EGL was supplied by Dongeui University Oriental Hospital (Busan, Korea). The freeze-dried and milled G. lucidum fruiting bodies (200 g) were extracted with 25% ethanol (4 liters) at room temperature for 10 h using a blender. The extracts were filtered through a Whatman no. 2 filter (Maidstone, UK), concentrated to 500 ml under vacuum conditions and then stored at −20°C (11). The EGL solution was directly diluted in medium prior to assay.

The murine BV2 cell line was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco-BRL, Grand Island, NY, USA) at 37°C in a humidified incubator with 5% CO₂. The cells were pre-treated with the indicated EGL concentrations for 1 h before adding LPS (0.5 μg/ml, Sigma-Aldrich, St. Louis, MO, USA). Cell viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich) reduction assay. In brief, cells (1×10⁵ cells/ml) were seeded and treated with EGL and/or LPS for the indicated time periods. Following treatment, the medium was removed and the cells were incubated with 0.5 mg/ml MTT solution. After a 3 h incubation at 37°C in 5% CO₂, the supernatant was removed and formation of formazan was measured at 540 nm with a microplate reader (Dynatech MR-7000; Dynatech Laboratories Inc., El Paso, TX, USA).

NO concentrations in culture supernatants were determined by measuring nitrite, which is a major stable product of NO, using Griess reagent (Sigma-Aldrich). Cells (5×10⁵ cells/ml) were stimulated in 24-well plates for 24 h, then 100 μl culture medium was mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride and 2.5% H₃PO₄). Nitrite levels were determined using an enzyme-linked immunosorbent assay (ELISA) plate reader at 540 nm and nitrite concentrations were calculated by reference to a standard curve generated using known concentrations of sodium nitrite (26).

BV2 cells were sub-cultured in 6-well plates (5×10⁵ cells/ml) and incubated with the indicated concentrations of EGL in the presence or absence of LPS (0.5 μ/ml) for 24 h. A 100 μl aliquot of the culture-medium supernatant was collected for determination of the PGE₂ concentration by ELISA (Cayman Chemical, Ann Arbor, MI, USA).

Total RNA was isolated from the cells using the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). The total RNA (1.0 μg) was reverse-transcribed using M-MLV reverse transcriptase (Promega Corporation, Madison, WI, USA) to produce cDNAs. The inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, IL-1β and TNF-α genes were amplified from the cDNA using PCR. The PCR primers were as follows: mouse iNOS, 5′-ATG TCC GAA GCA AAC ATC AC-3′ and 5′-TAA TGT CCA GGA AGT AGG TG-3′; COX-2, 5′-CAG CAA ATC CTT GCT GTT CC-3′ and 5′-TGG GCA AAG AAT GCA AAC ATC-3′; IL-1β, 5′-ATG GCA ACT GTT CCT GAA CTC AAC T-3′ and 5′-TTT CCT TTC TTA GAT ATG GAC AGG AC-3′; and TNF-α, 5′-ATG AGC ACA GAA AGC ATG ATC-3′ and 5′-TAC AGG CTT GTC ACT CGA ATT-3′. Following amplification, the PCR products were electrophoresed on 1% agarose gels and visualized by ethidium bromide (Sigma-Aldrich) staining. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control.

Cells were washed three times with phosphate-buffered saline (PBS) and lysed in lysis buffer (1% Triton X-100, 1% deoxycholate and 0.1% NaN3) containing protease inhibitors (Roche Diagnostics, Mannheim, Germany). In a parallel experiment, nuclear and cytosol proteins were prepared using NE-PER^® nuclear and cytoplasmic extraction reagents (Pierce Biotechnology Inc., Rockford, IL, USA) according to the manufacturer’s instructions. For the western blot analysis, equal amounts of protein were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes (Schleicher & Schuell Inc., Keene, NH, USA) by electroblotting. Blots were probed with the desired antibodies for 1 h, incubated with the diluted enzyme-linked secondary antibodies and visualized by enhanced chemiluminescence (Amersham Co., Arlington Heights, IL, USA) according to the manufacturer’s instructions. Actin and nucleolin were used as internal controls for the cytosolic and nuclear fractions, respectively. Antibodies against iNOS, COX-2, IκB-α, NF-κB p65, TLR4 and myeloid differentiation factor 88 (MyD88) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against actin and nucleolin were obtained from Sigma-Aldrich. Peroxidase-labeled goat anti-rabbit immunoglobulin and fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG were purchased from Amersham Co. and Sigma-Aldrich, respectively.

The levels of IL-1β and TNF-α were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Briefly, BV2 cells (5×10⁵ cells/ml) were plated in 24-well plates and pre-treated with the indicated EGL concentrations for 1 h prior to treatment with 0.5 μg/ml LPS for 24 h. A 100 μl aliquot of each culture-medium supernatant was collected for determination of the IL-1β and TNF-α concentrations by ELISA.

A total of 1×10⁶ BV2 cells were transfected with 2 μg NF-κB-luciferase reporter plasmids (BD Biosciences, San Jose, CA, USA) using Lipofectamine according to the manufacturer’s instructions (Gibco-BRL). Then, the cells were pre-incubated in the presence or absence of EGL before being stimulated with LPS for 6 h. Cells were washed twice with PBS and lysed with reporter lysis buffer (Promega Corporation). Following vortexing and centrifuging at 12,000 × g for 1 min at 4°C, the supernatant was stored at −70°C. For the luciferase assay, 20 μl cell extract was mixed with 100 μl luciferase assay reagent at room temperature, then, the mixture was analyzed using a LB96V microplate luminometer (Perkin-Elmer, Waltham, MA, USA) (27).

