The herbs that comprise Dojuksan were purchased from the University Oriental Herbal Drugstore (Iksan, Korea) in August 2011, and a voucher specimen was deposited at the Herbarium of the College of Pharmacy at Wonkwang University (Iksan, Korea). The Dojuksan water extract (DWE), and the 30% ethanol extract (DEE) were both deposited at the Standardized Material Bank for New Botanical Drugs (Wonkwang University). Dojuksan comprised Rehmanniae radix (8 g), Akebia caulis (8 g), Glycyrrhizae radix (8 g) and Phyllostachys folium (8 g). It (32 g) was extracted with either hot water or 30% ethanol (2 liters of each) for 2 h, and the extracts were concentrated in vacuo to obtain the 30% ethanol extract (NNMBS308). The 30% ethanol extract was subjected to C₁₈-functionalized silica gel flash column chromatography and eluted with a stepwise gradient of 0% (Fr. 1, DEE-1), 20% (Fr. 2, DEE-2), 40% (Fr. 3, DEE-3), 60% (Fr. 4, DEE-4), 80% (Fr. 5, DEE-5) and 100% (Fr. 6, DEE-6) (v/v) of MeOH in H₂O (4 ml each) (Fig. 4).

The solvents used for the extraction and flash column chromatography were reagent grade without further purification, whereas the solvents used for HPLC were of analytical grade. Flash column chromatography was performed using YMC octadecyl-functionalized silica gel (C₁₈). HPLC (YOUNGLIN-YL9100; Young Lin Instrument Co., Anyang, Korea) separation was performed using a Shiseido Capcell Pak C₁₈ column (4.6×250 mm and 5 µm particle size; Shiseido Co., Ltd., Tokyo, Japan) with a flow rate of 0.7 ml/min, and an injection volume of 20 µl. The mobile phase was composed of (A) water and (B) acetonitrile, with an applied gradient of 5% B increasing to 100% B within 60 min. The column was cleaned with 100% B for 10 min, and the system was then equilibrated for 20 min under starting conditions. The detection wavelengths were adjusted to ELSD, 210 and 254 nm.

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and all other tissue culture reagents used, were purchased from Gibco-BRL Co. (Grand Island, NY, USA) unless otherwise stated. Tin-protoporphyrin IX (SnPP), an inhibitor of HO activity, was obtained from Porphyrin Products (Logan, UT, USA). Lipofectamine™ 2000 was purchased from Invitrogen Life Technologies (Grand Island, NY, USA). Cobalt protophorphyrin IX (CoPP, Cat. no. C1900) and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise indicated. RAW264.7 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Small interfering RNA (siRNA) against Nrf2 and primary antibodies, including those against HO-1 (SC-10789), Nrf2 (SC-722), COX-2 (SC-1745) and iNOS (SC-650), and appropriate secondary antibodies [anti-goat (SC-2768), anti-mouse (SC-2005) and anti-rabbit (SC-2004)] for western blot analysis, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for PGE₂, TNF-α and IL-1β were purchased from R&D Systems (Minneapolis, MN, USA). TLC silica gel 60 F254 aluminum plates were purchased from Merck (Darmstadt, Germany).

Cell culture and viability were carried out as previously described (34). The RAW264.7 cells were maintained at 5×10⁵ cells/ml in DMEM supplemented with 10% heat-inactivated FBS, penicillin G (100 U/ml), streptomycin (100 mg/ml) and L-glutamine (2 mM), and were incubated at 37°C in a humidified atmosphere containing 5% CO₂ and 95% air. The effects of various experimental modulations on cell viability were evaluated by determining mitochondrial reductase function with an assay based on the reduction of tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) into formazan crystals. The synthesis of formazan is proportional to the number of functional mitochondria in living cells. For the determination of cell viability, 50 mg/ml MTT solution were added to 1 ml cell suspension (1×10⁵ cells/ml in 96-well plates) for 4 h. The formazan synthesized was dissolved in acidic 2-propanol, and the optical density was measured at 590 nm. The optical density of the formazan synthesized in the control (untreated) cells was considered to be 100% cell viability. The majority of experiments were performed using incubation with LPS and samples, alone and in combination, unless stated otherwise. Pretreatment with samples was performed, beginning at 12 h before the incubation period, and then stimulation with LPS (1 µg/ml) was undertaken.

The production of nitrite in the conditioned medium was determined using a method based on the Griess reaction, as described in a previous study (35). An aliquot (100 µl) of each supernatant was mixed with the same volume of Griess reagent [0.1% (w/v) N-(1-naphathyl)-ethylenediamine and 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid] for 10 min at room temperature. The absorbance of the final product was measured spectrophotometrically at 525 nm using an ELISA plate reader, and the nitrite concentration in the samples was determined from a standard curve of sodium nitrite made up in phenol red-free DMEM. The levels of PGE₂, TNF-α or IL-1β present in each sample were determined using a commercially available kit from R&D Systems (Abingdon, UK). The assay was performed according to the instructions provided by the manufacturer.

