Whole plants of Ixeris dentata family Compositae (Asteraceae) were collected in May 2006 from the herbarium at the Korea Research Institute of Chemical Technology (KRICT) and were authenticated by Dr Young Sup Kim. A voucher specimen (KR0472) was deposited into the herbarium at KRICT (27).

The air-dried whole plants (6 kg) of Ixeris dentata were soaked in methanol (MeOH) (2×40 l) at room temperature for 7 days. The MeOH extract was filtered and evaporated to dryness under reduced pressure. The concentrated extract (840 g) was suspended in 20 l of water and then extracted successively with an equal volume of dichloromethane (MC), ethyl acetate (EtOAc), and n-butanol (n-BuOH) to afford 160, 15 and 60 g of the compound in MC, EtOAc, and n-BuOH fractions, respectively (27). The suggested chemical structure of IXA is shown in Fig. 1.

RPMI-1640, penicillin, and streptomycin were obtained from HyClone Laboratories, Inc. (Logan, UT, USA). Bovine serum albumin and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for ERK, phosphorylated ERK, JNK, phosphorylated JNK, p38, phosphorylated p38, and β-actin, and peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies for human IL-6 and IL-8 and biotinylated antibodies for human IL-6 and IL-8 were purchased from BD Biosciences (San Jose, CA, USA). The RNeasy Mini kit and QuantiTect Reverse Transcription kit were purchased from Qiagen (Hilden, Germany). IL-6, IL-8, COX-2, and β-actin oligonucleotide primers were purchased from Bioneer Corp. (Daejeon, Korea).

HaCaT cells were grown in RPMI-1640 medium containing 5% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin sulfate. The cells were incubated in 5% humidified CO₂ atmosphere at 37°C. To stimulate the cells, the medium was replaced with fresh RPMI-1640 medium and exposed to UVB in the presence or absence of IXA for the indicated time points.

UVB irradiation was delivered using a closely spaced array of five sunlamps (G9T5E lamps; Sankyo Denki Co., Ltd., Hiratsuka, Japan). The distance between the sunlamps and the surface of the cell cultures was fixed at 7.5 cm, and the distance between the sunlamps and the surface of the cage was fixed at 30 cm. The energy output of the UVB (290–320 nm) lamps was measured using a UV radiometer (UVX; UVP Inc., Upland, CA, USA).

Cell viability was determined by the MTS assay. HaCaT cells were plated at a density of 3×10⁴ cells/well in 96-well plates (Thermo Scientific Nunc^®, Nunc AS, Copenhagen, Denmark). Each experiment had a non-treated group as the control. To determine the non-toxic concentration for the cells, IXA (2.5, 5, 10, and 20 μM) was added to each well. The plates were incubated for 24 h at 37°C under 5% CO₂. MTS solution (5 mg/ml) was added to each well, and the cells were cultured for another 2 h, followed by measuring their optical density at 490 nm. The cytotoxicity was calculated using the formula: 1 – (mean absorbance value of treated cells/mean absorbance value of untreated cells).

Cells were seeded at a density of 3×10⁴ cells/well in 48-well tissue culture plates and pre-treated with two concentrations of IXA (5 and 10 μM) for 24 h prior to UVB (100 mJ/cm²) stimulation. The culture supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) for IL-6 and IL-8 levels. To measure the cytokines, a modified ELISA method was used. First, a sandwich ELISA for IL-6 and IL-8 was conducted in duplicate in 96-well immuno plates (Nunc 439454; Thermo Scientific Nunc^®). The supernatant was decanted into a new microcentrifuge tube, and the cytokines were quantified by ELISA. ELISA plates were coated overnight at 4°C with anti-human IL-6 and IL-8 monoclonal antibodies diluted in coating buffer (0.1 M carbonate, pH 9.5), and then washed four times 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 at pH 7.0) for at least 1 h. After washing the plates again, the test sample or recombinant IL-6 and IL-8 standards was added. After incubation for 2 h, a working detector (biotinylated anti-human IL-6 and IL-8 monoclonal antibodies and streptavidin-horseradish peroxidase reagent) was added, and the mixture was incubated for 1 h. Subsequently, the substrate solution (tetramethylbenzidine) was added to the wells and incubated for 30 min in the dark before the reaction was quenched by adding 1 M H₃PO₄. The absorbance was read at 450 nm using an ELISA reader (Infinite M200; Tecan, Männedorf, Switzerland). Subsequent steps were conducted at room temperature, and the standards and samples were assayed in duplicate.

Protein expression was assessed by western blot analysis according to standard procedures. HaCaT cells were cultured in 60-mm-diameter culture dishes (4×10⁶ cells/well) and pre-treated with two concentrations of IXA (5 and 10 μM). After 30 min, 2 or 24 h, the cells were UVB-irradiated (100 mJ/cm²) and then incubated at 37°C. Following the incubation, the cells were washed twice in PBS (pH 7.4) and were resuspended in lysis buffer on ice for 20 min. The cell debris was removed by centrifugation (10,000 × g, 10 min, and 4°C). The protein concentrations were determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. Equal amounts of protein (20 μg) were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene membrane (Millipore, Bedford, MA, USA). The membrane was blocked using 5% non-fat milk in Tris-buffered saline with Tween-20 buffer (150 mM NaCl, 20 mM Tris-HCl, and 0.05% Tween-20, pH 7.4). After blocking, the membrane was incubated with primary antibodies for 18 h. Antibodies against ERK, phosphorylated ERK, JNK, phosphorylated JNK, p38, phosphorylated p38 and β-actin, and peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. The membrane was then washed with Tris-buffered saline containing Tween-20 and incubated with anti-mouse or anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibodies. The proteins were then supplemented with ECL Prime Western Blotting Detection Reagents and an ImageQuant LAS 4000 mini biomolecular imager (both from GE Healthcare, Cleveland, OH, USA) was used to evaluate the bands.

