A 95% ethanol extract (Code no.: FBM124-035) of the Garcinia subelliptica Merr. (Clusiaceae) was purchased from the International Biological Material Research Center (Daejeon, Korea). The RAW 264.7 macrophages, a mouse monocytic cell line, were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.), 50 unit/ml penicillin, and 50 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in humidified air containing 5% CO₂. The cytokines that were produced, including TNF-α and IL-6, were measured using enzyme-linked immunosorbent assay (ELISA) Ready-SET-Go!^® kits (eBioscience, Inc., San Diego, CA, USA). Rabbit anti-IκBα (sc-371) and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778) polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Mouse anti-phosphoylated (p)-IκBα (Ser32/36; 9246) and anti-p-extracellular signal-regulated kinase 1/2 (ERK 1/2; 9106) monoclonal antibodies; and rabbit anti-iNOS (2982), anti-COX-2 (4842), anti-p-p38 (9211), anti-p38 (9212), anti-ERK 1/2 (9102), anti-p-c-Jun N-terminal kinase (JNK; 9251) and anti-JNK (9252) polyclonal antibodies; and goat anti-rabbit horseradish peroxidase (HRP)-conjugated immunoglobulin (Ig)G (7074) and horse anti-mouse HRP-conjugated IgG (7076) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

RAW 264.7 macrophages were seeded in 96 well plates (4×10⁴/well). Following adhesion overnight, cells were incubated with 20, 50, 100, 200, and 400 µg/ml EGS and 1 µg/ml LPS for 24 h. Following incubation, cell viability was measured using an EZ-Cytox Cell Viability Assay kit (Daeil Lab Services Co., Ltd., Seoul, Korea). Briefly, the EZ-Cytox solution that contained a water soluble tetrazolium salt was added to each well for 2 h at 37°C and 100 µl of supernatants were transferred to 96 well plates. The absorbance was measured at 450 nm with a Synergy H1 Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA).

RAW 264.7 macrophages were incubated with 20, 50, 100 and 200 µg/ml EGS and 1 µg/ml LPS for 24 h. Following incubation the levels of NO synthesis were determined by assaying the culture supernatants for nitrite using the Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine dihydrochloride, and 2.5% phosphoric acid). Nitrate is the stable product of the reaction between NO and molecular oxygen. The absorbance was measured at 540 nm, using a Synergy H1 Microplate Reader, following incubation for 10 min.

RAW 264.7 macrophages were stimulated with 1 µg/ml LPS and 20, 50, 100 and 200 µg/ml EGS for 24 h. Following stimulation, the supernatants were obtained via centrifugation at 2,339 × g for 3 min at 4°C, and a sandwich ELISA was performed according to the manufacturer's instructions, using monoclonal antibodies specific to each mediator to determine the quantity of TNF-α and IL-6 in the culture supernatants. Prior to the application of samples, the plate was pre-coated with the coating antibody in the supplied buffer. Following incubation overnight at 4°C, the plate was washed with 1X phosphate-buffered saline (PBS)-Tween 20 (0.5%) and treated with 1X assay diluents for 1 h. Samples were loaded into each well and incubated for 2 h at room temperature. Following washing, the plate was treated with a biotinylated secondary antibody solution and HRP-streptavidin solution for 1.5 h, and the substrate solution was added to the washed-plate. Following 10 min incubation in dark conditions, 1 N phosphoric acid (H₃PO₄) was added and the optical density of the individual wells was measured at 450 nm using a Synergy Microplate Reader.

RAW 264.7 macrophages were stimulated with 50 ng/ml LPS and 20, 50, 100 and 200 µg/ml EGS for 6 h. Total RNA was extracted from the cells via isoproponal precipitation using Accuzol reagent (Bioneer Corporation, Daejeon, Korea), reverse-transcribed into complementary DNA (cDNA) using a TOPscript™ cDNA synthesis kit (Enzynomics, Daejeon, Korea) and then PCR amplified. Quantification of mRNA was performed using a real-time PCR reagent, iTaq™ Universal SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's instructions. The PCR was run for 40 cycles of denaturation at 94°C (5 sec) and annealing/extension at 60°C (30 sec) using a CFX Connect™ Real-Time Thermal Cycler (Bio-Rad Laboratories, Inc.). According to the 2^−∆∆Cq method (36), the results were normalized to the reference genes, β-actin and GAPDH, and were expressed as the ratio of gene expressions to the LPS treated group (100%). PCR primers were designed using Beacon Designer 7.0 (Premier Biosoft, Palo Alto, CA, USA) and subsequently synthesized by Bioneer Corporation. The sequences of PCR primers used are as follows: Mouse iNOS, sense 5′-TGG CCA CCA AGC TGA ACT-3′ and antisense 5′-TCA TGA TAA CGT TTC TGG CTCTT-3′; COX-2, sense 5′-GAT GCT CTT CCG AGC TGTG-3′ and antisense 5′-GGA TTG GAA CAG CAA GGA TTT-3′; TNF-α, sense 5′-CTG TAG CCC ACG TCG TAGC-3′ and antisense 5′-TTG AGA TCC ATG CCG TTG-3′; IL-6, sense 5′-TCT AAT TCA TAT CTT CAA CCA AGAGG-3′ and antisense 5′-TGG TCC TTA GCC ACT CCTTC-3′; IL-1β, sense 5′-TTG ACG GAC CCC AAA AGAT-3′ and antisense 5′-GAT GTG CTG CTG CGA GATT-3′; β-actin, sense 5′-CGT CAT ACT CCT GCT TGCTG-3′ and antisense 5′-CCA GAT CAT TGC TCC TCC TGA-3′; and GAPDH, sense 5′-GCT CTC TGC TCC TCC TGTTC-3′ and antisense 5′-ACG ACC AAA TCC GTT GACTC-3′.

