Skin specimens were obtained from six healthy male Chinese donors (mean age 6 years) who underwent circumcision in the Department of Urology of The First Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China) between December 2017 and January 2018. The present study followed the guidelines of the Helsinki Declaration (1964) and subsequent amendments. The study was approved by the Research Ethics Board of the First Affiliated Hospital of Xi'an Jiaotong University. The guardians of all healthy donors provided written informed consent prior to inclusion in the study.

Fresh tissues from children undergoing circumcision in the Department of Urology were obtained and digested for 16 h at 4°C with 0.25% dispase II (Roche Diagnostics, Indianapolis, IN, USA). The tissues were cut into pieces and 0.25% trypsin was added, followed by incubation at 37°C for 30 min and subsequent centrifugation for 5 min at 800 × g at room temperature. The cell suspension was filtered through a 70-mm cell strainer and seeded in a 10-cm cell culture dish at a density of 4-6×10⁶ cells/ml. Melanocyte culture medium (Epilife Medium 254, Cascade Biologics/Invitrogen, Portland, OR, USA) containing human melanocyte growth supplement was added to the melanocyte culture, which was incubated at 37°C with 5% CO₂. Primary melanocytes at passages three to eight were used in the present study.

The rabbit anti-human Nrf2 antibody (Abcam, Cambridge, MA, USA; cat. no. ab62352) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA; cat. no. A-11008) were used. Analytical grade H₂O₂ was from Tianjin Chemical Reagent Factory (Tianjin, China). GR, propidium iodide (PI) and ZnPP were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

Melanocyte viability was assessed using a CCK-8 cell proliferation test kit (KeyGen Biotech Co., Ltd., Nanjing, China), according to the manufacturer's instructions. Briefly, the primary melanocytes were seeded in 96-well plates at a density of 2.5×10⁴ cells/well. H₂O₂ (0.5 mM) was added to the plates at room temperature. Morphological changes in the melanocytes were observed after 24 h with a Nikon Eclipse TS2 fluorescence microscope (Nikon Corporation, Tokyo, Japan). The medium was replaced together with 10 µl/well cell proliferation and toxicity test solution, and the cells were cultured for 1-2 h at 37°C. The color change was monitored and measured at an absorbance at 450 nm using a microplate reader (Perkin-Elmer, Inc., Waltham, MA, USA).

The NHEMs were treated with 1 mM GR for 24 h in 6-well plates at a density of 1×10⁶ cells/well at room temperature. The culture medium was discarded and replaced with DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10 mM H2-DCFDA (Invitrogen; Thermo Fisher Scientific, Inc.), followed by incubation at 37°C for 30 min. The cells were then washed with PBS and assayed by flow cytometry (FACSCanto II) with BD FACSDiva v8.0.1 software (both from BD Biosciences, San Jose, CA, USA).

Nrf2 siRNA and control (scrambled) siRNA oligonucleotides were synthesized by Shanghai GenePharma Co. Ltd. (Shanghai, China). The sequences are listed in Table I. For siRNA transfection, cells were plated at a density of 1×10⁶ cells/well in 24-well plates. At 60% confluence, 30 nM Nrf2 siRNA and 250 µl Lipofectamine^® RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.) in Opti-MEM were mixed and added to cells in 24-well plates at room temperature. The knockdown efficiencies were assessed by immunoblotting 48 h following siRNA transfection.

The cells were lysed with RIPA lysis buffer and mixed with 5X loading buffer. The protein sample was quantified by Bradford assay and was boiled for 10 min and denatured. Each protein sample (50 µg) was subjected to 10% SDS-PAGE. The separated proteins were transferred onto a PVDF membrane. Following blocking with 5% (w/v) dry non-fat milk in PBS for 2 h at room temperature, the primary antibody was added to the PVDF membrane, followed by overnight incubation at 4°C. Blotting was performed with primary antibody against Nrf2 (cat. no. ab62352; Abcam; 1:1,000 dilution). An HRP-labeled goat-anti-rabbit secondary antibody (cat. no. 7074; Cell Signaling Technology, Inc., Danvers, MA, USA; 1:5,000 dilution) was then applied for 1 h at 37°C. β-actin (cat. no. 4967; Cell Signaling Technology, Inc.; 1:1,000 dilution) and Histone H3 (cat. no. 9715; Cell Signaling Technology, Inc.; 1:1,000 dilution) were used as controls. The membrane was washed three times with TBST, and the blots were developed using an Enhanced Chemiluminescence system (Pierce; Thermo Fisher Scientific, Inc.). ImageJ 1.8.0 (National Institutes of Health, Bethesda, MD, USA) was used to quantify the gray values of each blot.

