In vivo and in vitro anti-inflammatory and anti-nociceptive effects of the methanol extract of Inonotus obliquus

Journal of Ethnopharmacology 101 (2005) 120–128

In vivo and in vitro anti-inflammatory and anti-nociceptive effects of the methanol extract of Inonotus obliquus Young-Mi Park a , Jong-Heon Won a , Yang-Hee Kim a , Jong-Won Choi b , Hee-Juhn Park c , Kyung-Tae Lee a,∗ a

Department of Biochemistry, College of Pharmacy, Kyung-Hee University, Dongdaemun-Ku, Hoegi-Dong, Seoul 130-701, South Korea b College of Pharmacy, Kyung-Sung University, Pusan 608-736, South Korea c Division of Applied Plant Sciences, Sang-Ji University, Wonju 220-702, South Korea Received 28 February 2005; received in revised form 24 March 2005; accepted 7 April 2005 Available online 17 May 2005

Abstract The mushroom Inonotus obliquus (Fr.) Pil´at (Hymenochaetaceae), has been traditionally used for the treatment of gastrointestinal cancer, cardiovascular disease and diabetes in Russia, Poland and most of Baltic countries. This study was designed to investigate the anti-inflammatory and anti-nociceptive effects of the methanol extract from Inonotus obliquus (MEIO) in vivo and in vitro. MEIO (100 or 200 mg/(kg day), p.o.) reduced acute paw edema induced by carrageenin in rats, and showed analgesic activity, as determined by an acetic acid-induced abdominal constriction test and a hot plate test in mice. To reveal the mechanism of the anti-inflammatory effect of MEIO, we examined its effect on lipopolysaccharide (LPS)-induced responses in a murine macrophage cell line RAW 264.7. MEIO was found to significantly inhibit the productions of nitric oxide (NO), prostaglandin E2 (PGE2 ) and tumor necrosis factor-␣ (TNF-␣) in LPS-stimulated RAW 264.7 macrophages. Consistent with these observations, MEIO potently inhibited the protein and mRNA expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, MEIO inhibited the LPS-induced DNA binding activity of nuclear factor-␬B (NF-␬B), and this was associated with the prevention of inhibitor ␬B degradation and a reduction in nuclear p65 protein levels. Taken together, our data indicate that the anti-inflammatory and anti-nociceptive properties of MEIO may be due to the inhibition of iNOS and COX-2 expression via the down-regulation of NF-␬B binding activity. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Anti-inflammatory; Anti-nociceptive; Inonotus obliquus; Hymenochaetaceae; Nitric oxide; Prostaglandin E2 ; Nuclear factor-␬B

1. Introduction Mushrooms are a nutritionally functional food and a source of physiologically beneficial medicines. In Russian traditional medicine, an extract from the mushroom Inonotus obliquus (Fr.) Pil´at (Hymenochaetaceae) is used as an Abbreviations: ECL, enhanced chemiluminescence; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MTT, 3-(4,5dimethylthiazoyl-2-yl)-2,5-diphenyl tetrazolium bromide; NO, nitric oxide; TNF, tumor necrosis factor ∗ Corresponding author. Tel.: +82 2 9610860; fax: +82 2 9663885. E-mail address: [email protected] (K.-T. Lee). 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.04.003

anti-tumor medicine and diuretic (Huang, 2002). Moreover, it has been reported that Inonotus obliquus has therapeutic effects, such as anti-inflammatory, immuno-modulatory and hepatoprotective effects (Solomon and Alexander, 1999). Triterpenoids and steroids have been previously isolated from Inonotus obliquus (He et al., 2001), but their activities have not previously been reported. Nitric oxide (NO) and prostaglandin E2 (PGE2 ) are pleiotypic inflammatory mediators, which are produced by the inducible isoform of nitric oxide synthase (iNOS) and by cyclooxygenase (COX-2), respectively. Moreover, NO can also be formed by the conversion of l-arginine to l-citrulline by nitric oxide synthase (NOS) (Garry and

