Samples of S. muticum were collected from Udo (Jeju Island, Korea) and identified by Dr Dong Sam Kim (Jeju Biodiversity Research Institute, Jeju, Republic of Korea). A voucher specimen (A10-0000107) was deposited at the herbarium of Jeju Biodiversity Research Institute (Jeju, Korea). S. muticum was extracted with 80% ethanol under reflux. Subsequently, the 80% ethanol extract was suspended in distilled water and fractionated successively with n-hexane, dichloromethane, ethyl acetate and n-butanol (Merck Millipore, Darmstadt, Germany). The ethyl vacetate fraction of S. muticum (SME) was used for further experiments.

The primary antibody against MMP-1 was purchased from Epitomics (Burlingame, CA, USA), and the primary antibody against actin was obtained from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies against c-Jun and phosphorylated (phospho)-c-Jun were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). All other chemicals and reagents were of analytical grade, unless otherwise stated.

HR-1 strain hairless male mice (6 weeks old; 22–24 g) were purchased from Japan SLC, Inc. (Shizuoka, Japan) and allowed to acclimate to conditions in the facility for 1 week prior to experimentation. The animals were housed in climate-controlled quarters (24°C at 50% humidity) with a 12/12 h light/dark cycle and free access to standard rodent chow and water. Subsequent to the 1 week acclimation period, the mice were divided into three groups: An untreated control group (n=5), a UVB-treated vehicle group (n=5) and a UVB-treated SME group (n=5). The mice in the SME group were orally administered with SME (100 mg/kg body weight in 0.1 ml of water per day). SME was administered 5 days/week for 12 weeks. Exposure to UVB irradiation was then performed using an UVM-225D Mineralight UV display lamp (UVP, Inc., Phoenix, AZ, USA) emitting UV light at a wavelength of 302 nm. The UV strength was measured using a HD2102-2 UV meter (Delta OHM, Padova, Italy). UVB radiation was applied to the skin on the backs of the animals three times per week for 12 weeks, with the UVB dose progressively increased between 60 mJ/cm² per exposure in week 1 (one minimal erythematous dose=60 mJ/cm²) and 120 mJ/cm² per exposure in week 7. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Korea Institute of Oriental Medicine (Daejeon, Korea; approval no. 11-061). All experimental protocols were approved by the Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee (11-061).

The HaCaT keratinocyte cell line was obtained from Amore Pacific Company (Yongin, Korea). The cells were maintained at 37°C in an incubator with a humidified atmosphere of 5% CO₂, and were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% heat-inactivated fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.), streptomycin (100 µg/ml) and penicillin (100 U/ml). For irradiation, the cells were exposed to UVB light once at a dose of 30 mJ/cm². The CL-1000 M UV Crosslinker (UVP, Inc.) was used as the UVB source, which delivered a UVB energy spectrum of 280–320 nm.

Mice were anesthetized with intraperitoneal administration of a diluted solution (1:4 in phosphate-buffered saline) composed of a 2:1 mixture of 30 mg/kg Zoletil (Virbac, Carros, France) and 10 mg/kg Rompun (Bayer, Leverkusen, Germany) at the end of final oral administration. Mice were sacrificed using a CO₂ chamber subsequent to the final oral administration. Mouse dorsal skin replicas were obtained using a Repliflo Cartridge kit (CuDerm Corporation, Dallas, TX, USA). An image of the dorsal skin of each mouse was captured prior to the sacrifice of the animal. The impression replicas were placed on a horizontal sample stand, and wrinkle shadows were generated by illumination using a fixed-intensity optical light at an angle of 35°. Black and white images were recorded using a charge-coupled device camera and were analyzed using Skin-Visiometer^® VL 650 software (Courage + Khazaka Electronic GmbH, Cologne, Germany). The average wrinkle lengths and depths were measured, and used as parameters to assess skin wrinkling.

