Salidroside (chemical structure shown in Fig. 1) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO); the final concentration of DMSO was adjusted to <0.01% (v/v) in the culture medium. PMA and the calcium ionophore, A23187 (calcymycin; C₂₉H₃₇N₃O₆), were purchased from Sigma-Aldrich. The CellTiter 96^® AQueous One Solution Cell Proliferation assay (MTS) system was purchased from Promega (Madison, WI, USA). Iscove's modified Dulbecco's medium (IMDM) was obtained from Welgene (Daegu, Korea). Anti-human TNF-α (555212), anti-IL-6 (555220) and anti-IL-8 (555244) antibodies, biotinylated anti-human TNF-α (51-26372E), anti-IL-6 (51-26452E) and IL-8 (51-26542E) antibodies, and recombinant human TNF-α (51-26376E), IL-6 (51-26456E) and anti-IL-8 (51-26546E) antibodies were obtained from BD Pharmingen (San Diego, CA, USA). Anti-phosphorylated (p-)ERK1/2 (sc-7383), anti-p-JNK1/2 (sc-6254), anti-p-p38 (sc-7973), anti-ERK1/2 (sc-93), anti-JNK1/2 (sc-571), anti-p38 (sc-535), anti-β-actin (sc-47778), anti-NF-κB (sc-8008) and anti-rabbit antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The reverse transcription kit was purchased from Qiagen (Valencia, CA, USA), and nuclear and cytoplasmic extraction reagents were purchased from Thermo Scientific (Waltham, MA, USA).

The human leukemic mast cell line, HMC-1, was obtained from the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea) and grown in IMDM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 IU/ml penicillin, 50 μg/ml streptomycin and 1.2 mM α-thioglycerol at 37°C under 5% CO₂ in air.

For the analysis of cell viability by MTS assay, we used the manufacturer's procedure for the AG Protocol-CellTiter 96^® AQueous One Solution Cell Proliferation assay. Cell aliquots (5×10⁴) were seeded in microplate wells and treated with SAS (10, 25, 50 and 100 μM) for 30 min. The following day, the cells were incubated with 20 μl of MTS solution for 2 h at 37°C under 5% CO₂ and 95% air. An automatic microplate reader (Molecular Devices, Sunnyvale, CA, USA) was used to read the absorbance of each well at 490 nm.

The HMC-1 cells were treated with various concentrations of SAS (10, 25 and 50 μM) for 1 h prior to stimulation with PMA (50 nM) plus A23187 (1 μM). An ELISA was used to assay the protein levels of IL-6, IL-8 and TNF-8 in the culture supernatants. To measure the cytokine levels, we used a modified ELISA. First, we conducted a sandwich ELISA for IL-6, IL-8 and TNF-8 in triplicate in 96-well ELISA plates (Nunc, Roskilde, Denmark). The supernatant was then transferred to a new microcentrifuge tube, and the cytokines were quantified using ELISA. ELISA plates (Falcon; Becton-Dickinson Labware, Franklin Lakes, NJ, USA) were coated overnight at 4°C with anti-human IL-6, anti-IL-8 and anti-TNF-α monoclonal antibodies in coating buffer (0.1 M carbonate, pH 9.5) and then washed 3 imes with phosphate-buffered saline (PBS) containing 0.05% Tween-20. The non-specific protein binding sites were blocked with assay diluent (PBS containing 10% FBS, pH 7.0) for at least 1 h. After washing the plates again, the test sample or recombinant IL-6, IL-8 and TNF-α standards were added. Following incubation for 2 h, a working detector (biotinylated anti-human IL-6, anti-IL-8 and anti-TNF-α monoclonal antibodies and streptavidin-horseradish peroxidase reagent) were added followed by incubation for 1 h. The non-specific protein binding sites were blocked. Subsequently, substrate solution [tetramethylbenzidine (TMB)] was added to the wells followed by incubation for 30 min in the dark before the reaction was terminated by the addition of 1 M H₃PO₄. The absorbance was read at 450 nm. All subsequent steps were carried out at room temperature, and all standards and samples were assayed in triplicate.

Using an Easy Blue total RNA extraction kit (Intron Biotechnology, Gyeonggi-do, Korea) we isolated total RNA from the HMC-1 cells in accordance with the specifications of the manufacturer. The total RNA was dissolved in DEPC-treated distilled water. A spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) was used to assess RNA purity by measuring the ratio of the absorbance at 260 and 280 nm; only RNA samples with a value in the 1.6–2.0 range were used. A cDNA synthesis kit (Qiagen, Valencia, CA, USA) was used for 2 min at 42°C, 30 min at 42°C, and 30 min at 95°C to reverse transcribe each sample into cDNA. The primer sequences were as follows: IL-6 forward, 5′-GATGGATGCTTCCAATCTGGAT-3′ and reverse, 5′-AGTTCTCCATAGAGAACAACATA-3′; IL-8 forward, 5′-TGTGCTCTCCAAATTTTTTTTACTG-3′ and reverse, 5′-CTCTCTTTCCTCTTTAATGTCCAGC-3; TNF-α forward, 5′-CACCAGCTGGTTATCTCTCAGCTC-3′ and reverse, 5′-CGGGACGTGGAGCTGGCCGAGGAG-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward, 5′CCATGTTCGTCATGGGTGTGAACCA-3′ and reverse, 5′-GCCAGTAGAGGCAGGGATGATGTTC-3′. Finally, following electrophoresis on a 2% agarose gel, the expression levels were confirmed using a UV detector (ImageQuant LAS 500; GE Healthcare Life Science, Chicago, IL, USA).