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Csaba, 1996). Under pathological conditions, macrophages can greatly increase NO production. Thus, the inhibition of iNOS activity and/or of its expression and the inhibition of NO production are important anti-inflammatory goals. Cyclooxygenase (COX) represents a rate-limiting step in the synthesis of prostaglandins (PGs), and two COX isoforms (COX-1 and COX-2) have been described. COX-1 is constitutively expressed in mouse peritoneal macrophages and in RAW 264.7 cells, whereas COX-2 is overtly induced upon stimulation by lipopolysaccharide (LPS) (Mitchell et al., 1993; Guastadisegni et al., 2002). The use of isoform-selective COX inhibitors has revealed that the many anti-inflammatory benefits of non-steroidal anti-inflammatory drugs (NSAIDs) are derived from COX-2 inhibition, and that conversely, many undesirable side effects result from COX-1 inhibition (Jane et al., 1995; DeWitt, 1999). High levels of PGE2 , derived from COX-2 induced by many pro-inflammatory mediators including tumor necrosis factor-␣ (TNF-␣), interleukin-1␤ (IL-1␤) and lipopolysaccharide, have also been implicated in the pathogenesis of sepsis and inflammation (Xie et al., 1991; Habib et al., 1993). Nuclear factor kappa B (NF-␬B) is an important transcription factor and is associated with the expressions of several proinflammatory genes, such as, cytokines and inducible enzymes (Baeuerle and Henkel, 1994). NF-␬B is a key regulatory protein in the expression of a wide variety of genes involved in both innate and adaptive immunity (Karin and Ben-Neriah, 2000). In unstimulated cells, NF-␬B is generally found in the cytoplasm. This subcellular localization results from the efficient masking of the nuclear localization signal of NF-␬B by I␬B␤ and I␬B␧ (Li et al., 1997; MacMicking et al., 1997). Moreover, complexes binding I␬B␣ continuously shuttle between the cytoplasm and the nucleus. NF-␬B dependent gene transcription requires the phosphorylation of I␬B␣ by I␬B kinase (IKK), which releases this inhibitory component from the NF-␬B complex. This is followed by the migration of NF-␬B to the nucleus and by the binding to DNA sites of NF-␬B-responsive genes (Ghosh et al., 1998). In this study, we evaluated the in vivo anti-inflammatory and anti-nociceptive activities of orally administered Inonotus obliquus methanol extract and investigated the cell based anti-inflammatory activity of MEIO to clarify the mechanism involved.

2. Materials and methods 2.1. Fungus and extracts preparation The sporophores of Inonotus obliquus (Fr.) Pil´at (Hymenochaetaceae) were purchased from the Chun-Il Oriental Herbal Store in Wonju, Korea, and the fungus was identified by S.Y. Yun (Division of Applied Plant Sciences, Sangji University, Korea). A voucher specimen (#NATCHEM-28) was deposited in the Laboratory of Natural Products Analysis, Division of Applied Plant Sciences, Sangji University, Ko-

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rea. The crushed dry material (1.00 kg) was extracted under reflux with hot MeOH three times and then filtered. This extract was evaporated on a rotatory evaporator under reduced pressure and freeze-dried to give a powder (298 g). 2.2. Chemicals Dulbecco’s modified Eagle’s minimum essential medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). COX-2, iNOS, I␬B-␣, p65 monoclonal antibodies and the peroxidase-conjugated secondary antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The enzyme immunoassay (EIA) kits for prostaglandin E2 and tumor necrosis factor-␣ were obtained from R&D Systems (Minneapolis, MN, USA). NS-398, a COX-2 enzyme inhibitor, was from Calbiochem (CA, USA). RNA extraction kit was purchased from Intron Biotechnology. iNOS, COX-2, TNF-␣ and ␤-actin oligonucleotide primers were purchased from Bioneer (Seoul, Korea). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), aprotinin, leupeptin, phenylmethylsulfonylfluoride (PMSF), dithiothreitol, l-N6 -(1-iminoethyl)lysine (l-NIL), Escherichia coli LPS and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2.3. Animals ICR male mice weighing 20–25 g and Sprague–Dawley male rats weighing 100–120 g were purchased from the Daehan Biolink (Eumsung-Gun, Chungbuk) and maintained under constant conditions (temperature: 20 ± 2 ◦ C, humidity: 40–60%, 12-h light/12-h dark cycle) for 2 weeks or longer. Twenty-four hours before the experiment, only water was offered to the animals. Considering the variation of enzyme activity during 1 day, the animals were sacrificed at a fixed time (10:00–12:00 a.m.). These experiments were approved by the University of Kyungsung Animal Care and Use Committee. All procedures were conducted in accordance with the “Guide for the Care and Use of Laboratory Animals” published by the Korea National Institute of Health. 2.4. Carrageenin-induced paw edema in rats Pedal inflammation in male Sprague–Dawley rats (100–120 g) was produced according to the method described by Winter et al. (1962). Oedema was induced by subcutaneous injection of 0.1 ml of 1% solution of carrageenin into the right hind paw volume of the rats after the test samples had been administered orally. The test samples were first dissolved in 10% Tween 80 and diluted with saline. The control group received the vehicle. A test solution (100 or 200 mg/kg) was administered orally for the 7 consecutive days prior to injecting carrageenin. Paw volumes were measured up to 5 h after the carrageenin injection at intervals of 60 min, and the volume of the edema was measured with a plethysmometer.