The thickness of the epidermis was measured via light microscopy using an eyepiece micrometer (AX-70; Olympus Corporation, Tokyo, Japan). For this analysis, the dorsal skin (2 × 2 cm samples) was obtained under anesthesia and was fixed in 10% neutral-buffered formalin, embedded in paraffin and sectioned at a thickness of 5 µm. The sections were stained with hematoxylin and eosin to evaluate tissue morphology, or with Masson's trichrome stain for collagen fiber analysis.

The harvested HaCaT keratinocytes were lysed by incubating the cells on ice for 10 min in 100 µl PRO-PREP™ protein extraction solution (Intron Biotechnology (Seongnam, Korea). The cell lysates were centrifuged at 16,000 × g at 4°C for 5 min, and the supernatants were removed from the lysates. Protein concentrations were then determined using Quant-iT™ Protein Assay kit (Invitrogen; Thermo Fisher Scientific, Inc.). Aliquots of the lysates (40 µg of protein) were boiled for 5 min and electro-phoresed in a 10% sodium dodecyl sulfate-polyacrylamide gel. The proteins within the gel were then transferred onto nitrocellulose membranes, and the membranes were subsequently incubated with the rabbit monoclonal antibodies against MMP-1 (cat. no. #1973-1; 1:1,000) and phospho-c-Jun (Ser73; cat. no. D47G9; 1:1,000). Following incubation with primary antibodies, the membranes were further incubated with secondary anti-immunoglobulin G/horseradish peroxidase conjugates (Pierce Biotechnology, Inc., Rockford, IL, USA). Protein bands were visualized using an Enhanced Chemiluminescence Western Blotting Detection kit (GE Healthcare Life Sciences, Little Chalfont, UK).

MMP-1 activity was assessed using a Fluorokine^® E Human Active MMP-1 Fluorescent Assay kit (R&D Systems, Inc., Minneapolis, MN, USA). This assay uses a quenched fluorogenic substrate to detect enzyme activity. Production of the fluorescent cleavage product was measured using a fluorescence plate reader (BMG Labtech, Ortenberg, Germany) with excitation and emission wavelengths of 320 and 405 nm, respectively.

The ChIP assay was performed using a SimpleChIP™ Enzymatic Chromatin IP kit (Cell Signaling Technology, Inc.), according to the manufacturer's protocol with slight modification. Briefly, the HaCaT keratinocytes were crosslinked by the addition of 1% formaldehyde, and the prepared chromatin was enzymatically digested with nuclease for 20 min at 37°C. Primary antibody against the activator protein-1 (AP-1) family member, c-Jun rabbit monoclonal antibody (60A8; cat. no. #9165; 1:50; Cell Signaling Technology, Inc.), and normal rabbit polyclonal IgG (cat. no. #2729; 1:500; Cell Signaling Technology, Inc.) were added to the chromatin digest, which was then incubated overnight with constant rotation at 4°C. To capture the immunoprecipitated DNA/protein complexes, ChIP-grade protein G magnetic beads were added to the digest. Pellet protein G agarose was obtained by brief centrifugation (5,000 × g for 1 min at 4°C) and the supernatant fraction was removed. The protein G agarose-antibody/chromatin complex was washed by resuspending the beads in 1 ml each of the cold buffers in the order listed below, and incubation for 5 min on a rotating platform followed by brief centrifugation (5,000 × g for 1 min at 4°C) and careful removal of the supernatant fraction. The immunoprecipitates were eluted with ChIP elution buffer. The crosslinking was reversed by incubating the eluent at 65°C for 30 min, followed by the addition of proteinase K and further incubation at 65°C for 2 h.

