S. nodiflora was collected from the Slamet Mountains, Central Java, Indonesia. Plant samples were collected and identified by staff at the Center for Pharmaceutical and Medical Technology (PTFM), and verified at the Herbarium Bogoriense (LIPI). Voucher specimens recorded as KRIB 0039477 and PMT 1171, were deposited in the herbarium (KRIB) of the Korean Research Institute of Bioscience and Biotechnology (Daejeon, Korea) as well as in the Center for Pharmaceutical and Medical Technology (PTFM) and the Herbarium Bogoriense. The extract was deliquesced in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA) and added to the culture media to the final concentration as indicated. It was confirmed that S. nodiflora is not a protected or endangered species (37,38).

RAW 264.7 macrophages were purchased from ATCC. RAW 264.7 cells were cultured under the following condition: 10% fetal bovine serum (FBS, Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Inc.) in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C in 5% CO₂. LPS for activation of RAW 264.7 cells was purchased from Sigma-Aldrich; Merck KGaA.

RAW 264.7 cells were seeded in 96-well plates (4.5x10⁴ cells/well), pre-treated with MSN (100, 200, 300, 400 and 600 µg/ml) for 2 h, and then incubated with LPS (1 µg/ml) at 37˚C for 24 h. The cell viability was measured using the EZ-Cytox cell viability assay kit (Daeil Tech Co., Ltd.) according to the manufacturer's instructions. Cell viability was calculated following the absorbance for viable cells at 450 nm and reference absorbance at 650 nm (A₄₅₀-A₆₅₀) with the Synergy H1 Microplate Reader (BioTek Instruments, Inc.).

Cells (4.5x10⁴ cells/well; 96-well plate) were incubated with MSN (100, 200, 300 and 400 µg/ml) for 2 h and then with LPS (1 µg/ml) at 37˚C for 24 h. Nitrite assay was performed as described in a previous study (39).

RAW 264.7 cells (2x10⁵ cells/well; 12-well plate) were pre-treated with MSN (100, 200, 300 and 400 µg/ml) for 2 h and activated by LPS (1 µg/ml) for 3 h at 37℃. Total RNA preparation, cDNA synthesis, and quantification of mRNA were performed as previously described (39). Quantification of gene expression was analyzed using the 2^-∆∆Cq method (40). Calculated gene expression was normalized to reference genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-actin. The sequences of the PCR primers are listed in a previous study (41).

RAW 264.7 macrophages were seeded in 96-well plates (4.5x10⁴ cells/well) and incubated at 37˚C overnight. The cells were pre-treated with MSN (100, 200, 300 and 400 µg/ml) for 2 h and then incubated with LPS (1 µg/ml) at 37˚C for 24 h. Culture supernatants were collected by centrifugation at 1,500 x g for 1 min at room temperature (RT). ELISA kit for the detection of IL-6 (cat. no. 88-7064) and TNF-α (cat. no. 88-7324) were from eBioscience (Thermo Fisher Scientific, Inc.). TNF-α and IL-6 levels in cell supernatants were measured using sandwich ELISA with monoclonal antibodies specific to each mediator according to the manufacturer's instructions. Briefly, a 96-well-ELISA plate was pre-coated with the capture antibody at 4˚C for overnight. The plate was washed 4 times with 1X phosphate-buffered saline (PBS)/5% Tween 20 (PBST) and blocked with 1X assay diluent at RT for 1 h. Then, 100 µl of the sample was added to each well and incubated at RT for 2 h. Subsequently, a biotinylated detection antibody solution was added at RT for 1 h. After this, the plate was treated with HRP-streptavidin solution at RT for 30 min, and then 100 µl of 3,3',5,5'-tetramethylbenzidine was added for further incubation at RT for 10 min in the dark. The further reaction was blocked by adding 50 µl of 1N H₃PO₄. The absorbance was measured (450 nm) with a Synergy H1 Microplate Reader. The concentrations of the secreted cytokines were calculated based on a standard curve.