PCR primers were designed using Primer3 software (version 4.0.0), as previously described (37), and were subsequently synthesized by Bioneer Corporation. The sequences of the PCR primers used study are as follow: Mouse iNOS, sense 5′-GCA TGG AAC AGT ATA AGG CAA ACA-3′ and antisense 5′-GTT TCT GGT CGA TGT CAT GAG CAA-3′; COX-2, sense 5′-GCA TGG AAC AGT ATA AGG CAA ACA-3′ and antisense 5′-GTT TCT GGT CGA TGT CAT GAG CAA-3′; TNF-α, sense 5′-GTG CCA GCC GAT GGG TTG TACC-3′ and antisense 5-′AGG CCC ACA GTC CAG GTC ACTG-3′; IL-6, sense 5′-TCT TGG GAC TGA TGC TGG TGAC-3′ and antisense 5′-CAT AAC GCA CTA GGT TTG CCGA-3′; IL-1β, sense 5′-AGC TGT GGC AGC TAC CTGTG-3′ and antisense 5′-GCT CTG CTT GTG AGG TGCTG-3′; and GAPDH, sense 5′-GTC TTC ACC ACC ATG GAG AAGG-3′ and antisense 5′-CCT GCT TCA CCA CCT TCT TGCC-3′. The PCR was run for 20–25 cycles of 94°C (30 sec), 60°C (30 sec), and 72°C (30 sec) using a Genetouch thermal cycler (Bioer Technology Co., Ltd., Hangzhou, China). Following amplification, 10 µl of the RT-PCR products were separated in 1.5% (w/v) agarose gels and stained with ethidium bromide.

LPS-stimulated RAW 264.7 cells were treated with EGS and 1 µg/ml LPS for 15 min and washed with ice-cold PBS. The cells were lysed in lysis buffer containing 0.5% NP-40, 0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM ethylenediaminetetraacetic acid, 1% glycerol, 1 mM phenylmethylsulfonyl fluoride and 1 µg/ml aprotinin, and collected in microtubes prior to centrifugation at 15,814 x g for 30 min at 4°C. The supernatants were prepared in fresh microtubes.

Protein concentration was measured using the Bradford method (Bio-Rad Laboratories, Inc.). Aliquots (20 µg) of the cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel in a Mini-Protein II gel apparatus (both Bio-Rad Laboratories, Inc.) and transferred onto nitrocellulose membranes (GE Healthcare Life Sciences, Pittsburgh, WI, USA) with transfer buffer [192 mM glycine, 25 mM Tris-HCl (pH 8.8), and 20% MeOH (v/v)]. Following blocking of non-specific sites with 5% bovine serum albumin solution, the membrane was incubated overnight at 4°C with the primary antibodies (all 1:1,000). Subsequently, each membrane was incubated for 1 h at room temperature with secondary HRP-conjugated goat anti-rabbit or horse anti-mouse IgG (1:5,000). The target proteins were detected using an enhanced chemiluminescence solution (Daeil Lab Services Co., Ltd.). Protein levels were quantified by scanning the immunoblots and analyzing them with LabWorks software (version 4.6; UVP, Inc., Upland, CA, USA).

Data are presented as the mean ± standard error of the mean. Statistical differences between each result were assessed by comparing with the LPS-treated group using the Mann-Whitney U test. Mann-Whitney U test was performed using Prism 3.0 (GraphPad Software, Inc., San Diego, CA, USA) and P<0.01 was considered to indicate a statistically significant difference. The data from nine replicates were analyzed, including three independent experiments with three replicates in each.