Nuclear extraction reagent (NER) and cytoplasmic extraction reagent (CER) were used to extract the nucleus and cytoplasm. A cell volume of 20 μl corresponded to a volume ratio of CER I:CER II:NER (200:11:100 μl, respectively). A tube containing CER I was first vortexed vigorously on the highest setting for 1 sec to fully suspend the cell pellet. The tube was then incubated on ice for 10 min. Ice-cold CER II was then added to the tube, and the tube was vortexed for 5 sec on the highest setting. The tube was then incubated on ice for 1 min before being vortexed for 5 sec on the highest setting. The tube was then centrifuged at 13,000 rpm for 5 min on a microcentrifuge (Centrifuge 5415F; Eppendorf, Hamburg, Germany). The supernatant (cytoplasmic extract) was then immediately transferred to a clean, pre-chilled tube and stored until needed. Ice-cold NER was added to the pellet, and the tube was vortexed on the highest setting for 15 sec. The sample was placed on ice and vortexed for 15 sec every 10 min, for a total of 40 min. The tube was then centrifuged at 13,000 rpm for 5 min on a microcentrifuge. The supernatant (nuclear extract) was then immediately transferred to a clean, pre-chilled tube.

The HMC-1 cells (5×10⁶ cells/well) were stimulated with PMA (50 nM) plus A23187 (1 μM). The cell lysates were prepared in a sample buffer containing sodium dodecyl sulfate (SDS). The samples were heated at 95°C for 5 min and briefly cooled on ice. Following centrifugation at 13,000 rpm for 5 min, the proteins in the cell lysates were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was then blocked with 5% skim milk in PBS-Tween-20 for 1 h at room temperature and then incubated with anti-MAPKs, anti-β-actin and anti-NF-κB antibodies overnight. After washing the blot in Tris-buffered saline and Tween-20 (TBST) 3 times, it was incubated with a secondary antibody for 1 h, and then the antibody-specific proteins were visualized using an ECL™ prime western blotting detection reagent in accordance with the recommended procedure (Amersham Corp., Newark, NJ, USA).

Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett's t-test for multiple comparisons and the Student's t-test for single comparisons. The data from the experiments are presented as the means ± SEM. The numbers of independent experiments assessed are provided in the figure legends.

The cytotoxicity of SAS was evaluated by MTS assay. SAS was found to not affect the viability of the HMC-1 cells at concentrations of 10, 25, 50 and 100 μM. The cell viability of the cells treated with 100 μM SAS was 91.6% (Fig. 2).

To evaluate the effects of SAS on the production of IL-6, IL-8 and TNF-α, we treated the cells with SAS (10, 25 and 50 μM) prior to stimulation with PMA (50 nM) plus A23187 (1 μM) for 8 h and analyzed the levels using ELISA. The levels of IL-6, IL-8, and TNF-α in the HMC-1 cells significantly increased following stimulation with PMA plus A23187 (Figs. 3–5). Pre-treatment of the cells with SAS (10, 25 and 50 μM) significantly inhibited the increase in the levels of these cytokines in a concentration-dependent manner. The maximal inhibition of IL-6, IL-8 and TNF-α production by SAS (50 μM) was 88, 94 and 63%, respectively (Figs. 3–5).

The IL-6, IL-8 and TNF-α gene expression levels were analyzed by RT-PCR. The increase in the expression of IL-6, IL-8 and TNF-α induced by PMA plus A23187 was inhibited by pre-treatment of the cells with SAS. In particular, SAS (50 μM) significantly inhibited the PMA plus A23187-induced increase in the expression of IL-6 and IL-8 (Fig. 6).

In order to elucidate the mechanisms underlying the effect of SAS, we examined the effect of SAS on MAPK activation. The stimulation of the HMC-1 cells with PMA plus A23187 resulted in an increased phosphorylation of all three types of MAPKs (ERK, JNK and p38) after 1 h. SAS reduced the PMA plus A23187-induced expression of p-ERK1/2 and p-JNK1/2 (Fig. 7). However, SAS had no effect on the levels of p-p38 (Fig. 7).

To evaluate the mechanisms through which SAS affected the gene expression of pro-inflammatory cytokines, we examined the effects of SAS on NF-κB expression. The expression of pro-inflammatory cytokines is regulated by the transcription factor, NF-κB (8). Stimulation of the HMC-1 cells with PMA plus A23187 induced the nuclear translocation of NF-κB p65 after 2 h of incubation (8). SAS inhibited the PMA plus A23187-initiated nuclear translocation of NF-κB p65 (Fig. 8). In order to confirm the inhibitory effect of SAS on NF-κB expression, we examined the effect of SAS on NF-κB-dependent protein expression (Fig. 8).