3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and anti-β-actin antibody were purchased from Sigma (St. Louis, MO, USA). Primary antibodies for MMP-1 and MMP-3 were obtained from R&D Systems (Minneapolis, MN, USA). Dulbecco’s modified Eagle’s medium (DMEM) with high glucose level, Medium 154, growth supplement, fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were obtained from Gibco-BRL (Gaithersburg, ME, USA). Primary antibodies for p50, p65, IκBα, proliferating cell nuclear antigen (PCNA) and horseradish peroxidase (HRP)-conjugated IgG were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

The roots of A. gigas Nakai (Umbelliferae family) were extracted serially with methanol, ethylacetate and n-butanol and fractionated. From the ethyl-acetate fraction, decursin was isolated using silica gel column chromatography. After column chromatography, the structure of the purified coumarin compounds, decursin (C₁₉H₂₀O₅) and decursinol angelate (molecular weight, 328 g) were characterized by gas chromatography (Shimadzu, Kyoto, Japan), nuclear magnetic resonance (JEOL JNM-LA 400; Japan) and mass spectroscopy (JEOL-AX 505WA) at Daegu Haany University, Daegu, Korea.

HDFs were aseptically isolated from foreskin. The epidermis and dermis were separated by incubation in media with 0.9 U/ml dispase at 4°C for 16 h. After the epidermis and dermis were mechanically separated, the dermis was minced, attached to the surface of a tissue culture flask and incubated with DMEM containing 10% FBS for 1–2 weeks (20). Dermal fibroblasts that spread as radial outgrowths from the attached pieces of dermis were cultured in DMEM containing 10% FBS and 1% antibiotics at 37°C in a 5% CO₂ incubator.

HDFs were rinsed twice with PBS and irradiated using a UVB cross-linker (6×8 W, 312 nm; Model CL-508M; Vilber Lourmat, Paris, France) (20). HEKn were irradiated using a Stratalinker UV crosslinker (Model 2400; Agilent Technologies, Cold Spring, NY, USA). Immediately after irradiation, fresh serum-free medium was added to the HDFs, and complete growth medium was added to the HEKn. Responses were measured after incubation for each experimental condition. The same schedule of medium changes was followed for control cells.

The protective effect of decursin against UV-induced cytotoxicity of HDFs was determined using the MTT assay. Briefly, HDFs were seeded at a density of 3×10⁴ cells/plate and allowed to attach. After 24 h, the cells were treated with various concentrations of decursin (1, 5, 10, 30 and 50 μM). After incubation for 24 h, the cells were washed twice with PBS and MTT (0.5 mg/ml PBS) was added to each well. The plates were incubated at 37°C for 30 min. Formazan crystals that had formed were dissolved by adding DMSO (100 μl/well) and the absorbance was measured at 570 nm using a microplate reader (Model 3550; Bio-Rad, Richmond, CA, USA).

Cells were seeded onto a 10-cm dish and allowed to attach for 24 h. They were then treated with UVB at 25 mJ/cm². After 24 h, the cells were detached from the wells by treatment with trypsin, followed by staining with trypan blue; non stained cells were counted under an optical microscope with a hemocytometer.

HDFs (2×10⁶ cells) were irradiated with UVB (25 or 15 mJ/cm²); the cells were treated with decursin for 24 h and lysed using 40 μl of ice cold M-PER^® Mammalian Protein Extraction Reagent (Pierce Biotechnology, Inc., Rockford, IL, USA). Protein concentrations in the lysates were determined using the Bradford method (21). Samples were separated using 10% SDS-PAGE gels with 3% stacking gels; the resolved proteins were transferred to a Hybond™-PVDF membrane using a western blot apparatus (Bio-Rad). Polyvinylidene fluoride (PVDF) membranes were blotted with 1 μg/ml of primary antibodies for MMP-1, MMP-3, p50, p65, PCNA, or β-actin. HRP-conjugated IgG was used as a secondary antibody. Protein expression levels were determined by analyzing the signals captured on the PVDF membranes using an image analyzer (LAS-1000; Fuji Film, Japan).

Total RNA was extracted from cells using a FastPure™ RNA kit (Takara Bio, Inc., Shiga, Japan). RNA concentration and purity were determined by measuring the absorbance at both 260 and 280 nm. Then, cDNA was synthesized from 1 μg of total RNA using a PrimeScript™ RT reagent kit (Takara Bio, Inc.). MMP-1 and MMP-3 mRNA expression levels were analyzed using real-time PCR with the ABI PRISM 7900 sequence detection system and the SYBR-Green reagent (Applied Biosystems, Foster City, CA, USA). Primers used in the reaction were:MMP-1 (NM 002424.2) sense,5′-AGTGACTGGGAAACCGATGCTGA-3′ and antisense, 5′-CTCTTGGCAAATCTGGCCTGTAA-3′; MMP-3 (NM 002422) sense, 5′-ATTCCATGGAGCCAGGCTTTC-3′ and antisense, 5′-CATTTGGGTCAAACTCCAACTGTG-3′ and GAPDH (NM 002046) sense, 5′-ATGGAAATCCC ATCACCATCTT-3′ and antisense, 5′-CGCCCCACTTGA TTTTGG-3′. To control for variations in the mRNA concentration, all results were normalized to the housekeeping gene GAPDH. Relative quantitations were performed using the comparative ΔΔCt method according to the manufacturer’s instructions.

