The cytotoxic effects of RMO in RAW 264.7 cells were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As shown in Figure 1, RMO did not affect cell viability for 48 h at concentrations up to 200 μg/mL. For the subsequent experiments described in this study, non-toxic dose range (10 to 100 μg/mL) of RMO was used.

The inflammatory products NO and PGE₂ can be induced by the expression of iNOS and COX-2, respectively, as a part of the innate immune system, and the inhibition of NO and PGE₂over-production might be used as a therapeutic tool to treat inflammatory diseases [28]. Since there is a causal relationship between inflammation and cancer, iNOS and COX-2 are often considered as potential molecular targets for chemoprevention [29]. To examine whether RMO can suppress the inflammatory response, the inflammatory responses were induced in RAW 264.7 cells by LPS treatment. As shown in Figure 2A,B, treatment of the cells with LPS dramatically increased NO and PGE₂ productions; however, RMO significantly inhibited the production of these inflammatory molecules in a dose-dependent manner.

In particular, NO production decreased to near basal levels when the cells were treated with the highest concentration of RMO (100 μg/mL). In addition, RMO treatment at the doses over 50 μg/mL displayed stronger inhibition on NO production than the positive control aspirin, a well-known anti-inflammatory drug. The expressions of inflammatory enzymes iNOS and COX-2 were also strongly induced by LPS treatment while these over-expressions were suppressed by RMO in a concentration-dependent manner both at protein and mRNA levels (Figure 2C,D). Although there seems to be a dose-dependent tendency by RMO treatments in the expressions of these genes, the expression levels of protein and mRNA are not exactly same. The reason for this is not clear at this moment, but there might be an involvement of translational or post-translational modification of these genes and further studies are needed to elucidate the exact mechanisms. Anti-inflammatory agents decreasing NO and PGs productions by simultaneously inhibiting the iNOS and COX-2 genes have been considered to have a potentially therapeutic effect in the treatment of inflammatory and infectious diseases [30,31]. Our results suggest that RMO might inhibit the production of NO and PGE₂ by down-regulating the expression of iNOS and COX-2, which could be activated by inflammatory stimuli such as LPS.

Certain cytokines such as TNF-α, IL-1β and IL-6 have pro-inflammatory effects both in vitro and in vivo [32,33]. Moreover, TNF-α production is crucially required for the synergistic induction of NO synthesis in IFN-γ- and/or LPS-stimulated macrophages [34]. TNF-α elicits a number of physiological effects such a septic shock, inflammation, cachexia and cytotoxicity; IL-6 is believed to be an endogenous mediator of LPS-induced fever [35]. Therefore, the inhibition of the pro-inflammatory cytokines has been identified as targets for anti-inflammatory therapies. Since RMO was found to potently inhibit the pro-inflammatory mediators NO and PGE₂, we investigated its effects on LPS-induced TNF-α, IL-1β and IL-6 releases; this was done by enzyme immunoassay (EIA) and Western blot. As shown in Figure 3A,B, RMO concentration-dependently reduced the production of TNF-α, IL-1β and IL-6 and the corresponding protein expression levels.

Extracellular stimuli including LPS induce IKK/IκB/NFκB pathway to stimulate the production of inflammatory cytokines and pro-inflammatory enzymes [36,37,38]. iNOS and COX-2 genes are known to contain NFκB-binding sites in their promoter region [39]. Under unstimulated condition, NFκB is sequestered in the cytosol, associated with the inhibitory IκB protein. The activation of IKKs in response to extracellular stimuli leads to rapid phosphorylation and degradation of IκB, which makes NFκB translocate to the nucleus where it regulates gene transcription [40,41,42]. We examined whether the inhibitory effects of RMO on the production of inflammatory markers including NO, PGE₂, TNF-α, IL-1β and IL-6 are related to its blockade of the NFκB signaling pathway. Thus, we first assessed regulatory ability of RMO on the phosphorylation of p65 and IκBα. As illustrated in Figure 4A, LPS strongly induced the phosphorylation of p65 and IκBα whereas RMO dose-dependently inhibited these phosphorylations. Phosphorylation of the upstream kinase IKKα/β was also similarly diminished by RMO treatment but the protein level of IKKα/β was not affected by RMO. Therefore, RMO seems to reduce the production of inflammatory markers through inhibiting phosphorylations of IKK/IκB/NFκB signaling pathways.