RAW 264.7 cells were treated with MSN (100, 200, 300 and 400 µg/ml) at 37˚C for 2 h and then stimulated with LPS (1 µg/ml) at 37˚C for 3 min (for IκBα, Src, Syk, Akt, TAK1), 15 min (for MAPKs), or 24 h (for iNOS and COX-2), and subsequently washed twice with cold PBS (pH 7.4). Cells were collected and lysed in lysis buffer containing 150 mM NaCl, 20 mM trisaminomethane hydrochloride (Tris-HCl) (pH 8.0), 0.5% IGEPAL CA-630 (NP-40), 1 mM ethylenediaminetetraacetic acid, 0.5% Triton X-100, 1% glycerol, 10 mM sodium fluoride, 2 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate. The lysates were centrifuged at 15,814 x g at 4˚C for 30 min. Supernatants were transferred to a new tube.

Briefly, the protein concentration was measured using Bradford protein assay (Bio-Rad) according to the manufacturer's instructions. Aliquots of cell lysates were mixed with 5X sodium dodecyl sulfate (SDS)-polyacrylamide sample buffer including 12 mM Tris-HCl (pH 6.8), 5% glycerol, 1% β-mercaptoethanol, 0.4% SDS, and 0.02% bromophenol blue and boiled at 95˚C for 5 min. Samples were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (GE Healthcare). The membranes were blocked with 5% nonfat-dried skim milk in 1X TBST solution, followed by incubation at 4˚C overnight with the following primary antibodies: Mouse polyclonal anti-p38 (cat no. sc-7972), mouse monoclonal anti-c-Jun N-terminal kinase (JNK; cat no. sc-7345), rabbit polyclonal phosphorylated (p)-anti-IκBα (Ser32/36; cat no. sc-101713), mouse polyclonal anti-spleen tyrosine kinase (Syk; cat no. sc-1240), mouse monoclonal anti-c-proto-oncogene tyrosine-protein kinase Src (c-Src; cat no. sc-19), rabbit polyclonal anti-p-c-Src (Tyr424; cat no. sc-81521), rabbit polyclonal anti-Akt1/2/3 (cat no. sc-8312), rabbit polyclonal anti-p-Akt1/2/3 (Ser473; cat no. sc-7985), goat polyclonal anti-cyclooxygenase 2 (COX-2; cat no. sc-1745), rabbit polyclonal anti-inducible NO synthase (iNOS; cat no. sc-651) and mouse monoclonal anti-α-tubulin (cat no. sc-5286) antibodies were purchased from Santa Cruz Biotechnology, Inc. Rabbit polyclonal transforming growth factor-β-activated kinase 1 (TAK1; cat no. 4505), rabbit monoclonal anti-p-TAK1 (Thr184/187; cat no. 4508), rabbit polyclonal anti-p-p38 (Thr180/Tyr182; cat no. 9211), rabbit polyclonal anti-extracellular signal-regulated kinase (ERK; cat no. 9102), rabbit monoclonal anti-p-ERK (Thr202/Tyr204; cat no. 9106), rabbit polyclonal anti-p-JNK (Thr183/Tyr185; cat no. 9252) and rabbit polyclonal anti-p-Syk (Tyr525/526; cat no. 2711) antibodies were purchased from Cell Signaling Technology, Inc. All primary antibodies were diluted to 1:1,000 in 5% non-fat dried milk. Each membrane was washed 4 times with 1X TBST and incubated with the following secondary antibodies: Polyclonal anti-rabbit IgG-HRP (1:5,000; cat no. LF-SA8002) and polyclonal anti-mouse IgG Fc-HRP (1:5,000; cat no. LF-SA8001) were from AbFrontier; Young In Frontier Co., Ltd., at RT for 2 h. Each protein level was visualized using an enhanced chemiluminescence (ECL) immunoblotting detection reagent (Pierce; Thermo Fisher Scientific, Inc.). Protein levels were scanned and analyzed with LabWorks v.4.0 software (UVP Inc.).

All experiments were performed 3 times. The results are represented as means ± standard error of the mean (SEM). Differences between experimental conditions were assessed using one-way ANOVA followed by Tukey's test in Prism v.3.0 (GraphPad Software, Inc.). P<0.05 was concidered to indicate a statistically significant difference.

Since ethnopharmacological records indicate that S. nodiflora has been shown to exhibit potent anti-inflammatory effects (42), MSN was evaluated to identify its molecular mechanisms of action in the present study. To determine the maximal non-cytotoxic concentration of MSN, a cell viability assay was performed. RAW 264.7 cells were treated with MSN in the presence or absence of LPS. The cell viability was not altered until treatment with 400 µg/ml of MSN (Fig. 1A). When cells were treated with 600 µμg/ml of MSN, cell viability was significantly reduced. Therefore, concentrations <400 µg/ml of MSN were used in the subsequent experiments.

