Dimethyl sulfoxide (DMSO; cat. no. D8418), ascorbic acid (AA; cat. no. A5960) and MTT (cat. no. M2128) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM; cat. no. BE12-604F) and penicillin/streptomycin (P/S) cocktail (cat. no. BW17-718R) were purchased from Lonza Group Ltd., (Basel, Switzerland). Fetal bovine serum (FBS; cat. no. S001-01) was purchased from WELGENE, Inc., (Gyeongsan, Republic of Korea). The anti-Bcl-2 (cat. no. ab7973) and anti-Bax (cat. no. ab7977) primary antibodies were obtained from Abcam (Cambridge, MA, USA). The anti-β-actin (cat. no. sc-81178) primary antibody was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Horseradish peroxidase (HRP)- conjugated goat anti-rabbit immunoglobulin G (IgG; H+L; cat. no. 111-035-045) and goat anti-mouse IgG (H+L; cat. no. 115-035-062) secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). UV irradiation was performed with a UVA sunlamp (Sankyo Denki, Hiratsuka, Japan). An inverted microscope (CKX41; Olympus Corporation, Tokyo, Japan) was used for observation of cell growth and a CO₂ incubator (MCO-17AC; Sanyo Electric Co., Ltd., Osaka, Japan) was used for cell culture. A rotary vacuum concentrator (BÜCHI R-205; BÜCHI Labortechnik AG, Flawil, Switzerland) was used for sample extraction.

The root of Stachys riederi var. japonica was purchased from DONG UI Chosukjam Farmers, Inc. (Sancheong, Korea). A voucher specimen was deposited in the herbarium of our laboratory at the Department of Public Health, Faculty of Food and Health Sciences (Keimyung University, Daegu, Korea) with the plant identification no. KMU/J/638. A pulverized sample (50 g) was put into a flask and extracted in 500 ml 80% ethanol three times for 24 h each at 25°C. The extract was filtered and concentrated using rotary vacuum evaporator followed by lyophilization (yield 46.7%).

HDFs (Amore Pacific Company, Seoul, Korea) were grown in DMEM supplemented with 10% heat-inactivated FBS and 1% P/S cocktail in a humidified atmosphere of 95% air and 5% CO₂ at 37°C.

The effect of SREE on the viability of HDFs was assessed by MTT assay. HDFs were seeded in a 96-well plate (1×10⁴ cells/100 µl/well) overnight. HDFs were treated with different concentrations of SREE (25, 50, 100 and 200 µg/ml) for 48 h. Subsequently, MTT (0.5 mg/ml) was added to each well of the 96-well plate and the cells were incubated at 37°C for an additional 3 h. The 96-well plate was centrifuged 25°C at 168 × g for 10 min. Following removal of the supernatant, 200 µl DMSO was added to each well. Subsequent to dissolving the formazan with DSMO in the cells with a plate-shaker for 15 min, the absorbance at 540 nm was read using an ELISA plate reader.

HDFs were cultivated in a culture dish (1.5×10⁵ cells/ml) until they reached ~80% confluence. Following removal of the culture medium, HDFs were washed with PBS and exposed to 6.3 J/cm² UVA in DMEM without FBS prior to treatment with SREE or AA.

Cellular ROS levels were measured using dichlorodihydro-fluorescein-diacetate (DCFH-DA). Following UVA irradiation, HDFs were incubated at 37°C with freshly prepared pre-warmed 10 mM DCFH-DA for 20 min. HDFs were subsequently rinsed twice with PBS and levels of green fluorescence, corresponding to the levels of cellular ROS, were detected through a 520 nm long-pass filter on an FV-1000 laser fluorescence microscope (Olympus Corporation; magnification, ×100). The levels of cellular ROS were quantified by a fluoro-photometer with DataMax version 2.2 and GRAMS/32 software (HORIBA Jobin Yvon, Inc., Edison, NJ, USA), using an excitation wavelength of 480 nm and an emission wavelength of 530 nm.

