the relationship between ROS generation and NO production in the LPS-stimulated macrophages, N-acetyl-cysteine (NAC) was added into the LPS-stimulated RAW264.7 cells. NAC suppressed LPS-stimulated NO production in the macrophages in a concentration-dependent manner (Fig. 4B). In a similar

experiment, SP600125, a JNK inhibitor, significantly inhibited LPS-stimulated NO production and iNOS expression in the macrophages (Fig. 4C). These results suggest that ROS and JNK activity are required for LPS-stimulated NO production in the macrophages. The Gal4-c-Jun fusion protein consists of the transactivational domain (amino acid 1-223) of c-Jun fused to the binding domain of the yeast transcription factor Gal4. The RAW264.7 cells were transfected with the expression vector for Gal4-c-Jun. In the Gal4-c-Jun-expressing RAW264.7 cells, luciferase activity from the reporter plasmid significantly increased after the treatment with 1 ␮g/ml LPS for 24 h (Fig. 4D). However, this elevation was partly abolished by NAC (Fig. 4D). These results indicate that LPS increases NO production in a ROS-JNK-cJun-linked cascade in RAW264.7 macrophages. 3.4. Suppression by PL of LPS-stimulated ROS generation and JNK activation through HO-1 The role of HO-1 in LPS-stimulated ROS generation and JNK activation was examined in RAW264.7 macrophages. As shown in Fig. 5A, treatment with 1 ␮g/ml LPS for 30 min enhanced

Fig. 3. HO-1 mediates the suppression of LPS-stimulated iNOS expression and NO production in RAW264.7 cells following prolonged treatment with PL. (A) The mammalian cells were pretreated with varying amounts of PL for 1 h, and stimulated with 1 ␮g/ml LPS for 24 h. ** p < 0.01; * p < 0.05 vs. LPS alone. (B) The cells were pretreated with 0.5 mg/ml PL for 1 h, in the absence or presence of 5 ␮M Zn(II) protoporphyrin IX (ZnPP), and stimulated with 1 ␮g/ml LPS for 24 h. * p < 0.01 vs. LPS alone.

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Fig. 4. LPS induces iNOS expression and NO production through a ROS-JNK-linked cascade in RAW264.7 cells. (A) Variations in ROS level of LPS-stimulated RAW264.7 cells. The RAW264.7 cells were treated with 1 ␮g/ml LPS for varying time periods. (B) Suppression of NO production in LPS-stimulated RAW264.7 cells by N-acetyl-cysteine (NAC). The mammalian cells were pretreated with varying amounts of NAC for 30 min, and stimulated with 1 ␮g/ml LPS for 24 h. *** p < 0.001; ** p < 0.01; * p < 0.05 vs. LPS alone. (C) Suppression of NO production in LPS-stimulated RAW264.7 cells by SP600125, a JNK inhibitor. The mammalian cells were pretreated with varying amounts of SP600125 for 30 min, and stimulated with 1 ␮g/ml LPS for 24 h. ** p < 0.01; * p < 0.05 vs. LPS alone. (D) Suppression of c-Jun induction in LPS-stimulated RAW264.7 cells by NAC. The RAW264.7 cells, co-transfected with 1 ␮g of pFR-Luc, 0.2 ␮g of CMV-␤-GAL and 0.25 ␮g of GAL4-c-Jun, were pretreated with 5 mM NAC for 30 min, and stimulated with 1 ␮g/ml LPS for 24 h. * p < 0.01 vs. LPS alone.

Fig. 5. HO-1 mediates the suppression of LPS-stimulated ROS production and JNK activation in RAW264.7 cells following prolonged treatment with PL. (A) The mammalian cells were pretreated with 0.5 mg/ml PL in the absence or presence of 5 ␮M Zn(II) protoporphyrin IX (ZnPP), and stimulated with 1 ␮g/ml LPS for 30 min. (B) The cells were pretreated with varying amounts of PL for 6 h, in the absence or presence of 5 ␮M Zn(II) protoporphyrin IX (ZnPP), and stimulated with 1 ␮g/ml LPS for 30 min.