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Ibuprofen (100 mg/kg) was used as a reference drug (Choi et al., 2003). 2.5. Acetic acid-induced abdominal constriction test in mice The acetic acid-induced abdominal constriction test was performed as described by Whittle (1949). Vehicle, aspirin (100 mg/kg) and test solution (100 or 200 mg/kg) were orally administered 30 min before the experiment, and 0.1 ml/10 g of 0.7% acetic acid-saline was then injected i.p. 10 min after the injection. The number of writhings during the following 20 min period was counted. The anti-nociceptive effects of drugs were measured by calculating the mean reduction as compared to vehicle-administered controls. 2.6. Hot plate test in mice The hot plate test was used to measure the response latencies according to the method described previously by Eddy and Leimback (1953), with minor modifications. In these experiments, the hot plate (Ugo Basile, model-DS 37) was maintained at 56 ± 1 ◦ C. The reaction time was noted by observing either the licking of the hind paws or the jumping movements before and after drug administration. The cut-off time was 20 s and morphine sulphate (10 mg/kg) (Kuju Pharmaceutical Co), administered intraperitoneally, was used as a reference drug (Choi et al., 2003). 2.7. Cell culture and sample treatment The RAW 264.7 murine macrophage cell line was obtained from the Korean Cell Line Bank (Seoul, Korea). These cells were grown at 37 ◦ C in DMEM medium containing 10% heat-inactivated FBS, penicillin (100 units/ml) and streptomycin sulfate (100 ␮g/ml) in a humidified atmosphere of 5% CO2 . The cells were incubated with MEIO at various concentrations (45, 90 or 135 ␮g/ml) or 10 ␮M l-N6 -(1iminoethyl)lysine as a positive control of nitrite production and stimulated with 1 ␮g/ml LPS. 2.8. MTT assay for cell viability Cell viability was determined on the basis of mitochondrial-dependent reduction of MTT to formazan (Page et al., 1988). RAW 264.7 cells were cultured in 96-well plates (5 × 105 cells/ml) for 24 h. Cells were treated with LPS (1 ␮g/ml) in a volume of 200 ␮l in presence or absence of various concentrations (45, 90 or 135 ␮g/ml) of MEIO. After overnight incubation, cells were washed once before adding 50 ␮l of FBS-free medium containing MTT (5 mg/ml). After 4 h of incubation at 37 ◦ C, the medium was discarded and the formazan blue that formed in the cells was dissolved in DMSO (100 ␮l). The optical density was measured at 540 nm.