Following treatment, total RNA from the conditioned HDFs was isolated using TRIzol^® (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Equal amounts of total RNA (5 µg) were reverse transcribed in a 40 µl reaction mixture containing 8 µl Molony Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) 5X buffer, 3 µl 10 mM dNTPs, 0.45 µl 40 U/µl RNase inhibitor, 0.3 µl 200 U/µl M-MLV RT (Promega Corporation, Madison, WI, USA) and 3.75 µl 20 µM oligo dT (Bioneer Corporation, Daejeon, Korea). Single-stranded cDNA was amplified by PCR using 4 µl 5X Green Go-Taq^® Flexi reaction buffer, 0.4 µM 10 mM dNTPs, 0.1 µl 5 U/µl Taq polymerase, 1.2 µl 25 mM MgCl₂ (Promega Corporation), and 0.4 µl primer (20 pM/µl). The primer sequences used for PCR were as follows: Bcl-2 forward, 5′-CGACGACTTCTCCCGCCGCTACCGC-3′ and reverse, 5′-CCGCATGCTGGGCCGTACAGTTCC-3′; Bax forward, 5′GGCAATCTGACCTTCAACTG-3′ and reverse, 5′-AGTCTCTTGAGGACCCAACC-3′; β-actin forward, 5′-CCCACTAACATCAAATGGGG-3′ and reverse, 5′-ACACATTGGGGGTAGGAACA-3′. The PCR thermocycler conditions were as follows: Bcl-2, 38 cycles of denaturation at 94°C for 1 min, annealing at 56°C for 90 sec and extension at 72°C for 2 min; Bax, 35 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min and extension at 72°C for 2 min. The expression level of β-actin mRNA was used as an internal control to evaluate the relative mRNA expression of Bcl-2 and Bax. PCR products were visualized using ethidium bromide staining on a 1.2% agarose gel. DNA band density was semi-quantitatively analyzed using a Kodak Gel Logic 100 image analysis system with Kodak molecular imaging software, version 4.0 (Eastman Kodak Company, Rochester, NY, USA).

Following treatment, HDFs were washed with PBS and lysed on ice for 15 min using 0.1 M Tris-HCl (pH 7.2) buffer containing 1% Nonidet P-40, 0.01% SDS and a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Following centrifugation at 12,074 × g for 20 min at 4°C, the supernatants were collected, and protein concentrations were determined by bicinchoninic acid assay (Pierce; Thermo Fisher Scientific, Inc.).

Equal amounts of protein (10 µg) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). The membranes were washed with TBS (10 mM Tris-Cl, 150 mM NaCl; pH 7.5) with 0.05% (v/v) Tween-20 (TBST) followed by blocking for 2 h at 25°C with TBST containing 5% (w/v) non-fat dried milk. The membranes were incubated overnight with antibodies specific for Bcl-2 (1:1,000), Bax (1:500) or β-actin (1:10,000) at 4°C. The membranes were subsequently exposed to HRP-conjugated secondary antibodies of a goat anti-rabbit IgG (H+L; 1:2,000) or goat anti-mouse IgG (H+L; 1:2,000) for 2 h at room temperature and additionally washed three times with TBST. Immunoreactivity was detected using an enhanced chemiluminescence reagent according to the manufacturer's protocol (Amersham; GE Healthcare, Chicago, IL, USA). Band intensity was semi-quantified using ImageJ software (version 1.8.0; National Institutes of Health, Bethesda, MD, USA). β-actin was used as an internal control.

The ΔΨm was visualized by using 5,5′,6,6′-tetrachloro-1,1′3,3′-tetrathylbenzimidazolyl-carbocyanine iodide (JC-1) staining. JC-1 aggregates and monomers represent high and low ΔΨm, as indicated by red and green fluorescence, respectively. Following treatment, HDFs were incubated with 5 µg/ml JC-1 for 30 min at 37°C. JC-1 monomers and JC-1 aggregates were observed at an emission wavelength of 488 nm (green) and 561 nm (red), respectively. Images were acquired using an FV-1000 laser fluorescence microscope (Olympus Corporation; magnification, ×400).

Following treatment, caspase-3 activity in HDFs was determined using a colorimetric caspase-3 assay kit (cat. no. APT165; EMD Millipore) according to the manufacturer's protocol. Following UVA irradiation, HDFs were harvested and lysed in cell lysis buffer (cat. no. 90065) provided in the assay kit. Assays were performed in 96-well microtiter plates by incubating 20 µg cell lysates in 100 µl reaction buffer (1% NP-40, 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 10% glycerol) containing a caspase substrate (Asp-Glu-Val-Aspchromophore-p-nitroanilide). Lysates were incubated at 37°C for 90 min. Subsequently, the absorbance at 405 nm was measured with a spectrophotometer.

Following treatment, HDFs were washed with PBS and lysed in a lysis buffer [0.3 M Tris-HCl (pH 7.5), 0.1 M NaCl, 0.01 M EDTA and 0.2 M sucrose] followed by the addition of RNase A (0.5 µg/ml) for an additional 18 h at 55°C. The lysates were centrifuged at 25°C, 10,000 × g for 20 min, and nuclear DNA in the supernatant was extracted with an equal volume of neutral phenol-chloroform-isoamyl alcohol mixture (25:24:1) and analyzed by electrophoresis on a 1.7% agarose gel. DNA was visualized and images were captured under UV illumination following staining with ethidium bromide (0.1 µg/ml). All observations made concerning this data were made visually.

Data are presented as the mean ± standard deviation of three independent experiments. Differences between groups were evaluated by one-way analysis of variance followed by a Duncan multiple range test for post-hoc comparison using SPSS 21.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Initially, the effect of treatment with SREE at different concentrations for 48 h on the growth of HDFs was evaluated by an MTT assay. Notably, compared with the control (vehicle), treatment with SREE at 25 and 50 µg/ml slightly increased the viability of HDFs (Fig. 1). In addition, treatment with SREE at 100 and 200 µg/m only decreased the viability of HDFs by 2.7 and 9.9%, respectively. These results suggest that SREE at the concentrations examined has no or little cytotoxicity to HDFs.

