LPS and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against COX-2, iNOS, TNF-α, IL-1β, NF-κB p65 and IκB-α were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against phosphorylated PI3K (p-PI3K), PI3K, phosphorylated Akt (p-Akt) and Akt were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against nucleolin and actin were obtained from Sigma-Aldrich. Peroxidase-labeled goat anti-rabbit immunoglobulin was purchased from Koma Biotechnology (Seoul, Korea). Other chemicals were purchased from Sigma-Aldrich. To prepare EECO, the rhizomes of C. officinale, which were obtained from Dongeui University Oriental Hospital (Busan, Korea), were pulverized and extracted twice with 10 volumes of 80% ethanol at 85–90°C in a reflux condenser for 3 h, and then filtered with a 50 μm filter and concentrated by vacuum evaporation at 60°C. The solid form of the extract was dissolved in dimethyl sulfoxide.

BV2 microglial cells were cultured at 37°C in 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum and antibiotics (WelGENE Inc., Daegu, Korea). In all the experiments, the cells were pre-treated with the indicated concentrations of EECO for 1 h prior to the addition of LPS (500 ng/ml) in serum-free DMEM. Cell viability was measured based on the formation of blue formazan that was metabolized from colorless MTT by mitochondrial dehydrogenases, which are active only in live cells. In brief, the BV2 cells were seeded and treated with reagents for the indicated periods of time. Following treatment, the medium was removed, and the cells were incubated with 0.5 mg/ml of MTT solution for 2 h at 37°C and 5% CO₂, and then the supernatant was removed and the formation of formazan was measured at 540 nm using a microplate reader (Dynatech MR-7000; Dynatech Laboratories, Chantilly, VA, USA).

The concentration of NO generated by BV2 cells activated by LPS was detected using Griess reagent [1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride]. BV2 cells were cultured for 24 h in a 6-well culture plate, pre-treated with various concentrations of EECO for 1 h, and then treated again with LPS (500 ng/ml). After 24 h of culture, the cell culture medium was collected and the same quantity of Griess reagent was added to induce a reaction at room temperature. The optical density of the reaction solution was measured at 540 nm using a microplate reader and the quantity of NO generated by the cells was calculated based on the concentration of the sodium nitrite (NaNO₂) standard solution (standard curve).

To measure the quantity of PGE₂ generated by BV2 cells, medium from the cultures under the same conditions was collected and the quantity of PGE₂ generated was measured using a PGE₂ enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemical Co., Ann Arbor, MI, USA). The concentration (pg/ml) of PGE₂ in the cell culture medium was calculated based on the concentrations of the standard solution as previously described (25).

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 were loaded in 24-well plates and pre-treated with the indicated EECO concentrations for 1 h prior to stimulation with 500 ng/ml LPS for 24 h. A total of 100 μl of culture medium supernatant was collected to determine IL-1β and TNF-α concentration by ELISA.

RT-PCR was conducted to examine the effects of EECO on the expression of LPS-induced iNOS, COX-2, and inflammatory cytokines at the transcription level. Total RNA was separated from the BV2 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and reverse-transcribed using MMLV reverse transcriptase (Promega, Madison, WI, USA) to produce cDNA. The cDNA was amplified by PCR using specific primers: iNOS forward, 5′-CCT CCT CCA CCC TAC CAA GT-3′ and reverse, 5′-CAC CCA AAG TGC TTC AGT CA-3′; COX-2 forward, 5′-AAG ACT TGC CAG GCT GAA CT-3′ and reverse, 5′-CTT CTG CAG TCC AGG TTC AA-3′; IL-1β forward, 5′-ATG GCA ACT GTT CCT GAA CTC AAC T-3′ and reverse, 5′-TTT CCT TTC TTA GAT ATG GAC AGG AC-3′; TNF-α forward, 5′-GCG ACG TGG AAC TGG CAG AA-3′ and reverse, 5′-TCC ATG CCG TTG GCC AGG AG-3′; and GAPDH forward, 5′-ACC ACA GTC CAT GCC ATC AC-3′ and reverse, 5′-TCC ACC ACC CTG TTG CTG TA-3′. The following PCR conditions were applied: iNOS, COX-2, IL-1β and TNF-α: 25 cycles of denaturation at 94°C for 30 sec, annealing at 59°C for 30 sec, and extension at 72°C for 30 sec; GAPDH, 23 cycles of denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec and extension at 72°C for 30 sec. The PCR products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide. GAPDH was used as an internal control to evaluate relative expression.

The cells were washed three times with phosphate-buffered saline (PBS) and lysed in lysis buffer [1% Triton X-100, 1% deoxycholate, 0.1% sodium azide (NaN₃)] containing protease inhibitor cocktail tablets to isolate total protein (Roche Diagnostics GmbH, Mannheim, Germany). In a parallel experiment, cytoplasmic and nuclear extracts were prepared using NE-PER nuclear and cytosolic extraction reagents (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH, USA) by electroblotting. Proteins were detected using an enhanced chemiluminescence detection system (Pierce).