Western blot analysis was carried out as previously described (36). Western blot analysis was performed by lysing the cells in 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM PMSF, 5 mg/ml aprotinin, 5 mg/ml pepstatin A and 1 mg/ml chymostatin). The protein concentration was determined using a Lowry protein assay kit (P5626; Sigma Chemical Co.). An equal amount of protein for each sample was resolved by 12 or 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred onto a Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5%skimmed milk and sequentially incubated with primary antibody (Santa Cruz Biotechnology, Inc.) and horseradish peroxidase-conjugated secondary antibody, followed by ECL detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Preparation of cytosolic and nuclear fractions was carried out as previously described (37). The cells were collected and washed with phosphate-buffered saline (PBS) and suspended in 200 µl lysis buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF and a protease inhibitor cocktail]. The cells were allowed to swell on ice for 15 min; subsequently, 12.5 µl 10% NP-40 was added. The tubes were agitated on a vortex for 10 sec and then centrifuged for 5 min. The resulting supernatant represented the cytosolic extract. The nuclear pellets were resuspended in 50 µl ice-cold nuclear extraction buffer [20 mM HEPES (pH 7.9), 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM PMSF and a protease inhibitor cocktail] and incubated on ice for 1 h with intermittent vortexing. This nuclear extract was centrifuged for 10 min at 15,000 × g; the resulting supernatant represented the nuclear fraction.

As previously described (38), the DNA-binding activity of NF-κB in the nuclear extracts was measured using a TransAM kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's instructions. Briefly, 30 µl complete binding buffer (DTT, herring sperm DNA and binding buffer AM3) was added to each well. The samples were nuclear extracts of RAW264.7 macrophages that had been stimulated for 30 min with LPS and specific concentrations of DEE. Subsequently, the samples were diluted in complete lysis buffer and added to each well (20 µg nuclear extract diluted in complete lysis buffer to a final volume of 20 µl). The plates were incubated for 1 h at room temperature with mild agitation (100 rpm on a rocking platform). After washing each well with wash buffer, 100 µl of diluted NF-κB antibody (1:1,000 dilution in 1X antibody-binding buffer) was added to each well, and the plates were then further incubated, as previously described, for 1 h. After washing each well with wash buffer, 100 µl diluted HRP-conjugated antibody (1:1,000 dilution in 1X antibody-binding buffer) was added to each well followed by incubation for 1 h, as before. Developing solution (100 µl) was added to each well and left to react for 5 min, followed by the addition of a stop solution to each well. Finally, the absorbance of each sample was read at 450 nm on a spectrophotometer (microplate reader model 680, serial no. 19590, Bio-Rad) within 5 min of reaction termination.

Cells were transiently transfected with Nrf2 siRNA (Santa Cruz Biotechnology, Inc.) for 6 h using Lipofectamine 2000™ (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and then incubated in fresh media containing 10% FBS for 24 h before further manipulation.

Data are expressed as the means ± SD of at least 3 independent experiments. To compare three or more groups, one-way analysis of variance (ANOVA) followed by a Newman-Keuls post hoc test was used. Statistical analysis was performed using GraphPad Prism software, version 3.03 (GraphPad Software Inc., San Diego, CA, USA).

We found that the Dojuksan 30% ethanol extract (DEE) was significantly more effective in terms of its anti-inflammatory effects than DWE (data not shown), and that it exerted its anti-inflammatory effects by inhibiting the expression of the pro-inflammatory enzymes, iNOS and COX-2, and suppressing the secretion of the pro-inflammatory cytokines, NO, PGE₂, TNF-α and IL-1β, through the inhibition of NF-κB activation. Since DEE had more potent anti-inflammatory effects, it was used in our subsequent experiments. The cytotoxicity of DEE and DEE-5 in the RAW264.7 cells was determined by MTT assay. We found that cell viability was not significantly decreased by either DEE or DEE-5 at concentrations of up to 400 µg/ml (data not shown). Subsequently, we pre-treated the RAW264.7 cells with either DEE at non-cytotoxic concentrations (50–400 µg/ml) for 12 h and measured the production of NO, PGE₂, TNF-α and IL-1β following stimulation with LPS (1 µg/ml) for 18 h. With the increasing concentration of DEE, NO production decreased in a dose-dependent manner until it reached a level close to that of the control cells (which were not treated with LPS) at 400 µg/ml. We observed a similar effect on the production of PGE₂, TNF-α and IL-1β in the cells treated with DEE (Fig. 1).

The expression of the pro-inflammatory enzyme, iNOS, plays a crucial role in immune-activated macrophages through the production of NO (26,27). In addition, prostaglandins are an important mediator of the symptoms of inflammation, including fever and pain. Inducible COX-2 is the major source of prostaglandins (24,25). Thus, in order to examine the effects of DEE on the expression of iNOS and COX-2, the RAW264.7 cells were challenged with LPS (1 µg/ml) in combination with the indicated concentrations of DEE, and the protein expression levels of iNOS and COX-2 were measured by western blot analysis (Fig. 1E). DEE decreased both iNOS and COX-2 protein expression in a concentration-dependent manner.