We next investigated whether RMO could inhibit the nuclear translocation of p65 after its release from IκB using Western blot and confocal microscopy analyses. Upon exposure of cells to LPS, p-p65 was accumulated in the nuclear fraction whereas the nuclear accumulation of p-p65 was reduced to less than half after RMO treatment (Figure 4B). For the cytosolic fractions, the results were opposite. These results were confirmed by immunofluorescence staining assay and the results are displayed in Figure 4C. Cytoplasmic retention of p-p65 was observed in cells treated with RMO, while translocation of p-p65 into the nucleus was observed in LPS-induced cells. Taken together, these results clearly demonstrate that RMO inhibits NFκB activation by preventing the LPS-stimulated nuclear translocation of p-p65 in RAW 264.7 cells. Many studies have suggested that certain phytochemicals could suppress inflammatory responses by regulating the NFκB pathway [43,44,45]. These findings concur with our finding that the transcriptional inhibition of pro-inflammatory mediators by RMO is associated with the IKK/NFκB signal pathway.

Various intracellular pathways are involved in inflammatory actions through up-regulating the production of major mediators on inflammation [46]. Three families of MAPKs including ERK, JNK and p38 play critical roles in cell growth regulation and differentiation and in the control of cellular responses to cytokines and stressors [47]. MAPK phosphorylation activates the transcription of NFkB-mediated pro-inflammatory cytokines [48]; Therefore, MAPKs are often considered as molecular targets for the development of novel anti-inflammatory phytochemical. We investigated MAPKs phosphorylations in the presence or absence of RMO in LPS-stimulated cells (Figure 5A). The phosphorylations of ERK, JNK, and p38 were elevated when cells treated with LPS alone. RMO exclusively repressed p38 phosphorylation but did not affect ERK and JNK phosphorylations. p38 pathway inhibition has been reported to decrease pro-inflammatory cytokines production in monocytes/macrophages via transcriptional and post-transcriptional regulation [49].

Furthermore, the activation of MAPKs is known to require both tyrosine and theronine phosphorylation by the activated MAPKKs (MEK1/2, SEK1/MKK4, MKK7 or MKK3/6). Among the MAPKs, p38 MAPK is activated by dual phosphorylation on Thr180 and Tyr182 by upstream MAPK kinases: MAP2K6 or MAP2K3 (MKK3/6), which are activated by upstream MAPKKKs, and stimulated by a variety of stimuli. A MAPKK-independent mechanism of p38 activation involves transforming growth factor-β-activated protein kinase 1 (TAK1)-binding protein 1 (TAB1) [50,51]. To investigate the upstream kinases of p38 MAPK, we examined the modulatory effects of RMO on the phosphorylations of MAPKKs (MKK3/6, MEK1/2, MKK4, and MKK7) and MAPKKKs (TAK1, B-Raf, ASK1, and MLK3). RMO markedly suppressed the phosphorylations of MKK3/6 and TAK1 (Figure 5B,C), suggesting an involvement of RMO in TAK to MKK3/6 to p38 signaling pathways (Figure 6). Overall, our results imply that RMO might exert its anti-inflammatory ability through inhibiting p38 pathway which leads to NFκB inactivation and thereby suppressing expressions of inflammatory target proteins including proinflammatory cytokines and enzymes. Similar results have been reported with chemopreventive phytochemicals such as stercurensin [15], quercetin [52] and carnosol [53] which inhibit pro-inflammatory enzymes by targeting MAPK signaling pathways, including the p38 kinase pathway.

Phytosterols are plant sterols that are found in seeds, roots, stems, branches, leaves and blossoms of various plants, including medicinal herbs, edible plants, shrubs and trees [54]. The three major phytosterols in plants include sitosterol, campesterol and stigmasterol [55]. It has been widely reported that phytosterols lower serum cholesterol level in animals and humans [54]. Besides the hypocholesterolemic effects, phytosterols have recently been reported to have anti-inflmmatory properties through the inhibition of proinflammatory cytokines including IL-6 and TNF-α [55]. As a part of investigations on active ingredients in RMO, we analyzed the content and composition of RMO using gas chromatography (GC) analysis. As shown in Table 1, RMO contained about 108 mg of phytosterols per g RMO and sitosterol was by far the most abundant phytosterol which accounts for 84% of total phytosterols in RMO. So far, only a few studies have reported phytosterols in ginseng. Sitosterol has been found from the fruit of ginseng [56]. Beveridge et al. have reported phytosterol content in the seed oil of American ginseng [57]. In the ginseng seed oil, sitosterol was the most abundant phytosterol followed by stigmasterol and campesterol. There has been no report on the phytosterol content or composition of red ginseng. Although there have been positive correlations between phytosterols and anti-inflammation, it is not clear whether the anti-inflammatory properties of RMO come from its phytosterols at this moment. More studies on the anti-inflammatory mechanisms of RMO phytosterols and studies on the presence of other possible active ingredients should be followed. Volatile compounds profiles, other than the phytosterols, of RMO are under investigation and to be reported separately.

