HSFs were purchased from CoBioer Biosciences Co., Ltd. (Nanjing, China). The cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (ScienCell Research Laboratories, Inc., Carlsbad, CA USA), 1% penicillin and 1% streptomycin. The cells were incubated under 5% CO₂ at 37°C.

GSK126 was purchased from Selleck Chemicals Scientific, Inc., Houston, TX, USA and dissolved in dimethyl sulfoxide. GSK126 was stored at −20°C in a freezer and diluted with medium to a final concentration (2 µM) for experiments.

HSFs (1×10⁵ cells/well) were seeded in 6-well plates with DMEM supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin. Cells were incubated in an atmosphere containing 5% CO₂ at 37°C for 24 h. The medium was replaced with different concentrations (0, 2, 4 and 8 µM) of GSK126. Once the cells were further incubated for 24 h at 37°C, the cells were collected for RNA extraction and further experiments.

HSFs (1×10⁵ cells/well) were seeded in 6-well with DMEM containing 10% fetal bovine serum, 1% penicillin and 1% streptomycin. The cells were incubated in an atmosphere containing 5% CO₂ at 37°C for 24 h. Cells were divided into four groups: (−), cells without UVA and GSK126; GSK126 (2 µM); UVA (10 J/cm²); and UVA (10 J/cm²)+GSK126 (2 µM). The cell culture medium was replaced with an equal volume of sterile phosphate-buffered saline (PBS) in all groups (prior to radiation exposure in the irradiated groups). The radiation dose used was 10 J/cm² in the UVA (10 J/cm²) and UVA (10 J/cm²)+GSK126 (2 µM) groups. The PBS was discarded at the end of UVA irradiation, media containing serum and antibiotics were added to the (−) and UVA (10 J/cm²)+GSK126 (2 µM) groups, media containing serum and antibiotics and 2 µM GSK126 was added to GSK126 (2 µM) and UVA (10 J/cm²)+GSK126 (2 µM) groups, and cells were further incubated at 37°C for 24 h. Images of the cells were then captured with an optical microscope (magnification, ×40) to observe the cell growth and collected for RNA extraction and further experiments.

The ZF-1 Three Ultraviolet analyzer (Shanghai HQ Instruments Co., Ltd., Shanghai, China) was used as a radiation source, the wavelength was set to 365 nm and an UV radiation meter (Kühnast Strahlungstechnik, Dresden, Germany) was used to measure the radiation dose. According to the experiment, the appropriate number of HSFs were inoculated onto the corresponding orifice plates and, 24 h later, the desired radiation dose (J/cm²) was selected for UV irradiation. The cell culture medium was replaced with an equal volume of sterile PBS prior to radiation exposure. The cells were placed under the UV analyzer for irradiation under sterile conditions and the irradiation height was <2 cm. Following irradiation, PBS was replaced with fresh medium.

A cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) assay was used to determine cell viability, according to the manufacturer's instructions. It is based on dehydrogenase activity detection in viable cells. The formazan dye generated by dehydrogenases absorbs light at a wavelength of 450 nm. The amount of formazan dye in cells is directly proportional to the number of living cells.

HSFs were seeded into 96-well plates at a density of 0.5×10⁴ cells/well, with 200 µl of culture medium per well in triplicate wells for each experimental group. When cells were adherent (24 h post seeding), they were divided into four different UVA treatment groups: 0, 2.5, 5 and 10 J/cm². At specific time points after irradiation (0, 12 and 24 h), 20 µl of CCK-8 reagent was added to each well. After incubation for 1, 2, or 3 h at 37°C, absorbance was detected at 450 nm.

A Hoechst apoptosis staining kit (Beyotime Institute of Biotechnology, Haimen, China) was used to determine cellular apoptosis, according to the manufacturer's instructions. Under this staining, apoptotic nuclei demonstrate a dense white staining with fragmentation. Briefly, sterile cover slips were placed at the bottom of 6-well plates, and 0.3×10⁵ cells in 2 ml of DMEM supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin were added to each well. A total of 24 h after cell irradiation, the medium was removed and 0.5 ml of fixative solution was added. Cells were fixed for 10 min at room temperature. The cells were then washed twice with PBS for 3 min each time and 0.5 ml Hoechst 33258 staining solution was added. The cells were stained in a shaker for 5 min at room temperature and subsequently washed twice with PBS for 3 min each time. The cells were then carefully mounted onto a glass slide with a drop of fluorescence quenching mounting liquid. The cells were then observed under a fluorescence microscope.

