3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Anti-MMP-1 (cat. no. ab52631, 1:1,000) antibody was purchased from Abcam (Cambridge, UK). ERK1/2 (cat. no. 4377; 1:1,000), phosphorylated (p-) ERK1/2 (cat. no. 9101; 1:1,000), stress-activated protein kinase (SAPK)/JNK (cat. no. 9252; 1:1,000), p-SAPK/JNK (cat. no. 9251; 1:1,000), p38 MAPK (cat. no. 8690; 1:1,000), p-p38 MAPK (cat. no. 9215; 1:1,000), IκBα (cat. no. 2859; 1:1,000), p-IκBα (cat. no. 2078; 1:1,000), NF-κB p65 (cat. no. 9609; 1:1,000), p-NF-κB p65 (cat. no. 4887; 1:1,000), and β-actin (cat. no. 4967; 1:1,000) antibodies were obtained from Cell Signaling Technology, Inc., (Danvers, MA, USA). p38 inhibitor (cat. no. SB203580, 1:1,000) was purchased from Calbiochem (Merck KGaA).

HaCaT human keratinocytes were provided by Professor Moon Je Cho (Department of Biochemistry, National University, Cheju, Korea). Fibroblasts as human primary dermal cells were purchased from American Type Culture Collection (Manassas, VA, USA; cat. no. PCS-201-012TM). HaCaT cells and fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone; GE Healthcare Lifesciences, Logan, UT, USA) with 10% fetal bovine serum (FBS; PEAK, Colorado, USA) and 1% penicillin/streptomycin (10,000 U/100 µg/ml; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a 5% CO₂ humidified atmosphere incubator at 37°C. HaCaT cells were maintained until 80% confluence and then cultured for 24 h in medium without FBS. The cell medium was then replaced with 5 ml PBS and the cells were exposed to UVB light. The UVB doses were determined by irradiating the HaCaT cells with various doses of UVB (0, 20, 40, 60, 80 and 100 mJ/cm²) and optimizing the UVB light as 40 mJ/cm². Cells were cultured in DMEM medium containing 10% FBS. At 24 h post-irradiation, HaCaT-conditioned medium was collected and added on the fibroblasts. After 24 h incubation, the culture medium was collected. Fibroblasts were treated with various concentrations of scopoletin for 24 h in HaCaT-conditioned medium. Vehicle control was serum-free medium-treated fibroblasts.

To determine the effects of scopoletin on the NF-κB signaling pathway, fibroblasts were pretreated with scopoletin for 6 h, and then treated with HaCaT-conditioned medium (40 mJ/cm²) containing scopoletin (0, 30, 100 and 300 µM) for 15 min prior to western blot analyses. When examining the signaling pathway activation in fibroblasts, generally the phosphorylation reaction time is short, therefore pretreatment with scopoletin was performed in order to provide the required time to act on the fibroblasts.

A. capillaris was purchased from Hwasun-bul-minari Company (Hwasun, Korea). Dried A. capillaris (1,475 g) was extracted with 100% ethanol for 3 days at room temperature. The filtered extract was concentrated with a vacuum evaporator (EYELA Rotary evaporator, Tokyo, Japan) and was freeze-dried. The ethanol (EtOH) extract of A. capillaris (71.343 g) was dissolved in H₂O, and extracted with ethyl acetate (EtOAc). The EtOAc layer (38.56 g) was evaporated to dryness under vacuum and partitioned with 90% methanol (MeOH) and n-Hexane. The 90% MeOH layer (24.19 g) was fractionated by Waters MPLC system (Waters, Milford, MA, USA) using a YMC-DispoPackAT (SIL-25; 40 g) eluted with EtOAc and n-Hexane mixture in a gradient mode (EtOAc:n-Hexane, 2:8 to 10:0 in 60 min). The flow rate was 30 ml/min and the elution was monitored at UV 254 nm. The MMP-1 expression levels were evaluated in order to determine the effects of the fraction layers on HaCaT-conditioned medium-treated fibroblasts. Among the nine fractions eluted, the fraction with inhibitory activity on MMP-1 protein expression was further purified using a Waters prep-HPLC system using a YMC ODS A column (YMC-pack ODS-A; 5 µm, 20×250 mm). The column was eluted with 40% MeOH containing 0.2 mM ammonium acetate at a flow rate of 10 ml/min. The compounds were monitored by ultraviolet absorbance at 254 nm. The scopoletin structure was confirmed via nuclear magnetic resonance (NMR, 1D, 2D). ¹H- and ¹³C-NMR spectra were obtained on an Advance DPX 500 MHz NMR spectrometer (Bruker Corporation, Billerica, MA, USA), recorded in a deuterated chloroform (CDC13) solution (33). Scopoletin was dissolved in dimethyl sulfoxide (DMSO) and then diluted in serum-free medium for in vitro studies.