The prepared cells were washed twice with PBS and fixed for 15 min at 4°C using 4% paraformaldehyde. The cells were washed again with PBS, reactions were induced for 20 min at 4°C in PBS that contained 0.3% Triton X-100, and then reactions were induced for 1 h at room temperature in PBS containing 2% bovine serum albumin (BSA) to suppress non-specific reactions. The anti-NF-κB p65 antibody was then diluted to 1:200 in a PBS solution that contained 2% BSA to induce reactions for 2 h at room temperature. The cells were washed three times in PBS and fluorescein isothiocyanate (FITC)-conjugated IgG (Molecular Probes, Eugene, OR, USA), which is a secondary antibody, was diluted to 1:100 to induce reactions for 1 h at room temperature. Samples of the immunofluorescence-stained cells were observed under a confocal laser scanning microscope (Olympus, Tokyo, Japan). A wavelength of 488 nm was used for FITC, and the images were reassembled into final three-dimensional images according to the manufacturer’s instructions (Olympus Fluoview 300, Olympus).

All results are expressed as the means ± standard errors. Each experiment was repeated at least three times. Statistical significances were identified between each treated group by the paired Student’s t-test. A P-value <0.05 was considered to indicate a statistically significant difference.

To determine the inhibitory effects of EECO on LPS-induced NO and PGE₂ production, BV2 macroglial cells were incubated with the indicated concentrations of EECO in the presence or absence of LPS for 24 h, and the levels of NO and PGE₂ production were measured in the culture medium using Griess reagent and ELISA, respectively. LPS triggered an approximate 7-fold increase in NO production compared with that in the untreated control group; however, pre-treatment with EECO reduced LPS-induced NO production in a concentration-dependent manner (Fig. 1A). The amount of PGE₂ present in the culture medium also increased after 24 h of exposure to LPS alone; however, a concentration-dependent decrease was observed following pre-treatment with EECO (Fig. 1B).

We then investigated whether the inhibitory effects of EECO on NO and PGE₂ production are associated with decreased levels of iNOS and COX-2 expression, which are known to induce NO and PGE₂ production, using western blot analysis and RT-PCR. The mRNA levels of iNOS and COX-2 were markedly augmented in the presence of LPS alone; however, their expression levels were markedly upregulated in the presence of EECO (Fig. 2A). Western blot analyses also revealed that treatment with LPS increased iNOS and COX-2 protein expression, whereas pre-treatment of the cells with EECO attenuated the LPS-induced iNOS and COX-2 protein expression (Fig. 3B). These results indicate that EECO inhibits the LPS-induced release of NO and PGE₂ by suppressing iNOS and COX-2 expression at the transcriptional level.

We then determined the potential effects of EECO on the production of pro-inflammatory cytokines, such as TNF-α and IL-1β by ELISA. The levels of TNF-α significantly increased in the culture medium of LPS-stimulated BV2 cells; however, the levels decreased significantly in a dose-dependent manner following pre-treatment with EECO (Fig. 3A). IL-1β production increased following stimulation with LPS and EECO significantly decreased the levels of IL-1β in the supernatant of LPS-stimulated BV2 cells (Fig. 3B).

RT-PCR and western blot analysis were performed in parallel experiments to determine whether EECO inhibits TNF-α and IL-1β expression. The increased expression of TNF-α and IL-1β following treatment with LPS was markedly attenuated by pre-treatment with EECO at both the transcriptional and translational levels (Fig. 4). These results indicate that EECO is effective in suppressing pro-inflammatory cytokine production by altering the transcriptional levels of TNF-α and IL-1β in LPS-activated microglia.

As NF-κB is a central transcription factor that regulates the expression of a large number of inflammation-related genes (7,26), the effects of EECO on the LPS-stimulated nuclear translocation of NF-κB p65 subunits were examined. The western blot analysis results in Fig. 5A indicate that the levels of NF-κB p65 in the nucleus markedly increased within 15 min of exposure to LPS; however, the LPS-induced p65 levels in the nuclear fraction decreased following pre-treatment with EECO. In addition, IκB-α was markedly degraded 15 min following exposure to LPS; however, the LPS-induced IκB-α degradation was significantly reversed by EECO. We also investigated whether EECO interferes with the translocation of NF-κB in LPS-treated BV2 cells by immunofluorescence. The level of NF-κB p65 in the nucleus decreased significantly by EECO, indicating that EECO inhibits NF-κB activation in BV2 microglial cells by suppressing IκB degradation and the nuclear translocation of NF-κB (Fig. 4B).

As the activation of the PI3K/Akt signaling pathway leads to the production of inflammatory mediators and cytokines through the activation of NF-κB (9–12), we investigated the effects of EECO on the phosphorylation of PI3K and Akt proteins in LPS-stimulated BV2 cells. Using western blot analysis with anti-phospho-specific antibodies for PI3K and Akt, we found that EECO suppressed the LPS-induced phosphorylation of PI3K and Akt (Fig. 6), whereas the levels of non-phosphorylated PI3K and Akt were unaffected by either EECO or LPS treatment. These findings strongly suggest that the anti-inflammatory effects of EECO in LPS-stimulated BV2 cells are associated with the inactivation of the PI3K/Akt pathway.

We evaluated the viability of BV2 cells incubated with or without LPS in the absence or presence of EECO by MTT assay to determine the cytotoxic effects (if any) of EECO on BV2 microglia. The concentrations (12.5 to 100 μM/ml) of EECO used to inhibit LPS-induced inflammatory responses did not affect cell viability, confirming that the anti-inflammatory effects of EECO in LPS-stimulated BV2 cells are not due to the cytotoxicity of EECO (Fig. 7).