PF (purity>99.8%; Fig. 1A) was purchased from Chengdu Mansite Pharmaceutical Co., Ltd. (Chengdu, China). The HaCaT human keratinocyte cells were purchased from the China Center for Type Culture Collection (Wuhan, China), and were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 4 mM L-glutamine, 3.7 g/l sodium bicarbonate, 4.5 g/l glucose and 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were maintained in a humidified incubator containing 5% CO₂ at 37°C. WST-8 was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Propidium iodide (PI) and RNase A were purchased from Beyotime Institute of Biotechnology (Haimen, China). Antibodies against phosphorylated (P)-p38 (cat. no. 4511), p38 (cat. no. 8690), P-p53 (cat. no. 9284), p53 (cat. no. 2524), caspase 3 (cat. no. 9662), cleaved caspase 3 (cat. no. 9664) and β-actin (cat. no. 8457) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). NAC and SB203580 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

HaCaT cells were pretreated with various concentrations of PF (0, 25, 50 and 100 µM) for 2 h in serum-free medium. Subsequently, the cells were washed with phosphate-buffered saline (PBS) and covered with a thin layer of PBS. UV-B radiation was applied at a level of 60 mJ/cm² using a Philips TL-D/08 15W weathering lamp (wavelength 290–340 nm, peak 311 nm) (Philips, Amsterdam, The Netherlands). The radiation intensity was monitored using a UV-B radiometer (Beijing Normal University, Beijing, China). Following exposure to UV-B radiation, PBS was removed and DMEM containing PF (0, 25, 50 or 100 µM) was added to the cells, which were incubated at 37°C for a suitable period.

PF [dissolved in dimethyl sulfoxide (DMSO)] was used to treat the cells. The final concentration of DMSO used was <0.1% (v/v). Cell viability was measured using the WST-8 assay (Dojindo Molecular Technologies, Inc.), according to the manufacturer's protocol. Briefly, HaCaT cells were seeded at a density of 5×10³ cells/well in 96-well culture plates in DMEM, and were cultured in a humidified incubator at 37°C overnight. The cells were pretreated with PF (0, 25, 50, and 100 µM) for 2 h. Following UV-B radiation, the cells were incubated for a further 24 h, after which, 10 µl WST-8 was added to each well for 1 h. Subsequently, optical density (OD) was measured at a wavelength of 450 nm using a BIO-TEK MQX200 plate reader (Highland Park Efficiencies, Winooski, VT, USA). The percentage of viable cells was determined using the following formula: Ratio (%) = [OD(PF)-OD(Blank)/OD(Control)-OD (Blank)] × 100. Cell viability data are averages of three independent experiments each containing six replicates.

HaCaT cells were seeded at a density of 2×10⁵ cells/well in 6-well culture plates in DMEM, and were cultured in a humidified incubator at 37°C for 24 h. The cells were pretreated with 25 µM PF for 2 h. Following UV-B radiation, the cells were incubated for a further 24 h, after which, cells were collected and fixed in 70% ethanol for 24 h at 4°C. Subsequently, the cells were centrifuged at 300 × g for 5 min and the cell pellet was resuspended in 400 µl PBS containing RNase A (10 mg/ml, 50 µl) and PI (2 mg/ml, 10 µl). The mixture was incubated in the dark at 37°C for 30 min and data were acquired using a FACSCalibur flow cytometer (BD Biosciences, San Joe, CA, USA). The cell death data were analyzed using FlowJo software V6.0 (Tree Star, Inc., Ashland, OR, USA). The extent of cell death was determined by evaluating the sub G₁ fraction, or the percentage of cells with DNA content <2n. The experiment was replicated three times.