Cell viability was determined by MTT assay. Fibroblasts were seeded at 1.5×10⁴/well in a 24-well plate. After 24 h of incubation, the cell medium was replaced with serum-free medium and incubated for an additional 24 h. The cells were treated with scopoletin for 24 h in serum-free medium. MTT solution (5 mg/ml) was added to each of the wells, and the cells were incubated for 3 h at 37°C. The supernatants were removed, and DMSO was added to dissolve the formazan crystals. Absorbance at 570 nm was measured using an ELISA plate reader (Tecan group Ltd., Mannedorf, Switzerland).

Fibroblasts were lysed in RIPA buffer (Sigma-Aldrich, Merck KGaA) containing protease inhibitors and phosphatase inhibitors. The lysates were centrifuged at 3,000 × g for 15 min at 4°C, and the protein concentrations were determined using a BCA assay. The proteins (40 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were initially blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 30 min at room temperature and then incubated with primary antibodies at 4°C overnight. The membranes were washed with TBS-T and incubated with goat anti-rabbit antibody conjugated to horseradish peroxidase (cat. no. 1662408edu, 1:2,500, Bio-Rad Laboratories, Inc.) at room temperature for 1 h. The immunoreactive bands were visualized using Clarity Western ECL Substrate (cat no. 1705060, Bio-Rad Laboratories, Inc.) and quantified with Image J software (version 1.49v; National Institutes of Health, Bethesda, MD, USA) (34).

Total RNA samples from treated cells were isolated using an RNeasy Mini kit (Qiagen GmbH, Hilden, Germany). The cDNA samples were then synthesized using a Primescript 1st Strand cDNA Synthesis kit (Takara Bio, Inc., Otsu, Japan) following the manufacturer’s protocol. The reaction conditions were 42°C for 60 min and 95°C for 5 min. qPCR was performed with a SYBR Green Master Mix (Bio-Rad Laboratories, Inc.) and the following primers: IL-1α, forward 5′-CGC CAA TGA CTC AGA GGA AGA-3′ and reverse 5′-AGG GCG TCA TTC AGG ATG AA-3′ (120 bp); TNFα. Forward 5′-TCT TCT CGA ACC CCG AGT GA-3′ and reverse 5′-CCT CTG ATG GCA CCA CCA G-3′ (151 bp); GAPDH, forward 5′-TGC CAC CAG AAG ACT GTG G-3′ and reverse 5′-AGC TTC CCG TTC AGC TCA GG-3′. The thermocycling conditions were as follows: 10 min at 94°C, followed by a total of 45 cycles of 15 sec at 94°C and 1 min at 60°C. The expression levels of the genes presented as the quantification cycle (Cq) value was measured using the 2^−∆∆Cq relative quantitative analysis method (35), as automatically determined using the LightCycler 96 Software 1.1 (Roche Diagnostics, Basel, Switzerland).

All experiments were repeated at least three times, and results were presented as the mean ± standard deviation from three individual experiments. Statistical significance between two groups was examined with two-tailed Student’s t-test using the SPSS 19.0 software package (IBM Corps, Armonk, NY, USA). Multiple-group comparisons were performed using one-way analysis of variance followed by Dunnett’s post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of the ACE fractions on MMP-1 protein expression inhibition, fibroblasts were treated with the 9 fractions. The highest inhibition activity of MMP-1 protein expression was observed in the Fraction 5 (Fig. 1A). A bioassay-guided fraction aided in the isolation of a single compound from Fraction 5 that exhibited inhibition of MMP-1 protein expression. The NMR spectrum of this compound was identical to scopoletin, which has been reported to be isolated from ACE in a previous study (23). The structure of this compound is shown in Fig. 1B. To examine whether scopoletin may exhibit cytotoxicity, the cell viability of fibroblasts treated with scopoletin was evaluated using an MTT assay. The results indicated that scopoletin had no cytotoxicity, as tested at the doses of 0.5, 5, 15, 50, 150, 250 and 500 µM for 24 h (Fig. 1C).