2.9. Nitrite, PGE2 and TNF-α assay The nitrite which accumulated in culture medium was measured as an indicator of NO production according to the Griess reaction, as previously described (Misko et al., 1993). Briefly, 100 ␮l of cell culture medium (without phenol red) was mixed with 100 ␮l of Griess reagent [equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) naphthylethylenediamine-HCl], incubated at room temperature for 10 min, and then the absorbance at 550 nm was measured in a microplate reader. Fresh culture medium was used as the blank in all experiments. The amount of nitrite in the samples was measured with NaNO2 serial dilution standard curve and the nitrite production was measured. PGE2 and TNF-␣ levels in macrophage culture medium were quantified using EIA kits according to the manufacture’s instructions (R&D Systems). 2.10. Western blot analysis Protein expression was assessed by Western blot analysis as previously described (Li et al., 2000). Cellular proteins were extracted from both the control and MEIO-treated RAW 264.7 cells. Cells were collected by centrifugation and washed once with phosphate-buffered saline (PBS). The washed cell pellets were resuspended in extraction lysis buffer (50 mM HEPES, pH 7.0, 250 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol (DTT), 5 mM Na fluoride, 0.5 mM Na orthovanadate) containing 5 ␮g/ml each of leupeptin and aprotinin and incubated for 30 min at 4 ◦ C. Cell debris was removed by microcentrifugation, followed by quick freezing of the supernatants. The protein concentration was determined using the Bio-Rad protein assay reagent according to the manufacturer’s instruction. Forty micrograms of cellular protein from treated and untreated cell extracts were electroblotted onto a nitrocellulose membrane following separation using 8–12% SDS-polyacrylamide gel electrophoresis (PAGE). The immunoblot was incubated overnight with Tween 20/Tris-buffered saline (TTBS) containing 5% (w/v) non-fat milk at 4 ◦ C, followed by incubation for 4 h with a 1:500 dilution of monoclonal anti-iNOS antibody, 1:1000 dilution of anti-COX-2 antibody, 1:1000 dilution of anti-I␬B-␣ antibody and 1:500 dilution of anti-p65 antibody (Santa Cruz Biotechnology Inc.). Blots were washed two times with in TTBS and incubated with a 1:1000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Santa Cruz Biotechnology Inc.) for 1 h at room temperature. Blots were again washed three times with TTBS and then developed by enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL, USA). 2.11. RNA preparation and polymerase chain reaction RT-PCR analysis was performed similarly as previously described (Shin et al., 2004). Total cellular RNA was isolated

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using Easy Blue® kits (Intron Biotechnology) according to the manufacturer’s instructions. From each sample, 1 ␮g of RNA was reverse-transcribed (RT) using MuLV reverse transcriptase, 1 mM dNTP and oligo (dT12–18 ) 0.5 ␮g/␮l. Then PCR analyses were performed on the aliquots of the cDNA preparations to detect iNOS, COX-2, TNF-␣ and ␤-actin (as an internal standard) gene expression using a thermal cycler (Perkin-Elmer Cetus, Foster City, CA, USA). The reactions were carried out in a volume of 25 ␮l containing (final concentration) 1 units of Taq DNA polymerase, 0.2 mM dNTP, ×10 reaction buffer and 100 pmol of 5 and 3 primers. After initial denaturation for 2 min at 95 ◦ C, 30 amplification cycles were performed for iNOS (1 min of 95 ◦ C denaturation, 1 min of 60 ◦ C annealing and 1.5 min 72 ◦ C extension), COX-2 (1 min of 94 ◦ C denaturation, 1 min of 60 ◦ C annealing and 1 min 72 ◦ C extension) and TNF-␣ (1 min of 95 ◦ C denaturation, 1 min of 55 ◦ C annealing and 1 min 72 ◦ C extension). The PCR primers used in this study are listed below and were purchased from Bioneer (Seoul, Korea): sense strand iNOS, 5 -AATGGCAACATCAGGTCGGCCATCACT-3 ; anti-sense strand iNOS, 5 -GCTGTGTGTCACAGAAGTCTCGAACTC-3 ; sense strand COX-2, 5 -GGAGAGACTATCAA-GATAGT-3 ; anti-sense strand COX-2, 5 -ATGGTCAGTAGACTTTTACA-3 ; sense strand TNF-␣, 5 -ATGAGCACAGAAAGCATGATC-3 ; antisense strand TNF-␣, 5 -TACAGGCTTGTCACTCGAATT-3 ; sense strand ␤-actin, 5 -TCATGAAGTGTGACGTTGACATCCGT-3 ; anti-sense strand ␤-actin, 5 -CCTAGAAGCATTTGCGGTGCACGATG-3 . After amplification, portions of the PCR reactions were electrophoresed on 2% agarose gel and visualized by ethidium bromide staining and UV irradiation. 2.12. Preparation of nuclear extraction and electrophoretic mobility shift assay (EMSA) RAW 264.7 macrophages were plated in 100 mm dishes (5 × 106 cells). The cells were treated with various MEIO concentrations (45, 90 or 135 ␮g/ml), stimulated with LPS for 1 h, washed once with PBS, scraped into 1 ml of cold PBS and pelleted by centrifugation. Nuclear extracts and EMSA assay were prepared as described previously with slightly modification (Kim et al., 2003). The cell pellet was resuspended in hypotonic buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2 , 10 mM KCl, 0.2 mM PMSF, 0.5 mM DTT, 10 ␮g/ml aprotinin) and incubated on ice for 15 min. Then the cells were lysed by the addition of 0.1% Nonidet P-40 and vigorous vortexing for 10 s. The nuclei were pelleted by centrifugation at 12,000 × g for 1 min at 4 ◦ C and resuspended in high salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 400 mM KCl, 1.5 mM MgCl2 , 0.2 mM EDTA, 0.5 mM DTT, 1 mM NaF, 1 mM sodium orthovanadate). Cell debris were removed by microcentrifugation, followed by quick freezing of the supernatants. The protein concentration was also determined using the BioRad protein assay reagent according to the manufacture’s

