NHDFs (Lonza Group, Ltd., Basel, Switzerland) and the NF-κB Luciferase Reporter NIH3T3 Stable Cell Line (Signosis, Inc., Santa Clara, CA, USA) were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere containing 5% CO₂. H₂O₂ (Sigma-Aldrich; Merck KGaA) was diluted in phosphate-buffered saline (PBS) to obtain a 1 M stock solution. For the H₂O₂ treatment experiments, the H₂O₂ stock solution was diluted in cell culture media at the indicated concentrations. RA (Sigma-Aldrich; Merck KGaA) was dissolved in dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA).

Cytotoxicity was evaluated using a water-soluble tetrazolium salt (WST-1) assay (EZ-Cytox Cell Viability Assay kit; Itsbio, Seoul, Korea). NHDFs were seeded on 96-well culture plates at a density of 3×10³ cells and were incubated for 24 h, at 37°C. Subsequently, the cells were incubated with RA (0–50 µM) for 12 h, after which the cytotoxicity of RA was assessed. To examine the effects of RA against H₂O₂-induced cytotoxicity, NHDFs were incubated with the indicated concentration of RA (10, 20 and 30 µM) for 12 h prior to H₂O₂ treatment. After 12 h, 600 µM of H₂O₂ was added and incubated at 37°C for 2 h. 1/10 volume of WST-1 solution was then added to each well, and the cells were incubated at 37°C for 1 h. Cell viability was evaluated by measuring absorbance at 450 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The H₂O₂ scavenging activity of RA was evaluated as previously described (26). A solution containing H₂O₂ (40 mM in distilled water, pH 7.4) and varying concentrations of RA (0–1 mg/ml) was incubated at room temperature for 10 min. Scavenger activity was subsequently assessed by measuring the absorbance of the H₂O₂/RA solution at 230 nm using a UV spectrophotometer (Shimadzu Corporation, Kyoto, Japan). A solution lacking H₂O₂ was used as a control, and L-ascorbic acid was used as the experimental control treatment. The experiments were conducted in triplicate at each concentration of RA. Scavenger activity was calculated using the following formula: H₂O₂ scavenging activity (%) = (1 - sample/control) × 100.

Intracellular ROS production was evaluated using a 2′7′-dichlorofluorescein diacetate (DCF-DA; Sigma-Aldrich; Merck KGaA) staining assay. Briefly, cells were incubated with DCF-DA solution (100 µM) at 37°C for 1 h, after RA and H₂O₂ treatment. ROS levels were then analyzed using flow cytometry. The proportion of fluorescence-positive cells was measured using a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA, USA) with excitation and emission filters of 488 and 530 nm, respectively. N-acetyl-cysteine (NAC), ROS scavenger used as a positive control, was purchased from Sigma-Aldrich; Merck KGaA.

The NF-κB luciferase reporter assay was conducted as previously described (27) with some modifications. Briefly, NF-κB reporter NIH3T3 cells were seeded in 12-well cell culture plates at a density of 2×10⁵ cells/well. After 24 h incubation, cells were pretreated RA for 12 h at 10, 20 and 30 µM, and were then treated with 600 µM H₂O₂ for 2 h. At the end of the experiments, the cells were resuspended in Passive Lysis Buffer (Promega Corporation, Madison, WI, USA). Subsequently, luciferin (Sigma-Aldrich; Merck KGaA) was added to the cells, and luciferase activity was measured using a Veritas luminometer (Turner Designs, Sunnyvale, CA, USA). The luciferase activity was normalized to β-galactosidase (β-gal) activity, and relative activity is presented as the percentage activity of the control with standard deviation. The results represent the mean of three independent experiments.