To determine the optimal irradiation conditions, HaCaT cells were irradiated with various doses of UVB (0, 20, 40, 60, 80 and 100 mJ/cm²). After 24 h, HaCaT-conditioned medium was collected and then added to the fibroblasts. HaCaT-conditioned medium from 40 mJ/cm² irradiation produced the highest level of MMP-1 overexpression in fibroblasts (Fig. 2A), therefore 40 mJ/cm² was used in subsequent experiments. To investigate the effects of scopoletin on MMP-1 protein expression, fibroblasts were treated with various concentrations of scopoletin in conditioned medium from UVB-irradiated HaCaT cells. The protein expression levels for MMP-1 were determined by western blot analysis. MMP-1 protein expression was not altered when the fibroblasts were treated with serum-free medium containing scopoletin (300 µM; Fig. 2B). However, MMP-1 expression was significantly increased in fibroblasts treated with HaCaT conditioned medium. Scopoletin treatment decreased the MMP-1 protein expression levels with an inhibition rate of 44.84% at a concentration of 300 µM in the fibroblasts (Fig. 2B).

To examine whether UVB causes an increase in the mRNA expression of proinflammatory cytokines, the mRNA expression levels of IL-1α and TNFα were analyzed by RT-qPCR in HaCaT cells exposed to UVB for 24 h. The results demonstrated that 40 mJ/cm² UVB enhanced the mRNA levels of IL-1α and TNFα, by 2.66- and 1.85-fold, respectively (Fig. 2C and D). After collecting this conditioned medium, it was added on fibroblasts together with various concentrations of scopoletin (0, 30, 100 and 300 µM) for 24 h. In the fibroblasts treated with conditioned medium from the UVB-exposed HaCaT cells, the mRNA levels of IL-1α and TNFα were significantly increased compared with the control-exposed fibroblasts (Fig. 2E and F). Scopoletin inhibited this effect in a dose-dependent manner, in the range of 50-88.5% for IL-1α, and 15-80% for TNFα, compared with untreated, conditioned media-exposed fibroblasts (Fig. 2E and F).

To determine the effect of scopoletin on NF-κB activation, the phosphorylation levels of IκBα and p65 were examined by western blot analysis (Fig. 3). No phosphorylation of IκBα and p65 was observed when fibroblasts were treated with serum-free medium containing scopoletin (300 µM; Fig. 3A). However, IκBα and p65 phosphorylation levels were increased when the fibroblasts were treated with conditioned medium from UVB-exposed HaCaT cells (Fig. 3). While IκBα phosphorylation was unaffected (Fig. 3B), p65 phosphorylation was significantly inhibited by scopoletin treatment (Fig. 3C). The reduction of p65, in the rate of 11.54-21.92%, was observed at scopoletin concentration of 300 µM (Fig. 3C).

To determine whether scopoletin inhibited MMP-1 expression by blocking MAPK signaling, the phosphorylation levels of ERK, JNK, and p38 were examined by western blot analysis. No phosphorylation of ERK, JNK, and p38 was observed in fibroblasts with serum-free medium containing scopoletin (300 µM; Fig. 4A and B). By contrast, the phosphorylation of ERK, JNK, and p38 was markedly increased in the fibroblasts treated with conditioned medium from UVB-exposed HaCaT cells (Fig. 4). Scopoletin treatment significantly inhibited p38 phosphorylation, with a rate of 17.67-28.33% observed at the scopoletin concentration of 300 µM (Fig. 4A and C). No effect on reducing phosphorylation of ERK and JNK was observed following scopoletin treatment in fibroblasts treated with conditioned medium from UVB-exposed HaCaT cells (Fig. 4B, D and E).

First, the potential cytotoxicity of SB203580 was tested on the fibroblasts by MTT assay. The results indicated that SB203580 had no cytotoxicity at the concentration of 10 µM (Fig. 5A). Then, the inhibition effect of scopoletin on p38 phosphorylation and MMP-1 expression was confirmed by western blotting (Fig. 5B and C). To further explore this pathway, the effect of SB203580, a well-known p38 inhibitor (36-40), was assessed. The results demonstrated that treatment with SB203580 and scopoletin significantly inhibited the phosphorylation of p38 by 37.31 and 33.45% (Fig. 5B) and decreased the MMP-1 protein expression by 17.39 and 8.94%, respectively (Fig. 5C).