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instruction. A 10 ␮g sample of the nuclear extract was mixed with the double-stranded NF-␬B oligonucleotide (5 AGTTGAGGGGACTTTCCCAGGC-3 ) end-labeled by [␥32 P] dATP (underlying indicates a ␬B consensus sequence or a binding site for NF-␬B/cRel homodimeric and heterodimeric complex). Binding reactions were performed at 37 ◦ C for 30 min in 30 ␮l of reaction buffer at pH 7.5 containing 10 mM Tris–HCl, 100 mM NaCl, 1 mM EDTA, 4% glycerol, 1 ␮g of poly (dI-dC) and 1 mM DTT. The specificity of binding was examined by competition with the 80-fold unlabeled oligonucleotide. DNA-protein complexes were separated from the unbound DNA probe on native 5% polyacrylamide gels at 100 V in 0.5× TBE buffer. The gels were vacuum dried for 1 h at 80 ◦ C and exposed to X-ray film at −70 ◦ C for 24 h. The specificity of binding was examined by competition with the 80-fold unlabeled NF-␬B oligonucleotide. 2.13. Statistical analysis Values are expressed as mean ± S.D. Statistical significance was determined using the Student’s t-test. Values with p < 0.05 were considered significant.

3. Results 3.1. In vivo anti-inflammatory and anti-nociceptive effects When we examined the anti-inflammatory effect of MEIO in the carrageenin-induced edema model, we found that it exhibited an inhibitory effect on carrageenin-induced edema for 2–4 h, as shown in Table 1. The positive control drug, ibuprofen, showed significantly higher inhibitory effect than MEIO. The anti-nociceptive effects of test samples were assayed using two different models, i.e., by the acetic acid-induced abdominal constriction test and by the hot plate test in mice. In the former test, MEIO showed anti-nociceptive activity after oral administration of 100 or 200 mg/kg. Aspirin was also found to exert a significant protective effect (Table 2). Although the acetic acidinduced abdominal constriction test is non-specific (e.g. anti-cholinergic, anti-histaminic and other agents also show activity), it is widely used for analgesic screening and involves local peritoneal receptors (cholinergic and histamine receptors) and mediators of acetylcholine and histamine. The hot plate test was used to determine whether MEIO has any central analgesic effect, and as expected MEIO showed significant activity (Table 2). Morphine was used as a positive control in the hot plate test. These results show that MEIO has significant anti-inflammatory activity by carrageenin-induced paw edema assay, and that it had antinociceptive activity by abdominal constriction and hot plate assays.

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Table 1 Inhibitory effect of MEIO on carrageenin-induced hind paw edema in rats Group

Dose (mg/kg, p.o.)