A total of 1.0×10⁵ HSFs in 2 ml of DMEM supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin were added to each well of 6-well plates in triplicate wells for each experimental group. When cells were adherent for 24 h, they were exposed to different doses of irradiation (0, 2.5, 5 and 10 J/cm²). HSFs were transferred into 5-ml tubes and washed with PBS. An annexin V-FITC/PI apoptosis detection kit (Nanjing KGI Biological Technology Development Co., Ltd., Nanjing, China) was used to stain the nucleus according to the maunfacturer's instructions and the cells were resuspended in binding buffer (100 µl). Subsequently, cells were filtered into tubes through filter paper to remove cell clumps. Following this, 50 µl Annexin V-fluorescein isothiocyanate solution and 50 µl propidium iodide were added to each tube. The tubes containing the cells were then incubated in the dark at room temperature for 15 min. The cells were then analyzed by BD FACS Aria II SORP sorting flow cytometer (BD Biosciences, Franklin lake, NJ, USA). The excitation and emission wavelengths were 488 and 530 nm, respectively.

SA-β-gal activity was determined 24 h after UVA irradiation. A Senescence β-Galactosidase Staining Kit was performed, according to the manufacturer's instructions (Beyotime Institute of Biotechnology). A total of 0.3×10⁵ cells in 2 ml of culture medium were added to each well of 6-well plates in triplicate for each experimental group. A total of 24 h after cell irradiation, cells were washed once with PBS at room temperature, and 1 ml β-gal staining fixative solution was added to each well. The cells were fixed for 15 min at room temperature. The fixative solution was discarded and the cells were washed three times with PBS, 3 min for each wash. Staining fluid (1 ml) was subsequently added to each well. A plastic wrap was used to seal the 6-well plates and cells were incubated overnight at 37°C. The following day, the cells were observed under an optical microscope.

Human peripheral blood (6 ml) was collected and the plasma and leukocyte layer were separated following centrifugation at 566 × g at 4°C for 5 min. Following this, erythrocytes were washed three times with PBS, 3 min for each wash at 4°C. The erythrocytes were fixed with 10% formaldehyde solution (5:1; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) for 10 min at room temperature, then erythrocytes were washed three times with PBS, 3 min for each wash at 4°C and diluted to an erythrocyte suspension at a final concentration of 10⁷ cells/ml. Cells were seeded in 6-well plates at 0.5×10⁴ cells/well. Once the cells were irradiated for 24 h, 100 µl eythrocyte suspension was added to each well and the samples were incubated in an upright position for 5 min at 37°C. Fibroblasts were observed using a particle exclusion assay and images were captured using a Zeiss Axiovert phase-contrast microscope (22). Hyaluronidase (200 µl; 1 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added to the 0 J/cm² dose group, mixed gently, and incubated for 5 min at 37°C before fibroblasts were observed under a microscope and images were captured.

Total RNA was isolated from HSFs using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions, and quantified spectrophotometrically. Total RNA (1 µg) from each sample was subjected to first-strand cDNA synthesis using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China), according to the kit's instructions. cDNA was generated after the removal of genomic DNA by treatment at 42°C for 2 min and subjected to the following thermocycling conditions: 37°C for 15 min, 85°C for 5 sec; and maintenance at 4°C.

A Takara PCR amplification kit (Takara Biotechnology Co., Ltd.) was used according to the manufacturer's protocol with a CFX Real-Time PCR Detection system (Bio-Rad Laboratories, Inc.) for qPCR. The fluorescent dye used was SYBR Green (Takara PCR amplification kit; Takara Biotechnology Co., Ltd.). The reaction conditions were as follows: Initial denaturation at 95°C for 3 min; and 45 cycles of denaturation at 95°C for 15 sec, annealing at 54°C for 20 sec and extension at 72°C for 30 sec. The primers used are demonstrated in Table I. Gene expression was normalized to the level of GAPDH in a given sample. The expression level of genes and gene alternative splicing products were calculated and analyzed using the 2^−ΔΔCq relative quantification method (23). Each experimental treatment was conducted in triplicate.

All data are presented as mean ± standard deviation. Data was analyzed using SPSS software (version 22.0; IBM Corp., Armonk, NY, USA). Analysis of variance followed by Dunnett's test was performed. P<0.05 was considered to indicate a statistically significant difference.

As demonstrated in Fig. 1, the fibroblast proliferation activity in the 2.5, 5 and 10 J/cm² dose groups decreased gradually with the increase of UVA radiation dose. The 10 J/cm² UVA radiation dose had the greatest effect on cell viability, indicating that 10 J/cm² of UVA radiation is able to markedly inhibit fibroblast proliferation (Fig. 1).

Hoechst staining revealed that cells treated with UVA radiation demonstrated apoptotic nuclei, with a dense white dye or crushed nuclei shape (Fig. 2A). Statistical analysis demonstrated that all doses of UVA radiation (2.5, 5 and 10 J/cm²) significantly promoted fibroblast apoptosis (P<0.05) compared with the control group (0 J/cm²) in a dose-dependent manner. The 10 J/cm² dose promoted fibroblast apoptosis with the most significant effect (P<0.01; Fig. 2B). The 10 J/cm² UVA radiation dose demonstrated the most marked promotion of fibroblast apoptosis compared with the control group (Fig. 2C and D). The detection of apoptosis by flow cytometry demonstrated that the number of all UVA radiation groups significantly increased compared to the control group (P<0.05; Fig. 2E).