NF-κB is a key molecule in an important signaling pathway involved in inflammatory diseases, and it regulates iNOS and genes, such as COX-2 (39,40). Thus, to determine whether the NF-κB pathway is involved in the suppression of inflammatory responses by DEE, we measured NF-κB binding activity. DEE significantly decreased NF-κB binding activity in a concentration-dependent manner (Fig. 1D).

We examined whether HO-1 is the key player mediating the DEE-induced suppression of pro-inflammatory responses. The RAW264.7 cells were treated with the indicated concentrations of DEE for 12 h, or with 400 µg/ml of DEE for the indicated periods of time. We noted that DEE increased HO-1 expression in a concentration-dependent manner (Fig. 2A), and HO-1 expression began to increase 3 h following treatment with 400 µg/ml DEE (Fig. 2B). Nrf2 is as an indispensable regulator of the coordinated induction of phase II enzymes, including HO-1 (41). Therefore, we performed western blot analysis to determine whether treatment with DEE induces the nuclear translocation of Nrf2. When the cells were incubated with DEE for 0.5, 1.0 or 1.5 h at a concentration of 400 µg/ml, there was a gradual increase in the levels of nuclear Nrf2, with a concomitant decrease in the cytoplasmic levels (Fig. 2C). The role of Nrf2 in DEE-mediated HO-1 expression was confirmed using siRNA against Nrf2. The RAW264.7 cells were transiently transfected with Nrf2 siRNA, and then treated with 400 µg/ml DEE for 12 h. As shown in Fig. 2D, transient transfection with Nrf2 siRNA completely abolished DEE-induced HO-1 expression.

To confirm the suppressive effects of HO-1 on pro-inflammatory mediators, cytokines and the NF-κB pathway, we used SnPP, which is a competitive inhibitor of HO activity. Previously, imidazole-dioxolane compounds have been shown to act as inhibitors of HO activity (42,43), as well as specific HO isoenzymes (44). In the present study, RAW264.7 cells were pre-treated with DEE (400 µg/ml) for 12 h in the absence or presence of SnPP (20 µM). The inhibitory effects of DEE on the LPS-induced production of NO, PGE₂, TNF-α and IL-1β, as well as NF-κB binding activity, were partially reversed by SnPP (Fig. 3).

To further explore the differences in the anti-inflammatory effects of DEE, we generated 6 different fractions of DEE using a C₁₈ cartridge with a stepwise elution of MeOH in H₂O (Fig. 4), as the HPLC chromatograms of DEE showed unclear patterns (data not shown). We then performed a comparative analysis of the HPLC chromatograms of each of the 6 fractions of DEE (Fig. 5). Data from the HPLC analysis of DEE-5 was obtained in the form of chromatograms by monitoring detector responses at ELSD, 210 and 254 nm (Fig. 5). Of the 6 fractions, DEE-5 induced the greatest decrease in NO production in the LPS-stimulated RAW264.7 cells (data not shown). Therefore, we further examined the anti-inflammatory effects of the Dojuksan ethanol extract using DEE-5.

To confirm the inhibitory effects of DEE-5 on pro-inflammatory mediators and cytokines, we pre-treated the RAW264.7 cells with DEE-5 (100 or 200 µg/ml) for 12 h and then measured the protein expression of iNOS and COX-2, as well as the production of NO, PGE₂, TNF-α and IL-1β following stimulation with LPS (1 µg/ml) for 18 h. DEE-5 significantly decreased the protein expression of iNOS and COX-2, and the production of NO, PGE₂, TNF-α and IL-1β (Fig. 6).

We also examined whether DEE-5 is able to induce the expression of HO-1. The RAW264.7 cells were treated with the indicated concentrations of DEE-5 for 12 h, and we observed a significant increase in HO-1 expression at a concentration of 100–200 µM (Fig. 7A). The role of Nrf2 in DEE-5-mediated HO-1 expression was confirmed using siRNA against Nrf2. The RAW264.7 cells were transiently transfected with Nrf2 siRNA, and then treated with 200 µg/ml DEE-5 for 12 h. As shown in Fig. 7B, transient transfection with Nrf2 siRNA completely abolished DEE-5-induced HO-1 expression. To confirm the suppressive effects of HO-1 on pro-inflammatory mediators and cytokines, we used SnPP, which is a competitive inhibitor of HO activity. The RAW264.7 cells were pre-treated with DEE-5 (200 µg/ml) for 12 h in the absence or presence of SnPP. The inhibitory effects of DEE-5 on LPS-stimulated NO, PGE₂, TNF-α, and IL-1β production were partially reversed by SnPP (Fig. 7C–F).