To identify any antioxidant activity of SREE, the effect of treatment with SREE at different concentrations for 24 h on ROS production in UVA-irradiated HDFs was examined using fluorescence microscopy. For this, the fluorescein derivative DCFH-DA, a redox indicator, was used. As demonstrated in Fig. 2A, compared with the control (no UVA), HDFs exposed to UVA exhibited extensive green fluorescence staining, indicative of a marked increase in cellular ROS generation following UVA exposure. However, treatment with SREE led to a concentration-dependent decrease in green fluorescence staining in UVA-irradiated HDFs. As hypothesized, 100 µg/ml treatment with AA, used as an antioxidant positive control, additionally markedly decreased UVA-induced green fluorescence staining in HDFs. The quantitative fluorescence intensity is presented in Fig. 2B. Compared with UVA-treated HDFs, treatment with SREE at 50, 100 and 200 µg/ml decreased ROS generation by 6.7, 24.0 and 30.1%, respectively. It was additionally observed that 100 µg/ml AA treatment decreased ROS generation, similar to the inhibition by SREE at 200 µg/ml.

Given that disruption of the ΔΨm is crucial for UV-induced apoptosis of human keratinocytes (20), the present study aimed to examine whether UVA altered the ΔΨm in HDFs, and if SREE was involved, using fluorescence microscopy. The ΔΨm was visualized by JC-1 staining, in which high and low ΔΨm were indicated by JC-1 aggregates (red) and JC-1 monomers (green), respectively. As demonstrated in Fig. 3, control HDFs (no UVA) exhibited marked red staining. However, HDFs that were exposed to UVA exhibited extensive green staining, suggesting a decrease of the ΔΨm in UVA-irradiated cells. Notably, while treatment with SREE at 50 µg/ml had no effect on UVA-induced decrease of the ΔΨm in HDFs, SREE treatment at 100 or 200 µg/ml led to a concentration-dependent increase of red staining in the cells. At 100 µg/ml, AA treatment additionally markedly inhibited the UVA-induced decrease of the ΔΨm in HDFs.

The effect of SREE treatment at different concentrations for 24 h on the protein expression levels of Bcl-2 and Bax, mitochondrial membrane-associated proteins, was subsequently determined in UVA-irradiated HDFs using western blot analysis. As demonstrated in Fig. 4A, compared with the control (no UVA), UVA irradiation did not significantly affect the protein expression of Bcl-2 in HDFs. However, UVA irradiation markedly increased the protein expression of Bax in HDFs. Notably, SREE treatment concentration-dependently increased the protein expression of Bcl-2 in UVA-irradiated HDFs. In addition, SREE treatment concentration-dependently inhibited the ability of UVA to increase Bax protein expression in HDFs. At 100 µg/ml, treatment with AA additionally led to a slight increase in Bcl-2 protein expression and decreased expression levels of Bax protein in UVA-irradiated HDFs, compared with cells exposed to only UVA. RT-PCR assays were performed to examine whether SREE affected mRNA expression levels of Bcl-2 and Bax in UVA-irradiated HDFs. As presented in Fig. 4B, compared with the control (no UVA), UVA irradiation markedly downregulated mRNA expression of Bcl-2 while upregulated that of Bax in HDFs. Notably, SREE treatment at the concentrations examined markedly attenuated the ability of UVA to downregulate Bcl-2 mRNA expression in HDFs. In addition, SREE treatment concentration-dependently inhibited UVA-induced Bax mRNA upregulation in HDFs. Compared with the cells exposed to only UVA, AA treatment at 100 µg/ml additionally markedly inhibited the ability of UVA to decrease the mRNA expression of Bcl-2 and increase that of Bax in HDFs.

The effects of SREE treatment at different concentrations for 24 h on the activity of caspase-3, a member of the caspase family involved in UV-induced apoptosis (20), was investigated in UVA-irradiated HDFs. As demonstrated in Fig. 5, compared with the control (no UVA), UVA irradiation substantially increased the activity of caspase-3 in HDFs. SREE treatment at the concentrations examined significantly attenuated the ability of UVA to activate caspase-3 in HDFs. However, treatment with AA at 100 µg/ml did not significantly inhibit UVA activation of caspase-3 in HDFs.

Given that nuclear DNA fragmentation is a hallmark of apoptosis (7,21), whether UVA induces DNA fragmentation in HDFs, and whether SREE is involved in the mechanism, was examined using a DNA fragmentation assay. As indicated in Fig. 6, compared with the control (no UVA), UVA irradiation markedly induced DNA fragmentation in HDFs. Notably, SREE treatment inhibited UVA-induced DNA fragmentation in HDFs in a concentration-dependent manner. AA treatment at 100 mg/ml additionally markedly inhibited UVA-induced DNA fragmentation in HDFs.