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ROS level to about 1.43-fold compared to that in the untreated cells. Pretreatment with 0.5 mg/ml PL nearly prevented the elevation of ROS level in LPS-stimulated RAW264.7 macrophages (Fig. 5A). However, 5 ␮M ZnPP significantly restored the elevation of ROS level, in the presence of PL, in LPS-stimulated macrophages (Fig. 5A). These results indicate that suppression of ROS generation by PL in LPS-stimulated RAW264.7 cells is mediated by HO-1. The c-Jun N-terminal kinases (JNKs), belonging to the signaling network of mitogen-activated protein kinases (MAPKs), are involved in various physiological and pathological events such as mitosis, apoptosis and inflammation. JNK phosphorylates and activates c-Jun. JNK was significantly activated in LPS-stimulated macrophages, but the activation was weakened by PL (Fig. 5B). In the presence of ZnPP, the suppression by PL of JNK activation was notably prevented in LPS-stimulated macrophages (Fig. 5B). These findings suggest that HO-1 mediates the suppressive effect of PL in the LPS-stimulated JNK activation in RAW264.7 macrophages. 4. Discussion In the previous work, the n-BuOH subfraction (PL), prepared from fruiting bodies of medicinal mushroom Phellinus linteus, was shown to contain strong anti-inflammatory and related activities (Kim et al., 2004b). However, its anti-inflammatory mechanism remained to be explained. Induction of inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO), a pro-inflammatory mediator, is an important mechanism in the pathogenesis of inflammation. PL showed a significant inhibition in iNOS promoter activity, iNOS expression and NO production in LPS-stimulated RAW264.7 macrophages (Figs. 2A and 3A). These findings indicate that PL shows its antiinflammatory activity through ultimately diminishing the NO level. Heme oxygenase (HO)-1 is believed to beneficially play a cytoprotective role in a variety of pathological models such as inflammation. The anti-inflammatory properties of HO-1 are related with inhibition of adhesion molecule expression and reduction of oxidative stress, while exogenous carbon monoxide decreases the production of inflammatory mediators such as cytokines and NO (Sawle et al., 2005). Accordingly, COreleasing molecules are capable of modulating physiological functions via the liberation of CO (Sawle et al., 2005). Then, CO carriers can be used as an effective strategy to modulate inflammation. CO produced by HO-1 inhibits cytochrome P450 2E1 activity, subsequently diminishing oxidative injuries in HepG2 cells (Gong et al., 2004). PL induces expression of HO1 in non-stimulated RAW264.7 macrophages in concentrationand time-dependent manner (Fig. 1A and B). Since cycloheximide (CHX) abolished the induction of HO-1 by PL (Fig. 1C), the induction was considered to occur on a gene level. In order to find out the relationship between PL’s ability to induce HO-1 and its inhibition in NO production, a specific inhibitor of HO-1, ZnPP, was used. As shown in Fig. 2B, ZnPP significantly diminished PL’s ability to suppress iNOS

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promoter activity in LPS-stimulated RAW264.7 macrophages. In the similar experiments, ZnPP also inhibited PL’s ability to suppress expression of iNOS and production of NO in LPSactivated RAW264.7 macrophages (Fig. 3B). These results propose that PL shows an inhibitory activity in NO production in the LPS-stimulated macrophages through the mediation of HO-1. Several natural products have been identified to play their inhibitory roles in non-stimulated and stimulated cells. The aglycones of the flavonoids such as hesperidin and naringin inhibit LPS-stimulated NO production in accordance with the induction of HO-1 in mouse macrophage cell lines (Lin et al., 2005). Similarly, induction of HO-1 mediates anti-inflammatory properties of taurine in mammalian tissues (Olszanecki and Marcinkiewicz, 2004). Like hesperidin, naringin and taurine, PL is thought to play an anti-inflammatory role through the mediation of HO-1. However, the active principle(s) in PL remains unknown so far. LPS is known to enhance NO production in macrophage cells. When RAW264.7 macrophages were treated with LPS, the ROS level in the macrophages increased up to 30 min, and then dropped to the normal level (Fig. 4A). Our results confirm that the generation of ROS mediates production of NO and expression of c-Jun in LPS-stimulated macrophages (Fig. 4B and D). Separately, it was shown that SP600125 prevented expression of iNOS and production of NO in LPS-stimulated macrophages (Fig. 4C). The findings confirm that LPS enhances NO level via ROS generation, JNK activation, induction of c-Jun and iNOS in a sequential order. PL appeared to suppress the generation of ROS in LPS-stimulated macrophages through the mediation of HO-1, estimated from the fact that ZnPP markedly prevented PL’s suppression on the generation of ROS in LPS-stimulated macrophges (Fig. 5A). ZnPP inhibited PL’s suppression of JNK activation in LPS-stimulated macrophages (Fig. 5B), suggesting the participation of HO-1 in JNK activation. This effect might be based on the mediation of HO-1 in LPS-stimulated ROS generation. In this work, PL has been shown to diminish LPS-stimulated NO production via enhanced expression of HO-1. However, NO exerts antiproliferative and antiapoptotic effects on human T cells, and its suppressions of T cell proliferation and apoptosis are associated with an increased expression of HO-1 by NO (Pae et al., 2004). In ovariectomized rats, ZnPP significantly increases the plasma levels of NO metabolites, and the NO/iNOS system contributes to the induction of HO-1, which may subsequently suppress iNOS activity to modulate vasculoprotective effects after menopause (Lee et al., 2005a,b). However, LPS induces iNOS but not HO-1 in intestinal epithelial cells, suggesting that iNOS and HO-1 represent mutually exclusive survival mechanisms (Dijkstra et al., 2004). Interrelation between expression of HO-1 and production of NO might be dependent on cell types. Overexpression of HO-1 in Jurkat T cells gives rise to more resistance to Fas-mediated apoptosis than control cells (Choi et al., 2004a,b). Suppression of HO-1 by small interfering RNA (siRNA) enhances apoptosis in lung injury, via increased Fas expression and caspase 3 activity (Zhang et al., 2004). The HO-1 pathway of apoptosis resistance is associated with an increase in the levels of p21, and involves a p38 MAPK and ERK-mediated