Total RNA was extracted using a Universal RNA Extraction kit (Takara Bio, Inc., Otsu, Japan) and reverse transcribed into cDNA with PrimeScript RT Master Mix kit (Takara Bio, Inc., Otsu, Japan). The RT reaction was performed by incubating the samples at 37°C for 15 min, followed by incubation at 85°C for 5 sec and 4°C for 5 min. Equal quantities of cDNA were used for RT-PCR analysis with a SYBR Premix Ex Taq II analysis kit (Takara Bio, Inc.). The thermocycling conditions were as follows: 95°C for 5 min followed by 45 cycles of denaturation at 95°C for 5 sec, annealing at 60°C for 15 sec, and extension at 72°C for 30 sec. qPCR analyses were performed with the Bio-Rad CFX96™ Real-Time PCR detection system and analyzed using CFXTM manager 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The specific primers used are listed in Table II. Relative expression levels were normalized to the expression of GAPDH and were quantified using the comparative 2^-ΔΔCq method (30).

Following digestion with 0.25% trypsin, the primary melanocytes were centrifuged at 800 × g for 5 min at room temperature and resuspended with 100 µl binding buffer. Each sample was incubated for 30 min in the dark with 2 μl Annexin V-FITC and 2 μl PI staining buffer in binding buffer, following which 300 μl binding buffer was added to each sample. This was followed by analysis using the FACSCanto II flow cytometer.

The NHEMs were seeded on glass slides in a 12-well plate. The glass slides were removed from the 12-well plate and washed twice with PBS. The NHEMs were then fixed with 4% paraformaldehyde for 30 min at room temperature. Following permeabilization by incubation in 0.5% Triton X-100 for 15 min, the NHEMs were blocked with 5% BSA (Beyotime Institute of Biotechnology, Jiangsu, China) at 37°C for 1 h. The cells were then incubated with the primary anti-Nrf2 antibody (1:200 dilution) overnight at 4°C. The cells were washed with PBS five times (3 min each wash) and then incubated with the Alexa Fluor 488-labeled secondary antibody (1:200 dilution) at 37°C for 1 h. The nuclei were counterstained with DAPI. The cells was visualized under a confocal laser scanning microscope (TCS SP5 Leica GmbH, Wetzlar, Germany).

Data are presented as the mean ± SD Student's t-test was used to analyze differences between groups. Differences among multiple groups were analyzed by one-way ANOVA with Dunnett's post hoc or Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, C USA).

The present study investigated the effects of GR (Fig. 1A) on the survival rate and apoptosis of NHEMs treated with H₂O₂. Our previous study determined that treatment of melanocytes with 0.5 mM H₂O₂ for 24 optimal to induce oxidative stress (31). As shown in Fig. 1B there were no significant changes in cell viability at low doses of GR (0.25, 0.5 and 1 mM), whereas cell viability was significantly lower following treatment with a high dose GR (1.5 mM) compared with that in untreated cells. When the NHEMs were treated with 1.5 mM GR, the cell survival rate decreased to 48.05±5.35% compared with that in untreated cells (P<0.05). Therefore, 1 mM GR was used as the working concentration in the following experiments. The primary human melanocytes were treated with 0.5 mM H₂O in the presence or absence of 1 mM GR, and cell viability was then determined using Cell Counting Kit-8 (CCK-8) assays. Following treatment with 0.5 mM H₂O₂ for 24 h, morpho logical observation (Fig. 1D-a-d) revealed the melanocyte dendrites were shortened or had disappeared (Fig. 1D cell viability was decreased to 48.23±7.25% compared that in the control cells (Fig. 1C). However, pretreatment with 1 mM GR significantly attenuated H₂O₂-induced oxidative damage, as represented by an increase of cell viability to 82.7±5.45% compared with that in control cells (Fig. 1C) In addition, the NHEMs were pretreated with 1 mM GR for 24 h prior to 0.5 mM H₂O₂ treatment and melanocyte apoptosis was then examined using flow cytometry. As shown in Fig. 1E and F, following H₂O₂ treatment, the percentage of apoptotic cells increased to 17.5%, compared with 4.5% in the control group, whereas pretreatment with 1 mM GR markedly inhibited H₂O₂-induced apoptotic death, with the percentage of Annexin V-stained cells at 8.8%.