Swelling volume (ml) 1 (h)

2 (h)

3 (h)

4 (h)

5 (h)

1.24 ± 0.05

2.32 ± 0.06

2.99 ± 0.11

2.38 ± 0.04

1.89 ± 0.07

MEIO

100 200

1.28 ± 0.06 1.27 ± 0.05

2.21 ± 0.04 1.93 ± 0.07***

2.63 ± 0.08** 2.35 ± 0.09***

2.30 ± 0.04 2.13 ± 0.03***

1.86 ± 0.06 1.80 ± 0.05

Ibuprofen

100

0.74 ± 0.08***

1.20 ± 0.03***

1.43 ± 0.06***

1.13 ± 0.07***

0.90 ± 0.02***

Control

The assay procedure was described in the experimental methods. Values are expressed mean ± S.D. The number of animal used for each group was 10. ** p < 0.01. *** p < 0.001 vs. control group.

3.2. In vitro anti-inflammatory effect 3.2.1. Effect of MEIO on cell viability and LPS-induced NO and PGE2 production Cell viability, as examined by the MTT assay, was determined to exclude the possibility that the inhibitory effect of MEIO was due to cytoxicity. The viability of LPS-induced RAW 264.7 cells treated with or without MEIO at different concentrations was >95% (data not shown). Thus, we examined the effect of MEIO on NO synthesis in RAW 264.7 macrophages. MEIO showed an inhibitory effect on LPS-induced NO production in a dose-dependent manner, with an IC50 value of 89 ␮g/ml (Fig. 1A). l-N6 -(1-iminoethyl)lysine 10 ␮M, a competitive inhibitor of iNOS, was used as a positive inhibitor. As shown in Fig. 1B, the production of PGE2 was also significantly inhibited by MEIO in a dose-dependent manner.

B). A similar pattern was observed when the effect of MEIO on LPS-induced COX-2 expression was examined (Fig. 2A). Under the same conditions, COX-2 mRNA levels were also significantly reduced in a similar pattern (Fig. 2B). 3.2.3. Inhibition of LPS-induced TNF-α production and mRNA expression by MEIO To evaluate the effect of MEIO on the release of inflammatory cytokines that contribute to the sustained activation

3.2.2. Effect of MEIO on LPS-induced iNOS and COX-2 protein and mRNA expressions To elucidate the mechanism involved in the inhibitions of NO and PGE2 generation by MEIO in LPS-induced macrophages, we further studied the effect of MEIO on iNOS and COX-2 protein and gene expression. In LPSunstimulated macrophages, the protein and mRNA expressions of iNOS and COX-2 were undetectable. However, in response to LPS the expression of iNOS was markedly increased, and MEIO significantly inhibited iNOS protein and mRNA induction in a dose-dependent manner (Fig. 2A and Table 2 Anti-noceptive effect of MEIO by acetic acid-induced abdominal constriction and hot-plate test in mice Group

Dose (mg/kg, p.o.)

Stretching episodes (count/20 min)

Control

61.4 ± 1.8

MEIO

100 200

55.4 ± 1.8***

100 10

21.6 ± 4.3***

Aspirin Morphine

50.0 ± 1.6***

Action time (s) 8.7 ± 1.3 10.4 ± 1.1** 13.5 ± 1.3*** 22.8 ± 2.0***

The assay procedure was described in the experimental methods. Values are expressed mean ± S.D. The number of animal used for each group was 10. ** p < 0.01. *** p < 0.001 vs. control group.

Fig. 1. Effect of MEIO on nitrite (A) and PGE2 (B) production on LPSinduced RAW 264.7 cells. Results are expressed as means ± S.D. (n = 5). ** p < 0.01, *** p < 0.001 vs. LPS-treated group.

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3.2.4. Inhibition of LPS-induced NF-κB activation by MEIO To further investigate the mechanism of the MEIOmediated inhibition of iNOS, COX-2 and TNF-␣ transcription, we focused on NF-␬B, which is known to transactivate iNOS, COX-2, TNF-␣ and other genes (Baeuerle and Baltimore, 1996). Electrophoretic mobility shift assay (EMSA) analyses demonstrated that LPS-induced NF-␬BDNA binding activity in RAW 264.7 macrophages was significantly reduced by treatment with MEIO in a dose-dependent manner (Fig. 4). Moreover, the extent of this reduction was similar to those of iNOS and COX-2 protein and iNOS, COX2 and TNF-␣ mRNA expression.