LPS (Escherichia coli O127:B8), Triton X-100 and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

The supercritical CO₂ extract preparation was performed according to our previous report with some modifications [27]. In brief, dried red ginseng byproduct powder was placed into the extraction vessel of a pilot-scale supercritical fluid extraction system (Ilshin Autoclave Co., Ltd., Daejeong, Korea). Extractions with supercritical CO₂ were operated at 6,500 psi (relative to 450 bar) in combination with temperature at 65 °C. Extracted constituents were collected in a vial that was prefilled with a trapping solvent and maintained at 4 °C during the extraction step.

RAW264.7 murine macrophage cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured at 37 °C and 5% CO₂ in Dulbecco’s modified Eagles’s medium (DMEM) containing 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin (Hyclone, Logan, UT, USA).

The MTT [3-(4,5-dimethylthioazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay was performed with a CellTiter 96 aqueous nonradioactive cell proliferation assay kit (Promega Corp., Madison, WI, USA) according to the manufacturer’s instructions. Briefly, the cells were plated on 24-well plates at a density of 1 × 10⁵ cells/well. After 24 h of incubation, the cells were treated with different doses of each sample for 48 h. Then, media were removed, and culture media containing MTS and phenazine methosulfate solution were added. After 1 h, the absorbance was measured at 490 nm with a PowerWave XS microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). The data was expressed as percent cell viability compared to the vehicle control.

Nitrite, as an indicator of NO synthesis, was determined in cell culture supernatants by the Griess reaction. Briefly, RAW 264.7 cells were plated at 1 × 10⁵ cells/well in 24-well plates and incubated at 37 °C for 24 h and then treated with RMO with LPS. After incubation of cells for 24 h, the supernatants (0.1 mL) were added to a solution of 0.1 mL Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylene diaminedihydrochloride in 5% H₃PO₄) to form a purple azo dye. Using NaNO₂ to generate a standard curve, nitrite production was measured by spectrophotometry at 550 nm using a microplate reader (BioTek Instruments, Inc.).

The amount of PGE₂ produced from endogenous arachidonic acid was measured using a PGE₂ Parameter Assay Kit (R&D Systems, Minneapolis, MN, USA). RAW264.7 cells (1 × 10⁵ cells/well) were treated with different concentration of RMO for 1 h and stimulated with LPS for 24 h, the conditioned media was collected to perform PGE₂ enzyme immune-metric assay according to the manufacturer’s protocol. The concentration of PGE₂ was calculated according to the equation obtained from the standard curve plot using PGE₂ standard solution in the EIA kit.

RAW 264.7 cells were plated at 1 × 10⁵ cells/well, and stimulated with LPS for 24 h in the presence or absence of RMO. Culture supernatants were collected and the amount of TNF-α and IL-6 was determined by EIA using a protocol supplied by Amersham (Amersham Pharmacia Biosciences, Piscataway, NJ, USA).

After LPS stimulation (1 μg/mL) of RMO-exposed cells for 24 h, total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA using SuperscriptTM RNase H reverse transcriptase (Invitrogen) according to the manufacturer’s recommendations. Subsequent PCR analysis was carried out with aliquots of the cDNA preparation using a PCR System (Corbett Research, Sydney, Australia). The PCR conditions were as follows: predenaturation at 94 °C for 30 s, annealing at 55 °C (COX-2 and GAPDH) or 59 °C (iNOS) for 30 s and extension at 72 °C for 30 s. The PCR products were visualized in 2% agarose gels and documented using a Gel-Doc EQ system (Bio-Rad, Hercules, CA, USA). The primer sequences were as follows: mouse iNOS, 5'-GCC TTC AAC ACC AAG GTT GTC TGC A-3' (sense), 5'-TCA TTG TAC TCT GAG GGC TGA CAC A-3' (anti-sense); mouse COX-2, 5'-CTG GTG CCT GGT CTG ATG ATG-3' (sense), 5'-GGC AAT GCG GTT CTG ATA CTG-3' (anti-sense); mouse GAPDH (as an internal control for PCR), 5'-CAA TGC CAA GTA TGA TGA CAT-3' (sense), 5'-CCT GTT ATT ATG GGG GTC TG-3' (anti-sense).