NO is an essential cellular signaling molecule involved in diverse physiological and pathological processes in mammals (43). In the inflammatory response, NO levels are increased due to induced iNOS in cells, and the produced NO plays a dual role in immune and inflammatory responses (44). To investigate the anti-inflammatory effects of MSN, the MSN-regulated NO expression was examined in LPS-stimulated macrophages that showed inflammatory responses. MSN effectively inhibited NO production in a dose-dependent manner (Fig. 1B). Since iNOS is the essential enzyme of NO production, iNOS expression at the mRNA and protein levels following MSN treatment was evaluated. RT-qPCR analysis showed that upregulated iNOS expression by LPS stimulation was suppressed following MSN treatment (Fig. 1C). In addition, immunoblot analysis showed that increased protein expression of iNOS by LPS treatment was dose-dependently reduced by MSN (Fig. 1D). These results suggest that MSN negatively regulates NO production by suppression of iNOS at the transcriptional level.

Since COX-2 catalyzes the production of PGE₂, which causes fever, diarrhea, and excessive uterine contraction (45), the anti-inflammatory effects of MSN were assessed by evaluating mRNA and protein expression of COX-2. LPS-induced expression of COX-2 gene and COX-2 protein was suppressed by MSN in a dose-dependent manner (Fig. 2A and B). Therefore, these results suggest that MSN regulates COX-2 in LPS-activated macrophages through the inhibition of the COX-2 gene and COX-2 protein expression.

To further investigate the anti-inflammatory effects of MSN, the production of pro-inflammatory cytokines, which are induced by LPS in macrophages, was measured in the presence or absence of MSN. As shown in Fig. 2C and D, pro-inflammatory cytokines including IL-6 and TNF-α were significantly increased upon stimulation with LPS and decreased by MSN treatment. The effect of MSN on the gene expression of pro-inflammatory cytokines, including IL-6, IL-1β, and TNF-α, was also analyzed using RT-qPCR. The mRNA expression of the pro-inflammatory cytokine genes was inhibited by MSN in LPS-activated RAW 264.7 macrophages (Fig. 2E). Taken together, these results suggest that MSN suppresses the LPS-induced production of pro-inflammatory cytokines by inhibition of IL-6, IL-1β, and TNF-α transcription.

NF-κB and mitogen-activated protein kinase (MAPK) regulate the genes involved in inflammatory responses of RAW 264.7 cells (46). Phosphorylation at Ser32/36 of IκBα was found to lead to the degradation of IκBα bound to NF-κB, and freed and activated NF-κB translocated to the nucleus, inducing transcription of target inflammatory genes (47). We, therefore, measured the phosphorylation levels at Ser32/36 of IκBα and the protein level of IκBα to examine whether MSN caused a change in IκBα phosphorylation and protein levels. As presented in Fig. 3A, LPS induced the phosphorylation and degradation of IκBα, while the phosphorylation of IκBα decreased and IκBα levels increased in the MSN-treated groups. These results suggest that MSN inhibits NF-κB activation through the change in IκBα phosphorylation.

Since MAPK signaling, another LPS-induced inflammatory signaling pathway, regulates the production of inflammatory cytokines and mediators (48), the regulation of MAPK signaling by MSN was investigated in the present study. Phosphorylation levels of p38, JNK, and ERK in their activation loop were increased in RAW 264.7 cells upon exposure to LPS (Fig. 3B). When the cells were treated with MSN prior to LPS stimulation, the levels of p-ERK, p-JNK, and p-p38 were not altered, suggesting that MSN has little effect on MAPK signaling.

TAK1 is an upstream signaling kinase of NF-κB and MAPK signaling transduction (49). In addition, the TAK1-independent Syk/Src/Akt signaling pathway plays a vital role in NF-κB activation (50). The effect of MSN on the phosphorylation of TAK1/Syk/Src/Akt at their activation sites was investigated by immunoblot analysis to identify the targets of MSN in the regulation of NF-κB activation. The LPS-induced phosphorylation of Syk and Akt was reduced by MSN without changing the protein levels, whereas the phosphorylation of TAK1 and Src was not affected by MSN (Fig. 3C). These results suggest that MSN suppresses the phosphorylation of Syk and its downstream factors of the NF-κB pathway independently of TAK1 and Src.