As demonstrated in Fig. 3A, the blue precipitate indicated β-gal activity and the presence of senescent cells. UVA radiation induced changes in HSF cellular senescence. The 5 and 10 J/cm² dose radiation groups significantly induced cellular senescence compared with the control group (P<0.05). The 10 J/cm² dose had the most significant effect on cellular senescence, whereas the 2.5 J/cm² radiation group had no significant effect on senescence compared with the control (0 J/cm²) group (Fig. 3B).

A particle exclusion assay demonstrated that UVA radiation decreased HA content around HSF cells. Compared with the control group (0 J/cm²), HSF cell exclusion to erythrocytes in the 10 J/cm² dose group decreased. The exclusion in the 0 J/cm² group disappeared after adding 100 µl hyaluronidase (1 mg/ml), indicating that the exclusion effect of red blood cells was caused by HA formation (Fig. 4). As skin aging is predominantly a result of extracellular matrix reduction, of which, HA is one of the most important components, the decline of hyaluronan content is a sign of skin aging (24). Notably, fibroblasts are the primary generators of HA (8). In the present experiment, UVA radiation was indicated to reduce the synthesis of HA in fibroblasts, which demonstrated that a photoaging cell model was successfully established.

RT-qPCR results demonstrated that, 24 h after UVA radiation of HSF cells, BMI-1 and EZH2 mRNA expression levels in the 10 J/cm² dose group were significantly upregulated (P<0.05) compared with the control group (0 J/cm²; Fig. 5). However, the other dose groups demonstrated no significant differences compared with the control group. The 10 J/cm² dose therefore had the most significant effect on fibroblast proliferation, promoting apoptosis, inducing senescence, reducing the surrounding HA content and upregulating the mRNA expression levels of BMI-1 and EZH2. Based on these results, the 10 J/cm² dose was selected as the UVA treatment dose for the photoaging model.

GSK126 is a methyl transferase activity inhibitor that could selectively inhibit EZH2-methyl transferase activity. In the present study, the results demonstrated that GSK126 also inhibited the mRNA expression of EZH2. GSK126 at a concentration of 2 µM significantly inhibited EZH2 expression compared with the control group (0 µM GSK126) at 12 and 24 h (P<0.05). The mRNA expression of the EZH2 was significantly downregulated after treatment of HSF cells for 12 and 24 h (P<0.05; Fig. 6). Therefore, 2 µM GSK126 was selected as the inhibitor concentration for further analyses.

GSK126 inhibited the photoaging of fibroblasts as well as expression of the key genes in the PcG family. After treating cells for 48 h, cell growth state of the cells was observed under a microscope. It was demonstrated that the addition of 2 µM GSK126 did not significantly affect cell growth compared with the control group. However, a decrease in cell number, a poor growth state and UVA radiation-induced cellular senescence was observed in fibroblasts in the 10 J/cm² UVA irradiation group. Cell growth was largely unaffected in the UVA+GSK126 group, suggesting that inhibitors may resist UVA radiation-induced fibroblast senescence (Fig. 7A). RT-qPCR results demonstrated that the addition of GSK126 after UVA radiation significantly reduced the increase of EZH2 induced by UVA radiation (P<0.05; Fig. 7B). Following radiation, there was a significant increase in BMI-1 mRNA expression caused by the addition of GSK126 in the UVA+GSK126 group compared with the UVA radiation group (P<0.05; Fig. 7C). This may be closely linked to GSK126 resisting fibroblast senescence.

The effect of UVA radiation and GSK126 on HA anabolic family-related gene expression was analyzed. RT-qPCR analysis demonstrated that after 10 J/cm² UVA radiation on HSF cells, HA synthase 1 (HAS1), HAS2, HAS3, CD44 and hyaluronidase (Hyal-1) were upregulated, with the highest expression observed for HAS1, compared with the control group without any treatment (P<0.05; Fig. 8A-E). After adding GSK126 inhibitors, the upregulation of HAS1 and Hyal-1 in the UVA group was significantly inhibited (P<0.05; Fig. 8A). However, HAS2, HAS3, CD44 in the UVA+GSK126 group were significantly upregulated (P<0.05; Fig. 8B-E) compared with the UVA group, whereas Hyal-2 expression did not change significantly change (Fig. 8F).

Following this, the effect of UVA radiation and GSK126 on HSF cell photoaging molecular pathways was investigated (Fig. 9). RT-qPCR demonstrated that epidermal growth factor receptor (EGFR), smad2, smad4, P16 and matrix metalloproteinase-1 (MMP-1) mRNA expression levels were upregulated after 10 J/cm² UVA radiation on HSF cells. After the addition of GSK126, upregulation of smad2, smad4 and MMP-1 was significantly inhibited (P<0.05; Fig. 9C, D and F). However, the upregulation of P16 gene expression was further promoted with the addition of GSK126 (P<0.05; Fig. 9E). Addition of GSK126 had no significant effect on epidermal growth factor (EGF) and EGFR expression after UVA radiation (Fig. 9A and B).