RAW 264.7 cells grown at 2 × 10⁵ cells/well in 6-well plates were stimulated with RMO for 1 h following pretreatment in the presence of absence of 1 μg/mL LPS for various time periods. Whole-cell extracts were then prepared according to the manufacturer’s instructions using Cell lysis Buffer containing 20 mM Tris (pH 7.5), 135 mM NaCl, 2 mM EDTA, 2 mM DTT, 25 mM β-glycerophosphate, 2 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, 10 mM NaF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mM PMSF. The cytosolic and nuclear extracts were prepared as above using a kit (Pierce Biotechnology Inc, Rockford, IL, USA). The protein content was quantified by absorbance at 590 nm according to the BCA protein assay (Pierce Biotechnology).

Proteins (whole-cell extracts: 30 μg/lane, nuclear extracts: 10 μg/lane, cytosolic extracts: 30 μg/lane) were separated by electrophoresed on a 10% SDS-polyacrylamide gel, and transferred to PVDF membranes with a semidry transfer system (Bio-Rad). The membranes were blocked with 5% nonfat milk in PBST with 0.1% Tween 20 for 1 h at room temperature, and then incubated overnight with primary antibodies. After hybridization with primary antibodies, the membrane was washed five times with PBST for 5 min, then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at RT and washed five times with PBST for 5 min. Final detection was performed with Western Blotting Luminol reagents (SantaCruz Biotechnology, Santa Cruz, CA, USA). Monoclonal antibodies against IKK β, p-IκBα, IκBα, p-NFκB-p65, and ERK and polyclonal antibodies against TNF-α, NFκB-p65, p-IKK α/β, p-EKR, p-p38, p38, p-SAPK/JNK, SAPK/JNK, IKKα, p-MEK1/2, p-MKK3/MKK6, p-B-Raf, MLK3, SEK1/MKK4, p-MKK7, p-ASK1 and p-TAK1 were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The polyclonal antibodies against iNOS, COX-2, IL-1 β, IL-6, Lamin B and β -actin were purchased from Santa Cruz Biotechnology, Inc.

RAW264.7 cells were planted on the coverglass bottom dishes. After 24 h, the cells (2 × 10⁵cells/well) were pretreated with 100 μg/mL RMO for 2 h and then stimulated with 1 μg/mL LPS for 1 h. The cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 10 min, blocked with 3% BSA in PBS for 2 h, incubated with an anti-p65 primary antibody at room temperature for 2 h, sequentially incubated with an Alexa Fluor 555-conjugated secondary antibody (Cell Signaling) at room temperature for 2 h in the dark, and finally incubated with 1 μg/mL of 4',6'-diamidino-2-phenylindole (DAPI) at room temperature for 20 min in the dark. Images were obtained using a LSM 510 laser confocal microscope (Zeiss, Jena, Germany).

Ten g of RMO were saponified in a round-bottom flask containing 0.1 N ethanolic KOH (10 mL) and ethanol (40 mL). The mixture was refluxed for 1 h on a heating mantle at 95 °C. After saponification, the mixture was cooled to room temperature, mixed with saturated NaOH solution (50 mL) and transferred to a separatory funnel. The unsaponifiables were extracted 4 times with 100-mL portions of hexane. The combined hexane extracts were concentrated to dryness using a rotary evaporator and 25 mL of an internal standard solution (5α-cholestane, 1 mg/mL) was added to the concentrate. The unsaponifiables were analyzed by an Agilent 6890 gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a DB5 MS column (Agilent) and a flame ionization detector. The column temperature was programmed from 200 °C for 2 min, 200 °C to 300 °C at 5 °C/min, and held for 5 min. Nitrogen gas was the carrier gas at a flow rate of 3 mL/min and the spilt ratio was 10:1. The injection port and the detector temperatures were 270 °C and 300 °C, respectively.

The data were expressed as mean ± S.D. values. The values were compared with the control using analysis of variance followed by unpaired Student’s t tests. A p value of <0.05 was considered significant.