Fig. 2. Effect of MEIO on LPS-induced iNOS and COX-2 protein (A) and mRNA expression (B) in RAW 264.7 Cells. Data represent one of three similar results.

of macrophages, we investigated the effect of MEIO on LPSinduced TNF-␣ release and mRNA expression by using an enzyme immunoassay and RT-PCR. As shown in Fig. 3A and B, the production and mRNA expressions of TNF-␣ were slightly reduced in a dose-dependent manner when cells were treated with MEIO.

Fig. 3. Effect of MEIO on LPS-induced TNF-␣ release (A) and mRNA expression (B) in RAW 264.7 cells. The values shown represent means ± S.D. (n = 5). * p < 0.05, ** p < 0.01 vs. LPS-treated group.

3.2.5. Effect of MEIO on the degradation of IκB-α and the nuclear translocation of p65 In unstimulated cells, NF-␬B is sequestered in the cytosol by its inhibitor I␬B, which under conditions of LPS stimulation is phosphorylated by its inhibitor I␬B kinase, ubiquitinated, and rapidly degraded via the 26 S proteosome, releasing NF-␬B (Yasuyuki, 2001). We investigated whether MEIO (90 ␮g/ml) could inhibit the LPS-induced degradation of I␬B-␣ in Raw 264.7 cells by conducting a Western blot assay with anti-I␬B-␣ antibody. Fig. 5A shows that LPS induced the degradation of I␬B-␣ after 5–10 min and that this degradation was significantly blocked by pre-treatment with MEIO. We also investigated whether MEIO prevents

Fig. 4. Inhibition of NF-␬B-DNA binding by MEIO. Nuclear extracts were prepared and analyzed for NF-␬B binding to DNA by EMSA. The arrow indicates the position of NF-␬B and non-specific (N.S.) bands. Data represent one of three similar results.

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Fig. 5. Inhibition of I␬B-␣ degradation (A) and nuclear translocation of p65 (B) by MEIO.

the translocation of the subunit of NF-␬B, p65, from the cytosol to the nucleus after its release from I␬Bs. After treatment with MEIO, a reduction in the level of p65 in the nuclear fraction was detected in a concentration-dependent manner by Western blot analysis (Fig. 5B).

4. Discussion In recent years, mushroom polysaccharides have been found to have immunomodulatory and anti-tumour properties (Leung et al., 1997; Borchers et al., 1999; Wasser and Weis, 1999). Although it has been reported that Inonotus obliquus, and other mushrooms, has therapeutic effects, i.e., anti-inflammatory, anti-tumor, immuno-modulatory and hepatoprotective effects (Solomon and Alexander, 1999), the mechanism of the anti-inflammatory and anti-nociceptive effects of Inonotus obliquus has not been clearly elucidated. Whilst investigating the anti-inflammatory and antinociceptive effects of MEIO in vivo, we found that MEIO mildly reduced the edema induced by carrageenin, in which peak edema is characterized by the presence of PGs (Evangelos et al., 2002). The anti-nociceptive effects of test samples were assayed using two different models, i.e., by acetic acid-induced abdominal constriction test and by hot plate test in mice. The results obtained from the acetic acid-induced abdominal constriction tests with MEIO showed that it had a dose-response correlation at 100 and 200 mg/kg. A significant reduction of this abdominal constriction model indicates that MEIO’s mode of action is related to the sensitization of nociceptive receptors to prostaglandins (PGs). Hot plate test results indicated that MEIO significantly increased the latency of jumping response when treated at 100 or 200 mg/kg, without affecting the animals’ abilities to detect the thermal pain threshold (licking response), suggesting that MEIO has central