Total cellular RNA was isolated using an easy-BLUE™ RNA extraction kit (Intron Biotechnology, Inc., Seongnam, Republic of Korea) according to the manufacturer’s instructions. The total RNA (2 μg) was then converted to cDNA by treating it with 200 units of reverse transcriptase and 500 ng of oligo (dT) primer in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, and 1 mM deoxynucleotide triphosphates at 42°C for 1 h. The reaction was quenched by heating the samples at 70°C for 15 min, followed by enzymatic amplification of the cDNA mixture (3 μl). PCR was conducted in a reaction mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 0.2 mM deoxynucleotide triphosphates, 2.5 units of Taq DNA polymerase, and 0.1 μM each of IL-6, IL-8, COX-2, and GAPDH primers. PCR of GAPDH was conducted by subjecting the reaction mixtures to an initial denaturation of 92°C for 5 min, followed by 30 cycles of 92°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 2 min, with a final extension at 72°C for 5 min. PCR of IL-6 and IL-8 samples was conducted by heating the reaction mixtures to 95°C for 2 min, followed by 35 cycles at 95°C for 45 sec, 60°C for 45 sec, and 72°C for 90 sec, with a final extension at 72°C for 10 min. PCR of COX-2 samples was conducted by heating the reaction mixtures to 94°C for 5 min, followed by 30 cycles at 94°C for 1 min, 55°C for 30 sec, and 72°C for 1 min, with a final extension at 72°C for 7 min. The PCR products were then electrophoresed on 1% agarose gel and stained with ethidium bromide. The primer sequences are listed in Table I.

Statistical analysis was performed using one-way analysis of variance (ANOVA) or the Student’s t-test for single comparisons. Data are presented as the means ± standard error (SE), and the number of individual experiments conducted is mentioned in each figure legend. P<0.05 and P<0.005 were considered to indicate statistically significant differences.

The effect of IXA on cell viability following UVB irradiation was assessed on HaCaT cells. Cell viability was evaluated using the MTS assay (Fig. 2). When the cultures were incubated after UVB irradiation, UVB-induced toxicity increased compared to that in non-irradiated cells. Cell viability decreased depending on the dose of UVB irradiation and was markedly reduced at 24 h after UVB irradiation of 150 mJ/cm². Subsequently, an exposure dose of 100 mJ/cm² was selected to study cell toxicity in HaCaT cells treated with IXA 24 h after UVB irradiation (Fig. 3).

Initially, the cytotoxicity of IXA on HaCaT cells was examined using the MTS assay. The half maximal inhibitory concentration (IC₅₀) value of IXA was 50 μM (data not shown), and IXA did not show any cytotoxic effects up to 10 μM (Fig. 3). To evaluate the effect of IXA on the production of pro-inflammatory cytokines, the cells were pre-treated with IXA (5 and 10 μM) prior to stimulation by UVB (100 mJ/cm²) for 24 h and analyzed by ELISA. As shown in Fig. 4, the levels of IL-6 and IL-8 were considerably increased in HaCaT cells after stimulation with UVB (100 mJ/cm²). Pre-treatment of cells with IXA (5 or 10 μM) inhibited these increments in a concentration-dependent and statistically significant manner.

The pro-inflammatory cytokine gene expression was then analyzed using RT-PCR. Enhanced IL-6 and IL-8 mRNA expression induced by UVB (100 mJ/cm²) was inhibited by pre-treatment of the cells with IXA (Fig. 5). In particular, the pre-treatment with IXA at a concentration of 10 μM inhibited the UVB-induced gene expression of IL-6 and IL-8.

The effects of IXA on COX-2 expression were examined in UVB-irradiated HaCaT cells. The expression levels of COX-2 protein and mRNA were measured in HaCaT cells exposed to UVB (100 mJ/cm²) for 24 h. IXA effectively suppressed UVB-induced COX-2 expression. UVB (100 mJ/cm²) also increased COX-2 mRNA expression, which was inhibited in the presence of IXA (Fig. 6). Thus, IXA suppressed the expression of genes that were involved in the pathogenesis of inflammatory responses.

Inhibition of the ERK, JNK, and p38 MAPK pathways was recently found to attenuate pro-inflammatory cytokine secretion. The effect of IXA on UVB-induced MAPK phosphorylation in HaCaT cells was examined by incubating the cells with IXA 24 h prior to stimulation with UVB for 30 min, 1 or 2 h. UVB-induced phosphorylation of ERK, JNK, and p38 MAPK was then determined by western blot analysis. IXA pre-treatment significantly inhibited the UVB-induced phosphorylation of ERK, JNK, and p38 MAPK in a dose-dependent manner without affecting the total protein levels of these kinases (Fig. 7). These results indicated that the inhibitory effect of IXA on UVB-induced MAPK phosphorylation may result in blockage of the cytokine production and COX-2 expression in HaCaT cells.