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mechanism in papillary thyroid carcinoma cells (Chen et al., 2004). However, HO-1, which is induced by PL, was shown to play an anti-inflammatory role through the activation of JNK, in LPS-stimulated macrophages. In conclusion, the n-BuOH subfraction of Phellinus linteus shows its in vitro anti-inflammatory activity via induction of heme oxygenase-1 in RAW264.7 macrophages. The induced heme oxygenase-1 subsequently diminishes ROS generation, JNK activation, c-Jun induction and NO production in an in vitro inflammation model. Acknowledgements This study is supported by Kangwon BIO-NURI. Specially thanks to Mr. Sang-Ro Han, Chowon Sangwhang Microbiosis, Chuncheon, Korea for kindly donating the mushroom. References Abate, A., Yang, G., Wong, R.J., Schroder, H., Stevenson, D.K., Dennery, P.A., 2005. Apigenin decreases hemin-mediated heme oxygenase-1 induction. Free Radical Biology and Medicine 39, 711–718. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254. Chang, Y.C., Lai, C.C., Lin, L.F., Ni, W.F., Tsai, C.H., 2005. The upregulation of heme oxygenase-1 expression in human gingival fibroblast stimulated with nicotine. Journal of Periodontal Research 40, 252– 257. Chen, C.Y., Jang, J.H., Li, M.H., Surh, Y.J., 2005. Resveratrol upregulates heme oxygenase-1 expression via activation of NF-E2-related factor 2 in PC12 cells. Biochemical and Biophysical Research Communications 331, 993–1000. Chen, G.G., Liu, Z.M., Vlantis, A.C., Tse, G.M., Leung, B.C., van Hasselt, C.A., 2004. Heme oxygenase-1 protects against apoptosis induced by tumor necrosis factor-alpha and cycloheximide in papillary thyroid carcinoma cells. Journal of Cellular Biochemistry 92, 1246– 1256. Cho, J.-H., Cho, S.-D., Hu, H., Kim, S.-H., Lee, S.K., Lee, Y.-S., Kang, K.-S., 2002. The roles of ERK1/2 and p38 MAP kinase in the preventive mechanisms of mushroom Phellinus linteus against the inhibition of gap junction intercellular communication by hydrogen peroxide. Carcinogenesis 23, 1163–1169. Choi, Y.H., Huh, M.K., Ryu, C.H., Choi, B.T., Jeong, Y.K., 2004a. Induction of apoptotic cell death by mycelium extracts of Phellinus linteus in human neuroblastoma cells. International Journal of Molecular Medicine 14, 227–232. Choi, B.M., Pae, H.O., Jeong, Y.R., Oh, G.S., Jun, C.D., Kim, B.R., Kim, Y.M., Chung, H.T., 2004b. Overexpression of heme oxygenase (HO)-1 renders Jurkat T cells resistant to fas-mediated apoptosis: involvement of iron released by HO-1. Free Radical Biology and Medicine 36, 858– 871. Chow, J.-M., Shen, S.-C., Huan, S.K., Lin, H.-Y., Chen, Y.-C., 2005. Quercetin, but not rutin and quercitrin, prevention of H2 O2 -induced apoptosis via anti-oxidant activity and heme oxygenase-1 gene expression. Biochemical Pharmacology 69, 1839–1851. Dijkstra, G., Blokzijl, H., Bok, L., Homan, M., van Goor, H., Faber, K.N., Jansen, P.L.M., Moshage, H., 2004. Opposite effect of oxidative stress on inducible nitric oxide synthase and haem oxygenase-1 expression in intestinal inflammation: anti-inflammatory effect of carbon monoxide. Journal of Pathology 204, 296–303. Durante, W., 2003. Heme oxygenase-1 in growth control and its clinical application to vascular disease. Journal of Cellular Physiology 195, 373– 382.

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