The plant species was collected from Cantigi in Indonesia in 2008, and it was identified by the Center for Pharmaceutical and Medical Technology, Agency for Assessment and Application of Technology (Tangerang, Indonesia) and verified by Herbarium Bogoriense (Indonesian Institute of Sciences, Bogor, Indonesia). A voucher specimen (KRIB 0019989) has been deposited in the herbarium of the Korea Research Institute of Bioscience and Biotechnology, and also in the Center for Pharmaceutical and Medical Technology and Herbarium Bogoriense. The RA plant material was treated with MeOH and sonicated for 15 min, at 36.5 kHz using Ultrasonic Cleaning Bath (Branson Ultrasonics; Emerson Electric Co., St. Louis, MO, USA) and incubated for 2 h at room temperature. This procedure was repeated 30 times for 3 days to produce an extract. The methanol extract (360 mg) was suspended in distilled water, the same amount of n-hexane was added and mixed, and then the n-hexane soluble fraction and the water-soluble fraction were separated. This was performed three times, followed by cotton wool filtration and concentration under reduced pressure to obtain an n-hexane fraction (yield, 17.2 mg). Then, the n-hexane fraction was removed and the same amount of chloroform was added to the remaining water layer. A chloroform fraction (yield, 21.0 mg) was obtained in the same manner. The same amount of ethyl acetate was added to the remaining water later, and an ethyl acetate fraction (yield, 14.0 mg) was obtained in the same manner. Finally, an equivalent amount of butanol was added to the water layer, and a butanol fraction (yield, 47.3 mg) was obtained in the same manner. The remaining water layer was then concentrated to obtain a water fraction (yield, 235.5 mg).

The human keratinocyte HaCaT cell line (ATCC; American Type Culture Collection, Manassas, VA, USA) was cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) at 37°C in a humidified 5% CO₂ incubator. Prior to further association studies, the growth medium was changed to serum-free medium. Cells in the control group were treated with dimethyl sulfoxide (DMSO, 0.1% per 100 μl) alone. DMEM, FBS, penicillin, streptomycin, and PBS (pH 7.4) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Recombinant TNF-α (10 ng/ml) was purchased from Invitrogen; Thermo Fisher Scientific, Inc. and IFN-γ (10 ng/ml) was purchased from Merck KGaA (Darmstadt, Germany). The HaCaT cells were pretreated with RAME (2.5, 5, 10, 20 μg/ml) for 1 h and subsequently treated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml), and incubated for 24 h at 37°C. In another set of cultures, the cells were co-incubated with 5 μM Bay11-7082, a nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκB-α) inhibitor, 10 μM SB203580, a p38 inhibitor, and 10 μM SP600125, a c-Jun N-terminal kinase (JNK) inhibitor (Calbiochem; EMD Millipore, Billerica, MA, USA) for 1 h at 37°C in a humidified 5% CO₂ incubator.

To confirm the effect of RAME on HaCaT cells, the reduction of MTT (Amresco, Inc., Solon, OH, USA) by viable cells was measured. The cells were plated in 96-well culture plates (SPL Life Sciences, Pocheon, Korea) at a density of 1×10⁴ cells/well, and allowed to bind for 6 h. Cells were allowed to attach for 6 h, and the culture was continued for 24 h following addition of RAME (2.5, 5, 10, 20 μg/ml) at 37°C. The cells were cultured with 0.5 mg/ml MTT solution. After 4 h of incubation at 37°C in 5% CO₂, the supernatant was removed and DMSO was added. Absorbance was measured at 570 nm using a microplate reader (Tecan Group, Ltd., Männedorf, Switzerland). The percentage of viable cells was calculated by taking the optical density of the cells following a particular treatment and dividing that number with the optical density of untreated control cells, followed by multiplication by 100.

The culture medium was collected and the production of cytokines (IL-6 and IL-8) in the supernatant was measured using ELISA kits (IL-6; cat. no. 555220, BD Biosciences, San Jose, CA, USA; IL-8; cat. no. DY208, R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturers' protocols, as previously described (23,24).

The culture medium of the cells was harvested, and chemokine production (TARC and MDC) in the supernatant was measured using ELISA kits (cat nos. TARC cat. no. DY364 and MDC cat. no. DY336; R&D Systems, Inc.) according to the manufacturer's protocol.

Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc.) for RT-qPCR analysis. cDNA was synthesized using the AMPIGENE^® cDNA Synthesis kit (cat. no. ENZ-KIT106; Enzo Life Sciences, Inc., Farmingdale, NY, USA). qPCR was performed using SYBR Green PCR Master Mix (KAPA SYBR^® FAST qPCR kits; Kapa Biosystems, Inc., Wilmington, MA, USA). Reactions were run in a CFX96 Real-Time PCR System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using the following thermocycler conditions: Stage 1, 50°C for 2 min and 95°C for 10 min; stage 2, 95°C for 15 sec and 60°C for 1 min. Stage 2 was repeated for 40 cycles. Relative mRNA levels were calculated using the comparative threshold cycle 2^−ΔΔCq method and normalized to that of GAPDH (25). The primer sequences used in the present study are listed in Table I.

Briefly, the HaCaT cells were washed with cold PBS and then lysed in RIPA buffer (ELPIS Biotech, Inc., Daejeon, Korea) on ice for 20 min with a strong vortex. The cell lysate was centrifuged at 10,000 × g for 10 min at 4°C and protein concentrations were measured using the bicinchoninic acid method. Sample proteins (20 μg) were analyzed on 10% SDS-PADE and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane, following which the PVDF membrane was blocked in 5% non-fat dry milk in Tris-buffered saline (TBS, 20 mM Tris, 0.2 M NaCl, pH 7.5) containing 0.05% Tween-20 (TBS/T) for 1 h at room temperature. The PVDF membrane was incubated with primary antibodies for overnight at 4°C. Following washing three times with TBS/T, the membranes were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies, diluted 1:5,000 with 5% skim milk in TBS/T. The membranes were then visualized using SuperSignal West Pico Chemiluminescent Substrate (cat. no. 32106; Pierce; Thermo Fisher Scientific, Inc.). The primary antibodies used were as follows: anti-NF-κB p65 (cat. no. sc-8242; 1:1,000), anti-JNK (cat. no. sc-474; 1:1,000), anti-extracellular signal-regulated kinase (ERK; cat. no. sc-154; 1:1,000), anti-p38 (cat. no. sc-7149; 1:1,000), anti-p-p38 (cat. no. sc-7973; 1:1,000), anti-phosphorylated (p-) JAK1 (cat. no. sc-16773; 1:1,000), anti-JAK1 (cat. no. sc-376996; 1:1,000), anti-p-STAT1 (cat. no. sc-8394; 1:1,000), anti-STAT1 (cat. no. sc-464; 1:1,000; all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-p-ERK 1/2 (cat. no. 9101; 1:1,000), anti-p-IκB-α (cat. no. 2859; 1:1,000), anti-IκB-α (cat. no. 9242; 1:1,000), anti-NF-κB p-p65 (cat. no. 3033; 1:1,000; all from Cell Signaling Technology, Inc., Danvers, MA, USA), anti-p-JNK (cat. no. KAP-SA011; 1:1,000; Enzo Life Sciences, Inc.), and anti-β-actin (cat. no. 4967; 1:1,000; Cell Signaling Technology, Inc.). The secondary antibodies used were as follows: HRP-conjugated goat anti-rabbit IgG (cat. no. sc-2030; 1:5,000 in 5% skim milk; Santa Cruz Biotechnology, Inc.; used for detection of NF-κB p65, JNK, ERK, p38, p-p38, p-JNK, p-ERK, p-IκB-α, IκB-α, NF-κB p-p65, β-actin) and HRP-conjugated goat anti-mouse IgG (cat. no. sc-2005; 1:5,000 in 5% skim milk; Santa Cruz Biotechnology, Inc.; used for detection of JAK1, STAT1, p-JAK1, p-STAT1). Each protein was detected using an Enhanced Chemiluminescence detection system using ImageQuant LAS4000 (GE Healthcare Life Sciences, Little Chalfont, UK). Western blot bands were quantified using ImageJ software (version 1.6.0; National Institutes of Health, Bethesda, MD, USA).

HaCaT cells were transfected with 0.1 μg pGL4.32 (luc2P/NF-κB-RE/Hygro, Promega Corporation, Madison, WI, USA) plasmids, using Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Then, 20 h after transfection, the cells were stimulated with TNF-α/IFN-γ (10 ng/ml) for 24 h at 37°C, harvested, and then assessed for luciferase activity using the ONE-Glo™ luciferase reporter assay system (Promega Corporation) according to the manufacturer's protocol. Normalization was performed by comparison with Renilla luciferase activity.

