Red Ginseng Marc Oil Inhibits iNOS and COX-2 via NFκB and p38 Pathways in LPS-Stimulated RAW 264.7 Macrophages

In this study, we investigated the anti-inflammatory effects of red ginseng marc oil (RMO) in the RAW 264.7 macrophage cell line. RMO was prepared by a supercritical CO2 extraction of waste product generated after hot water extraction of red ginseng. RMO significantly inhibited the production of oxidative stress molecules such as nitric oxide and reactive oxygen species in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Levels of inflammatory targets including prostaglandin E2, tumor necrosis factor-α, interleukin (IL)-1β and IL-6 were also reduced after the treatment with RMO. In addition, RMO diminished the expressions of inducible nitric oxide synthase and cyclooxygenase 2 at both mRNA and protein levels. Blockade of nuclear translocation of the p65 subunit of nuclear factor κB (NFκB) was also observed after the treatment of RMO. Furthermore, RMO decreased the phosphorylations of p38 mitogen-activated protein kinase (MAPK) and its upstream kinases including MAPK kinases 3/6 (MKK3/6) and TAK 1 (TGF-β activated kinase 1). Gas chromatographic analysis on RMO revealed that RMO contained about 10% phytosterols including sitosterol, stigmasterol and campesterol which may contribute to the anti-inflammatory properties of RMO. Taken together, these results suggest that the anti-inflammatory effect of RMO in LPS-induced RAW 264.7 macrophages could be associated with the inhibition of NFκB transcriptional activity, possibly via blocking the p38 MAPK pathway.


Introduction
Inflammation, a biological response to physical or chemical injurious stimuli, is closely associated with the release of pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines such as interleukins (ILs) and tumor necrosis factor α (TNF-α) [1]. The expression of these inflammation-related genes is controlled by the intracellular signaling pathways such as nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs) at both transcriptional and post-transcriptional levels [2,3].
NFκB is a pivotal transcription factor regulating various genes expression involved in immune and acute phase inflammatory responses [4]. The transcription factor NFκB forms a cytoplasmic complex with its inhibitors, IκBs, under normal physiological conditions. Stimulation of inflammatory cells with lipopolysaccharide (LPS), TNF, irradiation or viral infection results in the activation of Toll-like receptor 4 and downstream inhibitor κB kinases (IKKs) which in turn phosphorylates IκB and leads to NFκB translocation into the nucleus where it regulates transcription of its target genes [5]. Recently, many studies have demonstrated the roles of phytochemicals in anti-inflammatory activity through down-regulation of the NFκB pathway [6][7][8][9].
Activation of the NFκB signaling pathway is closely linked to the activations of MAPKs, which activate downstream transcription factors that promote inflammatory gene expression [10,11]. Furthermore, MAPKs are phosphorylated by upstream MAPK kinases (MAPKKs), which are dual specificity kinases that can phosphorylate threonine and tyrosine residues, and MKKs are in turn activated by MAPK kinase kinases (MAPKKKs), which are related to serine/threonine protein kinases [12]. These activated transcription factors regulate various pro-inflammatory cytokines and mediator factors regulate various pro-inflammatory cytokines and mediator genes including iNOS, COX-2, interleukin and TNF-α [13][14][15].
Ginseng (Panax ginseng) has been used as a traditional remedy in oriental medicine. Ginseng in Korea is classified into three categories, including fresh, white and red ginseng [16]. Red ginseng has been heat steamed and dried. As a consequence of this process, red ginseng undergoes certain biochemical chances and acquires certain pharmacological properties such as antioxidant [9], anti-viral [17], anti-aging [18], anti-depressant [14], anti-obesity [19], anti-carcinogenic [20] and hepatoprotective [21] effects. These beneficial effects of red ginseng have been mostly studied using its water-soluble fractions, probably because red ginseng is widely consumed in the form of hot-water extract or its concentrates. Therefore, we prepared red ginseng marc oil (RMO) by supercritical CO 2 extraction of waste-products generated after hot water extraction of red ginseng. In recent years, there has been an increasing interest in extracted essential oils and lipid-soluble bioactive compounds from various herbs and plants compared to water-soluble bioactive compounds. Newly discovered properties of essential oils include antibacterial [22], antifungal [23], antiobesity [24], antioxidant [25] and anti-inflammatory activities [26]. The pharmacological properties of essential oils extracted from plants have been the focus of interest from both academia and the pharmaceutical industry. Recently, our research group documented the antioxidant and hepatoprotective effects of RMO in H 2 O 2 -treated HepG2 cell and CCl 4 -treated mice [27]. In the present study, we show that RMO from red ginseng byproducts has anti-inflammatory activity and elucidate its underlying molecular mechanism for the first time in LPS-induced RAW264.7 cells.