analgesic properties. The anti-nociceptive activity shown by MEIO in these models suggests that MEIO possesses peripheral and central mediated anti-nociceptive properties. To explore the mechanism underlying these potentially beneficial effects, the effects of MEIO on macrophage functions related to inflammation were investigated. We found that MEIO inhibited LPS-induced NO production in RAW 264.7 macrophages, dose-dependently. Generally, fungi are known as producers of polysaccharides, and these polysaccharide fractions have been shown to promote the production of pro-inflammatory cytokines, such as, IL-1, IL-10 and TNF-␣ (Yuh et al., 2001) through ligand–receptor binding complexes. However, our experimental data show that the methanol extract of Inonotus obliquus did not alter LPS-induced NO and TNF-␣ productions after pre-, co- or post-treating RAW 264.7 cells (data not shown), indicating that MEIO does not interfere with LPS to its receptor binding. From these results, we presumed that the inhibition of NO production in LPS-stimulated RAW 264.7 cells by MEIO occurred via the modulation of iNOS, because macrophages express significant amounts of iNOS upon stimulation by a variety of substances, including LPS (a bacterial endotoxin), which leads to increased NO production (Lowenstein et al., 1993). MEIO dose-dependently inhibited LPS-induced iNOS protein and mRNA expression. In addition to iNOS expression, the induction of COX-2 also represents an important patho-mechanism in diverse inflammatory processes. PGE2 is a pleiotropic mediator produced at inflammatory sites by COX-2, and causes pain, swelling, and stiffness (Makoto et al., 2002). Here, we found that MEIO dose-dependently inhibits LPS-induced PGE2 production that occurs in parallel with COX-2 protein and mRNA down regulation. TNF-␣, like NO and PGE2 is an important pro-inflammatory cytokine and is involved in normal physiological immune and inflammatory processes. However, when inappropriately expressed, TNF-␣ also plays a role in the development of

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chronic inflammation and in the development of associated diseases (Evangelos et al., 2002). Our data show that the inhibitory effects of MEIO on LPS-induced TNF-␣ secretion occurred through TNF-␣ mRNA expression and its measured accumulation. This action might also represent a crucial step in the anti-inflammatory action of MEIO. Interference with NF-␬B activation may also explain, at least in part, the inhibitory effects of MEIO observed in the present study, since this transcription factor appears to play a central role in the transcriptional regulations of iNOS, COX2 and TNF-␣ in macrophages (Drouet et al., 1991; Xie et al., 1994; Caivano et al., 2001; Young et al., 2001). This transcription factor (NF-␬B) is located in the cytoplasm and is associated with the inhibitory protein I␬B, which upon activation by inflammatory stimuli is phosphorylated and degraded. NF-␬B is then translocated to the nucleus and binds to the promoter regions of target genes (Boone et al., 2002). The present study, demonstrates that MEIO inhibits LPS-induced activation of NF-␬B via the inhibition of the degradation of I␬B␣. We also examined the expression of the NF-␬B p65 subunit by Western blot to determine whether the p65 subunit is directly concerned in transcription, because the most abundant form of NF-␬B is a p50/p65 heterodimer, in which p65 contains the transcriptional activation domain (Linda et al., 2002). In the present study, we found that MEIO blocked the LPS-induced activation of NF-␬B by inhibiting the degradation of I␬B. These results suggest that the inhibition of the LPS-induced expressions of the iNOS, COX-2 and TNF-␣ genes by MEIO occurs by blocking NF-␬B activation, although the inhibition of other factors, such as, AP-1, the interferon response element and the ␥-activated site may also be involved. Traditionally, Inonotus obliquus has been taken in the form of a hot water extract prepared from a small piece of the mushroom (1–2 g) or one tablespoon of crushed mushroom. This produces an aqueous extract, which is taken at a dose of three cups per day. In this study, we showed that the MEIO had the anti-inflammatory and anti-nociceptive effects after oral administration of 100 or 200 mg/kg and this amount of MEIO might be reasonable in comparison with the dose recommended in traditional medicine. Moreover, we did not find any toxic syndromes based on the body weight change during MEIO treatment. In conclusion, our results demonstrate that MEIO has antiinflammatory and anti-nociceptive effects in rats and mice. In addition, we found that MEIO is a potent inhibitor of LPSinduced NO, PGE2 and TNF-␣ production, and that this inhibition is caused by preventing NF-␬B activation in RAW 264.7 macrophages. Therefore, we conclude that MEIO appears to have potential as a treatment for inflammatory disease and as an analgesic.

Acknowledgments This research was supported by a grant (PF002104-07) from the Plant Diversity Research Center of the 21st

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Frontier Research Program funded by the Ministry of Science and Technology of the Korean government and by the Korea Science & Engineering Foundation (No. R13-2002-020-01002-0).

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