The DNA fragments recovered from the immunoprecipitated DNA/protein complex were purified on spin columns. DNA was amplified in a reaction containing primers, dNTPs, and 0.5 U Taq DNA polymerase (Intron Biotechnology) at a final volume of 25 µl. The PCR conditions were: 5 min at 95°C for initial denaturation, followed by 45 cycles of 30 sec at 94°C, 1 min at 49°C and 1 min at 72°C, and a final elongation period of 7 min at 72°C. PCR amplification was conducted in an Applied Biosystems 2720 thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers for the MMP-1 gene promoter were as follows: Sense 5′-CCTCTT GCTGCTCCAATATC-3′ and antisense 5′-TCTGCTAGGAGTCACCATTTC-3′ (Bioneer Corporation, Daejeon, Korea). The promoter region of the MMP-1 gene exists between −67 and +94 of the MMP-1 gene sequence, with reference to the transcription start site. The PCR products were separated on 2% agarose gels, and the DNA bands were visualized using RedSafe™ nucleic acid staining solution (Intron Biotechnology) and an ImageQuant 350 digital imaging system (GE Healthcare, Wankesha, WI, USA).

All measurements were obtained in triplicate, and all values are presented as the mean ± the standard error of the mean. The results were subjected to two-way analysis of variance followed by the F-test using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) to analyze differences between conditions. In all cases, P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of SME on UVB-induced wrinkle formation in vivo, the skin on the backs of HR-1 hairless mice was exposed to UVB light for a period of 12 weeks. UVB exposure resulted in macroscopic wrinkle formation in the dorsal skin of the vehicle-treated mice, compared with the unexposed control mice. However, wrinkling of the UVB-irradiated skin was markedly inhibited by SME pretreatment (Fig. 1A). To further evaluate the protective actions of SME against UVB-mediated skin damage, replicas of the mouse dorsal skin were obtained (Fig. 1B). SME visibly inhibited wrinkle formation in replica images of UVB-exposed skin, consistent with the macroscopic observations. Skin replicas were further evaluated using an image analysis system to quantify the degree of wrinkle formation. The mean length and depth of the wrinkles were significantly higher in the UVB-treated vehicle group, compared with those in the unexposed control group (Fig. 2A and B). However, the mean wrinkle length and depth were significantly decreased in the UVB-treated mice pretreated with SME.

As epidermal thickness is used as a quantitative parameter to assess inflammation and skin photoaging (16), the present study subsequently evaluated changes in the dermal thickness of the skin of UVB-irradiated mice, compared with unexposed control hairless mice. Measurement of the tissue sections indicated that the epidermal thickness of the dorsal skin was significantly increased following UVB irradiation in vehicle-pretreated mice. By contrast, epidermal hypertrophy was significantly reduced by SME pretreatment (Fig. 3A). Additionally, histological observations of hematoxylin and eosin-stained sections revealed that the epidermal thickness in the UVB-irradiated dorsal skin of the vehicle group was markedly increased. However, SME pretreatment inhibited the increase in epidermal thickness (Fig. 3B). The tissue sections were then subjected to Masson's trichrome staining to visualize changes in collagen fiber formation in the dermal areas of the UVB-exposed dorsal skin. Notably, the collagen fibers (blue) in the UVB-treated/SME-pretreated skin showed lower levels of damage and an increased extent of bundle formation, compared with those in the UVB-treated/vehicle-pretreated skin (Fig. 3C).

The cultured HaCaT keratinocytes were exposed to UVB radiation at a dose of 30 mJ/cm², and the protein expression level of MMP-1 was measured using western blot analysis. As shown in Fig. 4A, SME pretreatment inhibited the accumulation of UVB-induced MMP-1 protein 24 h following exposure. Furthermore, the activity of MMP-1 was significantly enhanced by UVB radiation, but reduced by SME pretreatment (Fig. 4B).

The MMP-1 promoter contains a binding site for the AP-1 transcription factor, and activation of AP-1 by UVB exposure increases the expression of MMP-1 (17). However, SME pretreatment inhibited UVB-induced binding of the AP-1 family member, c-Jun, to the MMP-1 promoter, as determined using a ChIP assay (Fig. 4C). Furthermore, UVB treatment markedly stimulated the expression of phospho-c-Jun, whereas SME treatment decreased its expression (Fig. 4D).