The present study subsequently examined whether glycyrrhizin protected melanocytes against oxidative stress. The effects of GR on ROS were measured in NHEMs treated with H₂O₂. The ROS level was measured using the probe H2-DCFH-DA. As shown in Fig. 2, following H₂O₂ treatment, there was an increase in the level of DCF fluorescence, and the ROS level in NHEMs was significantly reduced by GR pretreatment.

To examine the molecular mechanism underlying the effect of GR in reducing oxidative stress, the present study determined whether GR treatment activated the classical antioxidant pathway of Nrf2. As shown in Fig. 3A, Nrf2 was mainly localized in cytoplasm of the untreated NHEMs. However, in cells treated with GR for 24 h, the expression of Nrf2 in the nucleus was markedly increased. To confirm the nuclear translocation of Nrf2, cytoplasmic and nuclear protein fractions of NHEMs were extracted and immunoblotted with an antibody against Nrf2 (Fig. 3B and C). The expression of Nrf2 was increased significantly following GR treatment, which was consistent with the results of the immunofluorescence. Subsequently, the effects of GR on the expression levels of four downstream genes of the Nrf2 antioxidant pathway were examined. As shown in Fig. 3D, GR treatment increased the expression levels of the four genes, of which the expression of HO-1 was highly elevated.

Subsequently, the primary melanocytes were transfected with Nrf2 siRNAs. Three sequences of Nrf2 siRNAs were used for knockdown. The Nrf2 knockdown efficiency was evaluated by western blotting and RT-qPCR analysis (Fig. 4). The most efficient sequence was the Nrf2 (3) siRNA. The expression of HO-1 was significantly decreased following transfection with Nrf2 (3) siRNA, whereas the expression of the three other genes was not decreased significantly. Therefore, GR exerted antioxidant effects on melanocytes mainly through the Nrf2-mediated induction of HO-1.

The present study then examined whether Nrf2 has a direct role in the antioxidant effect of GR. The NHEMs were first transfected with Nrf2 siRNA or scrambled siRNA for 48 h, followed by 0.5 mM H₂O₂ treatment in the presence of 1 mM GR for 24 h. As shown in Fig. 5A, the viability of the NHEMs was significantly decreased following silencing of Nrf2 compared with that of cells in the control group. Transfection with scrambled siRNA had no cytoprotective effect; cell viability was comparable with that of cells treated with H₂O₂ and GR. GR pretreatment had a significant protective effect on NHEMs, and the apoptotic rate of the cells was decreased. When the Nrf2 gene was knocked down, the protective effect of GR was decreased significantly (Fig. 5B and C). The cells treated with scrambled siRNA exhibited no changes in GR-induced apoptotic rate under H₂O₂ treatment.

To determine the role of the increased expression of HO-1 in the antioxidative effect of GR on melanocytes, the NHEMs were treated with ZnPP, an inhibitor of HO-1, for 24 h, followed by treatment with H₂O₂ for 24 h. The cells were then treated with GR for another 24 h. As shown in Fig. 6, pretreatment with GR increased the viability of the NHEMs and resulted in a significant decrease of apoptotic cells, whereas the addition of ZnPP reversed the effect of GR by reducing the viability of the NHEMs and increasing apoptotic cells (Fig. 6A-C).