HaCaT cells were cultured in Lab-Tek™ chamber slides (Thermo Fisher Scientific, Inc.) and immobilized in ethanol at 4°C for 15 min. Slides were washed three times with PBS and blocked with 2% (w/v) bovine serum albumin (BSA100, Bovogen Biologicals Pty Ltd., Melbourne, Australia) in PBS for an additional 1 h at room temperature. Slides were then incubated with anti-NF-κB p65 subunit (cat. no. sc-8242; rabbit polyclonal IgG; 1:200, Santa Cruz Biotechnology) and anti-STAT1 (cat. no. sc-464; mouse monoclonal IgG; 1:100, Santa Cruz Biotechnology, Inc.) antibodies for 24 h at 4°C. Following washing with PBS three times to remove excess primary antibody, the slides were further incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (cat. no. A-11034; 1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.) or Texas Red conjugated goat anti-mouse IgG secondary antibodies (cat. no. A-11003; 1:1,000; Invitrogen; Thermo Fisher Scientific, Inc.) for 2 h at room temperature, washed with PBS, and stained with Gold Antifade reagent containing DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) for 5 min prior to the position and quantification of ProLong nuclei. Subsequently, the slides were coverslipped and visualized using confocal laser scanning microscopy (LSM 510 m; Carl Zeiss AG, Oberkochen, Germany). All samples were quantified from the images obtained, which were taken under the same exposure conditions.

The data represent the mean ± standard error of the mean. Statistical differences among groups were determined by one-way analysis of variance with repeated measures followed by Student-Newman–Keuls testing in SPSS 14.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The effect of RAME on cell viability was confirmed using HaCaT cells and an MTT assay. As presented in Fig. 1, RAME did not result in a significant cytotoxic effect, failing to affect cell viability even at a relatively high concentration of 20 μg/ml for a treatment period of 24 h. Therefore, RAME was experimentally confirmed to not show toxicity, even when HaCaT cells were treated with 20 μg/ml. In addition, the effect of RAME and solvent fraction (n-hexane, chloroform, ethyl acetate, butanol, water layer) on cell viability was confirmed using HaCaT cells and MTT assays. RAME and solvent fraction did not affect cell viability, even at a relatively high concentration of 20 μg/ml for a 24 h treatment period (data not shown).

Next, the effect of suppressing TARC and MDC production by RAME in HaCaT cells stimulated with TNF-α/IFN-γ was investigated using ELISA and RT-qPCR (Fig. 2). TARC and MDC were significantly increased in the group treated with TNF-α/IFN-γ compared with the untreated group. In addition, when HaCaT cells were treated with TNF-α/IFN-γ following RAME treatment, TARC and MDC mRNA expression decreased in a concentration-dependent manner, compared with the group treated with TNF-α/IFN-γ. Furthermore, RAME treatment inhibited the expression of TARC and MDC protein in the HaCaT cells stimulated with TNF-α/IFN-γ.

Next, the effect of inhibiting the production of IL-6 and IL-8 through RAME treatment was investigated in HaCaT cells stimulated with TNF-α/IFN-γ using ELISA and RT-qPCR (Fig. 3). The results revealed that IL-6 and IL-8 were significantly increased in the group treated with TNF-α/IFN-γ compared with the untreated group. In addition, when HaCaT cells were treated with TNF-α/IFN-γ following RAME treatment, IL-6 and IL-8 mRNA expression decreased in a concentration-dependent manner compared with the group treated with TNF-α/IFN-γ. Furthermore, RAME treatment inhibited the expression of IL-6 and IL-8 protein in the HaCaT cells stimulated with TNF-α/IFN-γ.

TNF-α/IFN-γ activate JNK, ERK and p38 expression (24). Therefore, the effect of RAME on JNK, ERK and p38 protein expression in HaCaT cells was investigated (Fig. 4). HaCaT cells were treated with TNF-α/IFN-γ and RAME was added after 2 h. Phosphorylation changes to JNK, ERK, and p38, which are MAPKs that are known to be important in the atopy pathway, were confirmed. The results revealed an increase in the phosphorylation of JNK, ERK, and p38 in HaCaT cells stimulated for 1 h with TNF-α/IFN-γ, compared with unstimulated HaCaT cells. Furthermore, this experiment confirmed that when the cells were pretreated with RAME, the phos-phorylation of JNK, ERK, and p38 was markedly suppressed as compared with the group treated with TNF-α/IFN-γ alone. Consequently, RAME exerted an inhibitory effect on the expression of p-JNK, p-ERK, and p-p38 following TNF-α/IFN-γ stimulation.

NF-κB signaling is critical in skin inflammatory AD responses induced by MDC and TARC (26). To examine whether RAME downregulated TNF-α/IFN-γ-mediated NF-κB activation in the keratinocyte cells, the expression of associated proteins was examined by western blot analysis using specific antibodies, ICC, and luciferase assays. TNF-α/IFN-γ significantly increased the phosphorylation of NF-κB p65 at 1 h. RAME decreased the phosphorylation of NF-κB p65 in TNF-α/IFN-γ-induced HaCaT cells (Fig. 5A). TNF-α/IFN-γ-induced nuclear translocation of p65 was inhibited by treatment with RAME at 10 μg/ml (Fig. 5B). Furthermore, the inhibitory effect of RAME on TNF-α/IFN-γ-stimulated nuclear translocation of NF-κB p65 was also observed during ICC analysis (Fig. 5C). These results revealed that RAME inhibited the activation of NF-κB in HaCaT cells.