Data are presented as mean ± standard deviation. Statistical significance was determined using an analysis of variance followed by the Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.

The potential anti-inflammatory properties of EGL were evaluated against the production of two major inflammatory mediators, NO and PGE₂, in LPS-stimulated BV2 microglia. To determine the levels of NO and PGE₂ production, the amounts of nitrite and PGE₂ released into the culture medium were measured using Griess reagent and ELISA, respectively. According to the NO detection assay result, LPS alone markedly induced NO production from cells compared with the control (Fig. 1A). However, pre-treatment with EGL significantly repressed the levels of NO production in the LPS-stimulated BV2 microglia in a concentration-dependent manner, up to 1 μg/ml. Under the same conditions, stimulating the cells with LPS also resulted in a significant increase in PGE₂ production; however, this was markedly repressed by pre-treatment with EGL (Fig. 1B).

The cells were exposed to EGL for 24 h in the presence or absence of LPS to exclude a cytotoxic effect of EGL on BV2 cell growth. Cell viability was then measured by the MTT assay. As indicated in Fig. 2, the concentrations of EGL (0.1–1 mg/ml) used to inhibit NO and PGE₂ production did not affect cell viability. The results clearly indicate that the inhibition of NO and PGE₂ production in the LPS-stimulated BV2 cells was not due to a cytotoxic effect of EGL.

RT-PCR and western blot analyses were carried out to determine whether the inhibition of NO and PGE₂ production by EGL in LPS-stimulated BV2 cells was associated with reduced levels of iNOS and COX-2 expression. As shown in Fig. 3, iNOS and COX-2 protein levels were markedly upregulated following 24 h of treatment with LPS (0.5 μg/ml) alone; however, EGL significantly inhibited the iNOS and COX-2 protein expression in the LPS-stimulated BV2 microglia in a concentration-dependent manner (Fig. 3A). The effects of EGL on iNOS and COX-2 mRNA expression were evaluated to investigate whether EGL suppressed the LPS-mediated induction of iNOS and COX-2 at the pre-translational level. RT-PCR data revealed that the reduction in iNOS and COX-2 mRNAs correlated with the reduction in the corresponding protein levels (Fig. 3B). These results suggest that EGL-induced reductions in iNOS and COX-2 expression were the cause of the reduced NO and PGE₂ production levels.

The effects of EGL on the production of pro-inflammatory cytokines, including IL-1β and TNF-α, were analyzed using ELISA. BV2 cells were incubated with various concentrations of EGL in the presence or absence of LPS (0.5 μg/ml) for 24 h and cytokine levels in the culture media were measured. As shown in Fig. 4, the IL-1β and TNF-α levels were markedly increased in the culture media of the LPS-stimulated BV2 microglia. However, pre-treatment with EGL significantly reduced the release of these pro-inflammatory cytokines in a concentration-dependent manner. In a parallel experiment, the effects of EGL on LPS-induced IL-1β and TNF-α mRNA expression were studied using RT-PCR. As shown in Fig. 5, IL-1β and TNF-α mRNA transcription levels also decreased following EGL treatment. These results suggest that EGL was effective in suppressing pro-inflammatory cytokine production by altering IL-1β and TNF-α transcription levels in activated microglia.

Activation of NF-κB is crucial for the induction of iNOS, COX-2, TNF-α and IL-1β genes in activated BV2 microglia (28,29). To further characterize the mechanism through which EGL inhibits pro-inflammatory and/or cytotoxic factor expression, the ability of EGL to prevent translocation of the NF-κB p65 subunit to the nucleus was first examined. Western blot analysis revealed that the amount of NF-κB p65 in the nucleus markedly increased following exposure to LPS alone; however, the LPS-induced p65 level in the nuclear fraction was reduced following EGL pre-treatment (Fig. 6A). In addition, the ability of EGL to block the LPS-stimulated degradation of IκB-α was investigated by western blotting. As shown in Fig. 6A, IκB-α was markedly degraded at 30 min after LPS treatment; however, this LPS-induced IκB-α degradation was significantly reversed by EGL. Furthermore, the inhibition of LPS-induced NF-κB activation by EGL was confirmed using a luciferase assay. BV2 cells transfected with NF-κB-luciferase reporter plasmids were pre-treated with EGL for 1 h then, following stimulation with LPS for 6 h, luciferase activity was measured. As shown in Fig. 6B, LPS significantly enhanced the NF-κB activity up to ∼8-fold over the basal level, whereas EGL significantly inhibited the LPS-induced NF-κB activity. These findings show that the anti-inflammatory effect of EGL in LPS-stimulated BV2 cells involves the NF-κB pathway.

Previous studies established that the inflammatory response to LPS completely relies on the presence of TLR4, which triggers the intracellular association of MyD88 with its cytosolic domain (30–32). To investigate whether the anti-inflammatory activity of EGL is associated with the modulation of these proteins, the effects of EGL on LPS-induced upregulation of TLR4 and MyD88 in BV2 cells were examined. As shown in Fig. 7, EGL concentration-dependently attenuated the LPS-stimulated increase in TLR4 and MyD88 expression. This finding indicates that EGL is capable of disrupting key signal transduction pathways, including TLR signaling pathways activated by LPS in BV2 microglia, which subsequently prevents the production of pro-inflammatory mediators and cytokines.