Selective regulation of inflammatory mediators and the identification of specific target signaling molecules are valuable strategies for the development of novel anti-inflammatory reagents. Based on this concept, the current study investigated a number of natural plant extracts, which are known traditionally to have pharmacological effects, and evaluated their inhibitory effects on inflammation by measuring the production of NO, a major inflammatory mediator, in LPS-stimulated RAW 264.7 macrophages. Of the extracts that exerted an inhibitory effect on NO production, EGS was selected as Garcinia species have been used traditionally to treat numerous inflammatory diseases. Considering that the anti-inflammatory effects of EGS should be studied at concentrations that do not influence cell viability, the non-cytotoxic and maximally effective concentrations of EGS were identified by the dose-dependent treatment of LPS-stimulated RAW 264.7 macrophages. Cell viability was determined by measuring the ability of cells to metabolically reduce a water-soluble tetrazolium salt to a formazan dye. As shown in Fig. 1, the cell viability of the RAW 264.7 macrophages was not reduced by EGS treatment at doses of ≤200 µg/ml in the presence of 1 µg/ml LPS. However, a number of dead cells (31.79±3.14%) were detected when the cells were treated with 400 µg/ml EGS. These results suggest that a low dose of EGS (200 µg/ml) does not affect the viability of RAW 264.7 macrophages. Therefore, concentrations below 200 µg/ml were used in subsequent experiments.

To evaluate the anti-inflammatory function of EGS in macrophages, the profile of NO production was measured. As expected, RAW 264.7 macrophages stimulated with LPS (1 µg/ml) produced a large quantity of NO compared with the non-treated control group. When the cells were treated with EGS, the enhanced production of NO by LPS was inhibited in a dose-dependent manner, and the LPS-induced NO production was nearly abolished at a dose of 200 µg/ml (Fig. 2A). Considering that NO production is tightly regulated by iNOS expression, the effects of EGS on the expression of iNOS mRNA and protein were investigated using PCR and immunoblot analysis. The RT-qPCR results revealed that LPS notably induces iNOS expression, which was inhibited by EGS in a dose-dependent manner (Fig. 2B). Semi-quantitative PCR data indicated a similar profile of expression as the RT-qPCR data, which supported the inhibitory effect of EGS on the expression of iNOS mRNA (Fig. 2C). In addition, LPS-induced expression of iNOS protein was notably reduced by EGS in a dose-dependent manner, and the maximum dose of EGS resulted in a similar iNOS expression profile as the non-treated control group (Fig. 2D). These results imply that EGS inhibits NO production in activated macrophages by modulating iNOS expression.

As COX-2 is the enzyme responsible for the production of PGE₂ in activated macrophages, the present study explored the effect of EGS on the expression levels of COX-2 mRNA and protein to measure its anti-inflammatory function. As shown in Fig. 3A, induction of COX-2 mRNA expression by LPS was detected by RT-qPCR analysis. EGS treatment notably inhibited the enhanced expression of COX-2 mRNA at concentrations of 100 and 200 µg/ml. Furthermore, the inhibitory effect of EGS on the COX-2 mRNA expression detected by RT-qPCR was supported by semi-quantitative PCR (Fig. 3B). To examine the effect of EGS on the expression of COX-2 protein in activated macrophages, RAW 264.7 macrophages were treated with EGS in the presence of LPS, and total cell lysates were prepared. The increased levels of COX-2 protein expression mediated by LPS were reduced markedly by EGS in a dose-dependent manner (Fig. 3C). From these data, COX-2 expression was observed to be downregulated by EGS, and the subsequent production of PGE₂ may therefore be regulated by EGS.

To assess the anti-inflammatory properties of EGS in activated macrophages, the effects of EGS on the production of pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α, were measured in LPS-stimulated RAW 264.7 macrophages. Production of IL-6 and TNF-α was increased notably following LPS stimulation (Fig. 4A and B). However, EGS treatment inhibited the production of IL-6 whilst not altering the expression of TNF-α (Fig. 4A and B). The mRNA expression of pro-inflammatory cytokines was assessed to determine whether the production of pro-inflammatory cytokines is regulated at the transcriptional level. As presented in Fig. 4C, RT-qPCR analysis indicated that EGS treatment inhibited the LPS-stimulated expression of IL-6 and IL-1β but not TNF-α. Similarly, semi-quantitative PCR analysis showed that EGS selectively reduced the mRNA expression levels of IL-6 and IL-1β (Fig. 4D). Taken together, these results suggest that EGS negatively regulates the LPS-induced production of pro-inflammatory cytokines by transcriptional repression of IL-6 and IL-1β however, not of TNF-α.

The phosphorylation levels of JNK and IκB were measured to investigate which inflammatory signaling pathways are associated with the inhibitory effects of EGS in LPS-stimulated macrophages. As shown in Fig. 5A, LPS treatment altered the expression of p-IκBα and IκBα. However, EGS did not affect the expression profiles of p-IκBα and IκBα. In addition, the modulation of JNK phosphorylation by EGS was measured. Immunoblot analyses revealed that EGS inhibited the phosphorylation of JNK without affecting the protein levels of JNK. EGS did not influence the phosphorylation or protein expression of additional MAPK pathway mediators, including ERK and p38. Collectively, these results suggest that EGS exerts anti-inflammatory effects by inhibiting the activation of JNK.