The p65 subunit is an important element of activated NF-κB. Activation of NF-κB in TNF-α/IFN-γ induced HaCaT cells was studied by measuring the phosphorylation of NF-κB subunit p65 by western blot analysis. Pretreatment with RAME and Bay11-7082 decreased the levels of p-IκB-α and p-p65 compared with the TNF-α/IFN-γ treatment only, while the TNF-α/IFN-γ treatment group increased the levels of p-IκB-α and p-p65 compared with the negative control (Fig. 6A). Stimulation of HaCaT cells with TNF-α/IFN-γ induced nuclear translocation of p65 NF-κB and degradation of IκB-α. NF-κB-dependent gene reporter analysis was used to confirm the inhibitory effect of RAME and Bay11-7082 on NF-κB activation. RAME and Bay11-7082 suppressed TNF-α/IFN-γ-stimulated nuclear translocation of p65 NF-κB (Fig. 6B). As presented in Fig. 6C, the TNF-α/IFN-γ stimulated NF-κB promoter activity in HaCaT cells was significantly reduced by RAME and Bay11-7082 inhibitors. In addition, stimulation of HaCaT cells with TNF-α/IFN-γ induced the expression of chemokines and pro-inflammatory cytokines. RAME and Bay11-7082 treatment suppressed TNF-α/IFN-γ-stimulated expression of TARC, MDC, IL-6, and IL-8 (Fig. 7A-D). As presented in Fig. 7, the expression of TNF-α/IFN-γ stimulated chemokines (TARC and MDC) and pro-inflammatory cytokines (IL-6 and IL-8) in HaCaT cells was significantly decreased by treatment with RAME and Bay11-7082 inhibitors.

The translocation of NF-κB into the nucleus was reduced in RAME-treated HaCaT cells (Fig. 5). The MAPKs may be activated by TNF-α/IFN-γ, a key stimulator of the skin inflammation response in keratinocytes, as well as multiple other types of cell. As presented in Fig. 4, RAME markedly decreased the expression levels of p-p38, p-ERK, and p-JNK in TNF-α/IFN-γ-induced HaCaT cells compared with untreated cells. These data suggested that RAME suppresses the inflammatory response via partial regulation of NF-κB and MAPK signaling. To assess the function of NF-κB and MAPKs in the production of TNF-α/IFN-γ-induced inflammatory mediators, the effects of selective NF-κB and MAPK inhibitors on TNF-α/IFN-γ-induced chemokine and cytokine production were investigated. ELISA analysis revealed that Bay11-7082 (IκB-α inhibitor) SP600125 (JNK inhibitor), PD98059 (ERK inhibitor) and SB203580 (p38 inhibitor) markedly inhibited TARC, MDC, IL-6, and IL-8 expression, respectively. Furthermore, treatment with RAME and inhibitors were demonstrated to markedly inhibit TNF-α/IFN-γ-mediated expression of TARC (Fig. 7A), MDC (Fig. 7B), IL-8 (Fig. 7C), and IL-6 (Fig. 7D). These results suggested that RAME-mediated inactivation of NF-κB p65, JNK, ERK, and p38 MAPK may, at least in part, be responsible for the suppression of TARC, MDC, IL-8 and IL-6 in HaCaT cells. In addition, TNF-α/IFN-γ stimulation induced an increase in the activation of NF-κB and MAPKs, while treatment with RAME inhibited this effect.

The JAK/STAT pathway is important to the immune system. To investigate whether RAME treatment resulted in the downregulation of TNF-α/IFN-γ-mediated JAK/STAT activation in HaCaT cells, western blot analysis and immunocytochemistry using specific antibodies were utilized (Fig. 8). RAME pre-treatment 1 h prior to TNF-α/IFN-γ exposure in the HaCaT cells. TNF-α/IFN-γ increased the phosphorylation of JAK/STAT signaling for 1 h. As a result, RAME was revealed to inhibit the phosphorylation of the JAK/STAT pathway in TNF-α/IFN-γ-stimulated HaCAT cells (Fig. 8A). In addition, RAME treatment was revealed to decrease translocation of STAT1 in HaCaT cells by immunocytochemistry (Fig. 8B). These results revealed that RAME inhibited the activation of JAK/STAT in HaCaT cells.