β-gal expression was used as a marker of senescence (28). β-gal expression levels were determined using an SA-β-gal staining kit (Biovision, Inc., Milpitas, CA, USA) according to the manufacturer's instructions. NHDFs were seeded at a density of 1×10⁶ cells/well in 60-mm cell culture plates and were incubated until cell confluence reached 90%. Later, cells were pretreated RA for 12 h at 10, 20 and 30 µM, and were then treated with 600 µM H₂O₂ for 2 h. After treatment, the cells were washed in PBS and incubated in 0.5 ml fixative solution (4% formaldehyde, 0.5% glutaraldehyde in PBS buffer, pH 7.2) for 10 min at room temperature. The fixed cells were incubated in a staining solution mixture [staining solution (470 µl), staining supplement (5 µl) and 20 mg/ml X-Gal in dimethylformamide (25 µl)] for 24 h at 37°C. Subsequently, 70% glycerol (1 ml) was added to the cells and blue SA-β-gal-positive cells were counted under a microscope (Olympus IX51; Olympus Corporation, Tokyo, Japan).

Before RNA extraction, cells were treated with RA (10, 20 and 30 µM) for 12 h and H₂O₂ (600 µM) for 2 h, in a consecutive manner. Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. RNA purity and concentration were evaluated using a MaestroNano^® microspectrophotometer (Maestrogen, Las Vegas, NV, USA). Total RNA was reverse transcribed into cDNA using the oiligo d(T)₂₃ primer and ProtoScript First Strand cDNA Synthesis kit (New England BioLabs, Inc., Ipswich, MA, USA) following the manufacturer's instructions. HO-1, SOD1, CAT, SIRT1, TNF-α and IL-6 mRNA expression levels were evaluated using 5× HOT FIREPOL^® EvaGreen^® qPCR mix (Solis BioDyne, Tartu, Estonia), a StepOnePlus Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and gene-specific primers. Each thermo-cycling condition was same in denaturation (95°C) and extension (72°C), except the annealing temperature which differed when different primers were used. The Cq value associated with each gene was normalized to the expression of the β-actin housekeeping gene. The sequences of the qPCR primers are provided in Table I. The 2^−ΔΔCq method (29) was used to calculate the relative expression levels of each gene.

Transcriptional activity of NRF2 was determined using an antioxidant ARE Reporter kit (cat. no. 60514; BPS Bioscience, San Diego, CA, USA). The luciferase activity was quantitatively assessed according to the manufacturer's instructions. Briefly, NHDFs were cotransfected with NRF2-ARE-luciferase reporter plasmid (BPS Bioscience) and a plasmid that constitutively expressed Renilla luciferase using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The transfection was conducted according to the manufacturer's instructions with some modifications. The Lipofectamine reagent was mixed with serum-free Opti-MEM (Invitrogen; Thermo Fisher Scientific, Inc.) for 5 min, and then added to the plasmid with gently flicking for 20 min, at room temperature. The transfection mixture was added to the cells and incubated at 37°C. After 24 h of transfection, cells were pretreated for 12 h with the indicated doses of RA (10, 20 and 30 µM), and were then exposed to 600 µM H₂O₂ for 2 h. The cells were re-suspended in Passive lysis buffer (Promega Corporation) and luciferase activity was measured using a Dual-Luciferase Reporter Assay system (Promega Corporation). The luciferase activity of ARE was normalized to Renilla luciferase activity.

All results are presented as the mean ± standard deviation of three independent experiments. Statistical analysis was performed using SPSS software version 17.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance followed by Tukey post hoc test was conducted for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The present study initially determined the dose range of RA that was not markedly cytotoxic to NHDFs. The cells were treated with increasing concentrations of RA (0–50 µM) for 12 h. Concentrations of RA <50 µM were not were not significantly cytotoxic; therefore, 10–30 µM RA was used for subsequent experiments. To determine if RA affects the viability of NHDFs exposed to H₂O₂-induced cellular stress, NHDFs were sequentially treated with RA and H₂O₂, and cytotoxicity was evaluated using a WST-1 assay. As presented in Fig. 1, cell viability decreased to 76% of the control in cells treated with 600 µM H₂O₂ for 12 h. Pretreatment with 10–30 µM RA for 12 h significantly inhibited H₂O₂-induced cytotoxicity (Fig. 1B).