HDFs were seeded in 100-mm culture dishes at a density of 2×10⁶ cells/dish and then irradiated with UVB (25 mJ/cm²). Following 24 h of incubation, the culture supernatants were collected and centrifuged at 10,000 × g for 5 min to remove the particulate matter and stored at −80°C in fresh tubes. The protein concentration in the supernatants was determined using the Bradford method (21). The active MMP-1 in culture supernatants was quantified by fluorescent assay, using the Fluorokine E Human Active MMP-1 Fluorescent assay kit, and MMP-3 in the cell culture supernatants was then determined using Quantikine ELISA kits (all from R&D Systems), according to the manufacturer’s protocol.

HDFs (2×10⁶ cells) were irradiated with 25 mJ/cm² UVB and then treated with decursin for 3 h. Cells were immediately washed twice, scraped into 1.5 ml of ice cold PBS (pH 7.9) and then pelleted at 12,000 × g for 30 sec. Cytoplasmic and nuclear extracts were prepared from cells using NE-PER^® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology).

Activation of NF-κB and AP-1 was assayed with a gel mobility shift assay using nuclear extracts. An oligonucleotide containing the κ-chain (κB, 5′-CCGGTTAACAGAGGGGGCTTTCCGAG-3′) or AP-1 (5′-CGCTTGATGAGTCAGCCGGAA-3′) binding site was synthesized and used as a probe for the gel retardation assay. The two complementary strands were annealed and labeled with [α-32P]dCTP. Labeled oligonucleotides (10,000 cpm), 10 μg of nuclear extracts and binding buffer [10 mM Tris-HCl (pH 7.6), 500 mM KCl, 10 mM EDTA, 50% glycerol, 100 ng poly(dI·dC), 1 mM dithiothreitol] were then incubated for 30 min at room temperature in a final volume of 20 μl. The reaction mixtures were analyzed by electrophoresis on 4% polyacrylamide gels in 0.5X Tris-borate buffer. The gels were dried and examined by autoradiography. Specific binding was controlled by competition with a 50-fold excess and cold AP-1 oligonucleotide.

Statistical analysis was performed using analysis of variance (ANOVA) and Duncan’s test. A P-value <0.05 was considered to indicate a statistically significant result.

The structure of decursin is shown in Fig. 1A. To investigate the cytotoxicity of decursin, HDFs were treated with various concentrations of decursin for 24 h. Cell viability was determined using the MTT assay. Decursin did not cause a significant change in the viability of HDFs up to 50 μM (Fig. 1B). To investigate the cell protective effect of decursin on UVB-induced cytotoxicity, cells were incubated with the indicated concentrations of decursin for 24 h in the presence of UVB. UVB-induced cytotoxicity was determined using the trypan blue exclusion test. Decursin (10 and 30 μM) significantly inhibited cell toxicity induced by UVB irradiation (Fig. 1C).

UVB activates MMP secretion, which is a hallmark of skin aging (4,5,22). We examined the effects of decursin on UVB-induced expression of MMP-1 and MMP-3. Western blot analysis revealed that irradiation of HDFs with UVB (25 mJ/cm²) markedly increased MMP-1 and MMP-3 levels (Fig. 2A). The UVB-induced increase in MMP levels was significantly reduced by treatment with decursin. Consistent with these results, real-time PCR analysis also showed an increase in expression of MMP-1 and MMP-3 mRNA after UVB irradiation while treatment of HDFs with decursin suppressed this UVB-induced increase in MMP-1 and MMP-3 expression (Fig. 2B). We also determined the effect of decursin on UVB-induced MMP secretion with ELISA. UVB irradiation of HDFs resulted in an increase in MMP-1 and MMP-3 secretion, while decursin significantly diminished the UVB-induced MMP-1 and MMP-3 secretion (Fig. 2C). Decursin itself had no effects on expression and secretion of MMP-1 and MMP-3 in HDFs. These results indicate that decursin inhibits the UVB-induced expression and secretion of MMP-1 and MMP-3 in HDFs.

To clarify the mechanism of decursin-mediated inhibition of MMP-1 and MMP-3 expression, the effect of decursin on UVB-induced activation of NF-κB and AP-1 was evaluated using EMSA and western blot analysis. As shown in Fig. 3A and B, pre-treatment with decursin inhibited UVB-induced DNA binding activity of NF-κB, but not AP-1. Decursin itself had no effect on the DNA binding activity of NF-κB or AP-1. Additionally, we determined the levels of p65, p50, p-c-Jun in the nuclear fraction. Cell treatment with UVB resulted in increased levels of p65, p50 and p-c-Jun; however, decursin blocked the UVB-induced translocation of p65 and p50 to the nucleus (Fig. 3C). These results suggest that decursin specifically blocks NF-κB activation in HDFs. The IκB kinase (IKK) enzyme complex is part of the signal transduction cascade upstream of NF-κB. IKK specifically phosphorylates the inhibitory IκB protein. Under basal conditions, the cytoplasmic protein IκB directly binds to p65 and p50 subunits and represses their nuclear translocation. IKK phosphorylation results in the dissociation of IκB from NF-κB and thereby activates NF-κB (23–26). Therefore, we determined the changes in the levels of p-IKKαβ and p-IκBα in the cytoplasmic fraction. The cytoplasmic fraction of UVB-stimulated HDFs showed higher levels of p-IKKαβ and p-IκBα than in unstimulated cells; however, the UVB-induced increase in the levels of p-IKKαβ and p-IκBα was significantly suppressed by treatment with decursin (Fig. 3D).

Since MAP kinase is an upstream regulator of NF-κB and AP-1, the role of MAP kinase (ERK, p38 and JNK) in the activation of MMP expression is fairly well understood (27,28). We investigated the effect of decursin on UVB-induced activation of MAP kinase. Decursin showed no effects on MAPK (Fig. 4). These results suggest that the MAPK pathway is not involved in the regulation of UVB-induced expression of MMP by decursin.