Effects of RMO on Cell Viability
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.

RMO Inhibits NO and PGE 2 Production by Suppressing the Expression of iNOS and COX-2 in LPS-Stimulated RAW 264.7 Cells
The inflammatory products NO and PGE 2 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 PGE 2 over-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 PGE 2 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 PGE 2 by down-regulating the expression of iNOS and COX-2, which could be activated by inflammatory stimuli such as LPS.

RMO Inhibits the Protein Expression of Pro-Inflammatory Cytokines in LPS-Stimulated RAW 264.7 Cells
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 PGE 2 , 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.

RMO Suppresses IKK/IκB/NFκB Signals and NFkB Nuclear Translocation in LPS-Induced in LPS-Stimulated RAW 264.7 Cells
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, PGE 2 , 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.

RMO Inhibits Phosphorylation of MAPK, MAPKK, and MAPKKK in LPS-Stimulated RAW 264.7 Cells
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].

Phytosterols in RMO
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.

Preparation of RMO
The supercritical CO 2 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 CO 2 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.

Analysis of Cell Viability
The MTT [3-(4,5-dimethylthioazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, 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 × 10 5 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.

Measurement of Nitrite Oxide Formation
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 × 10 5 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% H 3 PO 4 ) to form a purple azo dye. Using NaNO 2 to generate a standard curve, nitrite production was measured by spectrophotometry at 550 nm using a microplate reader (BioTek Instruments, Inc.).

Measurement of PGE 2 Production
The amount of PGE 2 produced from endogenous arachidonic acid was measured using a PGE 2 Parameter Assay Kit (R&D Systems, Minneapolis, MN, USA). RAW264.7 cells (1 × 10 5 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 PGE 2 enzyme immune-metric assay according to the manufacturer's protocol. The concentration of PGE 2 was calculated according to the equation obtained from the standard curve plot using PGE 2 standard solution in the EIA kit.

Measurement of TNF-α and IL-6 Production
RAW 264.7 cells were plated at 1 × 10 5 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).

Preparation of Whole-Cell, Cytoxolic, and Nuclear Extracts
RAW 264.7 cells grown at 2 × 10 5 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. Wholecell 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).

Confocal Microscopy Analysis
RAW264.7 cells were planted on the coverglass bottom dishes. After 24 h, the cells (2 × 10 5 cells/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).

Analysis of Phytosterol Content
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.

Statistical Analysis
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.

Conclusions
RMO suppressed the LPS-stimulated inflammatory responses including NO and PGE2 generation, secretion of pro-inflammatory cytokines of TNF-α, IL-1β and IL-6 and the expression of pro-inflammatory mediators such as iNOS and COX-2 in RAW264.7 macrophages. Furthermore, RMO enhanced the inhibitory effects on inflammation via inactivation of NFκB and p38/ MKK3/6/TAK1 signaling pathways. RMO contained about 10% phytosterols including sitosterol, stigmasterol and campesterol which may contribute to the anti-inflammatory properties of RMO. Taken together, our data suggest 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. Our findings suggest for the first time the potential of red ginseng marc oil as a potent natural anti-inflammatory agent.