HaCaT cells were seeded at a density of 5×10⁵ cells/well in 6-well culture plates in DMEM, and were cultured in a humidified incubator at 37°C for 24 h. The cells were pretreated with 25 µM PF or 5 µM SB203580 for 2 h. Following UV-B radiation, the cells were incubated for a further 24 h, after which, cells were collected and resuspended in lysis buffer [150 mmol/l NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mmol/l Tris-Cl, pH 8.0] containing 2 µg/ml aprotinin, 2 µg/ml leupeptin 40 mg/ml phenylmethylsulfonyl fluoride and 2 mmol/l DTT. The cells were centrifuged at 12,000 × g for 15 min, in order to remove nuclei and cell debris. Supernatants were then immediately frozen at −80°C, until further use. Protein concentrations were determined using the Bradford Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and 30 µg cellular proteins were separated by 10% SDS-polyacrylamide gel electrophoresis followed by electro-blotting onto polyvinylidene difluoride membranes (45 µm; EMD Millipore, Billerica, MA, USA). The membranes were blocked for 1 h with 5% milk at room temperature, followed by an overnight incubation at 4°C with the following primary antibodies: P-p38, P-p53, p38, p53, cleaved caspase 3, caspase 3 and β-actin (all 1:1,000 dilution). Blots were washed twice with 0.1% Tween 20/Tris-buffered saline (TTBS) prior to incubation with a horseradish peroxidase-conjugated secondary antibody (1:1,000 dilution; Cell Signaling Technology, Inc.) for 1 h at room temperature. Blots were further washed with TTBS and were developed by enhanced chemiluminescence using Supersignal West Femto Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.). Band intensities were quantified using UN-SCAN-IT Gel Analysis software (version 6; Silk Scientific, Inc., Orem, UT, USA). The OD for target proteins was indicated as a proportion of β-actin OD. Western blotting was replicated three times.

ROS were detected using the cell-permeable fluorescent probe 2,-7,-dichlorofluorescein-diacetate (H₂DCFDA) (Sigma-Aldrich), a non-fluorescent compound, which is converted into highly fluorescent dichlorofluorescein (DCF) by cellular peroxides. The cells were exposed to 25 µM PF or 2 mM NAC for 2 h. Following radiation, the cells were incubated for a further 6 h, after which, the cells were treated with H₂DCFDA (10 µM) in serum-free DMEM. Following incubation at 37°C for 30 min, the cells were washed with PBS and fluorescence was monitored by flow cytometry or fluorescence microscopy (DM1000; Leica Camera, Wetzlar, Germany). The mean fluorescence intensity (MFI) data were analyzed by FlowJo software V6.0 (Tree Star, Inc.). The MFI data were replicated three times.

All data are presented as the mean ± standard deviation. Data analysis was performed using SPSS version 20.0 (IBM SPSS, Amronk, NY, USA). Statistical significance was detected using one-way analysis of variance. For comparisons between two groups, Student's t-test was used. P<0.05 was considered to indicate a statistically significant difference.

Cell viability was evaluated using the WST-8 assay. Exposure of HaCaT cells to UV-B resulted in a significant reduction in the percentage of viable cells (45%) at 24 h, as compared with the medium control group (P<0.01). Treatment with PF (25, 50 or 100 µM) alone did not affect cell viability; however, cells pretreated with PF (25, 50, and 100 µM) prior to UV-B radiation exhibited a significant increase in the percentage of viable cells (60%). No dose-dependent effects were observed following PF treatment (Fig. 1B).

It is well known that UV-B exposure induces apoptosis. As demonstrated by flow cytometry, UV-B (60 mJ/cm²) radiation significantly increased cell death (9.45%) at 24 h, as compared with in the medium control group (2.58%), whereas PF pretreatment markedly reduced UV-B-induced cell death (6%) (Fig. 2A and B). Caspase 3 is expressed in an inactive pro-form, procaspase 3. In apoptosis, procaspase 3 is activated and generates two active subunits, cleaved caspase 3 (17). As shown in Fig. 3, UV radiation resulted in a significant decrease in the expression levels of procaspase 3 and an increase in cleaved caspase 3 expression. PF pretreatment partially counteracted the effects of UV-B on procaspase 3 and cleaved caspase 3 expression.

UV-B exposure results in the generation of ROS, which induce cell damage. The MFI of DCF was used to evaluate the production of ROS. As shown in Fig. 4, ROS production was significantly increased following UV-B radiation. Pretreatment with 25 µM PF or 2 mM NAC (a ROS scavenger), markedly inhibited the production of ROS after UV-B radiation.

The activation of p38 and p53 is associated with UV-B-induced cell damage. Western blotting (Fig. 5) demonstrated that UV-B radiation significantly increased the expression levels of P-p38 and P-p53. Conversely, PF pretreatment markedly reduced the expression levels of P-p38 and P-p53 following UV-B-radiation. Both UV radiation and PF pretreatment did not alter the expression levels of total p38 and p53. It has previously been reported that p38 kinase mediates UV-induced phosphorylation of p53 protein (10). As shown in Fig. 5C and D, SB203580, a p38 inhibitor, significantly reduced the expression levels of P-p53 after UV-B radiation.