H₂O₂-mediated ROS activates endogenous cellular responses to oxidative stress (30). However, when this defense system collapses, superoxide anion, hydroxyl radical and singlet oxygen production induces cellular senescence and apoptosis (31–33). In the present study, H₂O₂ induced cellular senescence in NHDFs, as demonstrated by an SA-β-gal assay (Fig. 2). Notably, RA markedly inhibited cellular senescence in a concentration-dependent manner in H₂O₂-exposed NHDFs (Fig. 2), thus suggesting that RA protects NHDFs from H₂O₂-mediated induction of SA-β-gal activity.

Similar to previous studies, the present study investigated whether the protective effects of RA were associated with its oxidative properties (23–25). To evaluate the ROS scavenging activity of RA, H₂O₂ scavenging activity, DCF-DA fluorescence intensity, NRF2 activity and the expression of genes that regulate oxidative stress (HO-1, SOD1 and CAT) were assessed. RA showed an increase in H₂O₂ scavenging activity and ascorbic acid was used as positive control (Fig. 3A). Although the free radical scavenging activity of RA was less than the positive control (L-ascorbic acid), the half maximal inhibitory concentration of RA was 0.56 mg/ml, and the radical scavenging activity of RA occurred in a dose-dependent manner. DCF fluorescence intensity, which is an indicator of intracellular ROS levels, was evaluated in RA-pretreated NHDFs with or without H₂O₂ exposure. DCF intensity increased >8-fold in H₂O₂-exposed cells compared with the nonexposed control group (Fig. 2B). However, this effect was strongly reduced in NHDFs pretreated with either RA or the positive control antioxidant NAC (Fig. 3B). NRF2 serves a key role in the regulation of antioxidant mechanisms by activating the transcription of genes encoding antioxidant enzymes, including HO-1, SOD1 and CAT, via the ARE in the target gene promoter (5–7). Therefore, the present study analyzed the transcriptional activity of NRF2 using the ARE-luciferase assay. As presented in Fig. 3C, RA markedly enhanced luciferase activity in a concentration-dependent manner, thus suggesting that RA activates NRF2 activity in NHDFs. In addition, the expression levels of the NRF2 target genes, HO-1, SOD1 and CAT, were detected. Notably, RA markedly upregulated the target genes against H₂O₂-induced oxidative stress (Fig. 3D). These results suggested that RA exerts a protective effect on H₂O₂-induced oxidative stress in NHDFs via NRF2-associated antioxidant mechanisms.

The mammalian Sir2 ortholog, SIRT1, is a histone deacetylase that targets histones and nonhistone substrates, including NF-κB and p53 (8,9,34). Previous studies have demonstrated that SIRT1 is involved in the regulation of numerous cellular processes, including inflammation, apoptosis and autophagy, and that SIRT1 expression is downregulated in response to oxidative stress (10–12,35). Therefore, the present study evaluated the expression levels of SIRT1 and various inflammatory cytokines, including NF-κB, TNF-α and IL-6 in NHDFs. SIRT1 levels were decreased in NHDFs exposed to 600 µM H₂O₂, whereas RA inhibited this effect in a concentration-dependent manner (Fig. 4A). To determine the effects of H₂O₂ and RA on NF-κB expression, an NF-κB luciferase reporter stable NIH-3T3 cell line was used. As presented in Fig. 4B, relative luciferase activity in H₂O₂-exposed NHDFs increased 5.71±0.42-fold compared with the control group (P<0.05). Notably, pretreatment with 20 and 30 µM RA significantly inhibited this effect (P<0.05). To further evaluate the effects of H₂O₂ and RA on the activity of NF-κB, the expression levels of the NF-κB target genes, TNF-α and IL-6, were determined. H₂O₂ upregulated TNF-α and IL-6 expression, whereas RA significantly inhibited this effect in a concentration-dependent manner (Fig. 4C). Taken together, these results indicated that RA exerts an anti-inflammatory effect on NHDFs under conditions of excessive oxidative stress.