Crocin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was dissolved in sterile PBS solution at a concentration of 10 mM, stored at −20°C and then freshly thawed at room temperature prior to each use.

To test the antioxidant effects of crocin, 1,1-diphenyl-2-picrylhydrazyl (DPPH; Sigma-Aldrich; Merck KGaA) radical scavenging activity was measured as previously described (9). Reaction mixtures (200 µl) containing DPPH and a serial dilution of crocin (100–800 µM) were placed in a 96-well plate at room temperature in the dark for 30 min, then the absorbance was measured at 515 nm using a Varioskan Flash Spectral Scanning Multimode Reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The inhibition rate was determined using the following equation: Inhibition rate (%)=[1-(A_sample/A_control)] ×100. Where A refers to the absorbance measured at 515 nm.

Dermal fibroblasts were isolated from human foreskin specimens. Samples were obtained from five donors (age, 6–12 years) who underwent a routine circumcision procedure at Shanghai 9th People's Hospital between January 2016 and January 2017. Written informed consent was obtained. The present study was approved by The Ethics Committee of Shanghai 9th People's Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai, China). A single-cell suspension was obtained as previously described (10). Cells were then suspended in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 300 µg/ml L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (all Sigma-Aldrich; Merck KGaA). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ and passaged by trypsinization with 0.25% trypsin-EDTA (Gibco; Thermo Fisher Scientific, Inc.) every 3–5 days. Cells at passage 3–5 were used in the following experiments.

Fibroblasts were seeded in a 96-well plate at a density of 2,000 cells/well and treated with different concentrations of crocin (0, 12.5, 50 and 100 µM) and subsequently maintained at 37°C in a humidified atmosphere containing 5% CO₂ for 72 h. Cell proliferation was then measured using a Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology, Haimen, China), according to the manufacturer's instructions. Untreated cells served as the control.

Fibroblasts were cultured in 96-well (2,000 cells/well) or 6-well plates (1×10⁵ cells/well) in the aforementioned supplemented DMEM mixture, and maintained at 37°C for 24 h. Following a further 24 h of incubation at 37°C with or without crocin (0, 12.5, 50 and 100 µM), culture medium was replaced with PBS. Cells were then exposed to UVB light (Philips 311 nm, TL 20W/01; Philips Lighting Holding B.V., Eindhoven, The Netherlands) at a total dose of 100 mJ/cm². Following irradiation, the medium was replaced with culture medium with or without crocin, and cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ for 24 or 72 h for further analyses. Cells treated with UVB irradiation and 150 µM vitamin C (Vit C; Sigma-Aldrich; Merck KGaA) served as a positive control. The following experiments were then performed.

Cell proliferation was measured at 72 h post-irradiation using a CCK-8 kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Results are expressed as the relative cell proliferation (%) with respect to the control cells (cells without UVB irradiation or crocin treatment).

At 24 h post-UVB radiation, 5×10⁵ cells were harvested by 0.25% Trypsin-EDTA and then fixed in 70% ethanol overnight at 4°C. Fixed cells were washed twice with PBS and then incubated with 1.5 mg/l RNase A (Sigma-Aldrich; Merck KGaA) for 1 h at 37°C, followed by staining with 5 µl of propidium iodide (Sigma-Aldrich; Merck KGaA) for 20 min on ice. DNA content was assessed using an Epics Altra Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA), and analyzed with Modi Fit LT v2.0 software (Verity Software House, Inc., Topsham, ME, USA).

To measure the cell-aging rate, SA-β-gal staining was performed at 72 h post-irradiation using a Senescence β-Galactosidase staining kit purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; cat. no. 9860). Cells were washed in PBS, fixed for 10 min at room temperature in 4% paraformaldehyde and stained according to manufacturer's instructions. Five random fields from each sample (n=3 samples/group) were selected to observe under a light microscope (Nikon Eclipse 90i; Nikon Corporation, Tokyo, Japan), and the number of SA-β-gal-positive cells was counted using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). The aging rate was determined as the percentage of positive cells out of the total number of cells.

Intracellular ROS levels were determined by 2′,7′-dichlorodihydrofluoresce in diacetate (DCFH₂-DA; Sigma-Aldrich; Merck KGaA) staining. Briefly, immediately following UVB irradiation, cells were incubated with DCFH₂-DA (10 mM) at 37°C for 20 min. Following this, half of the cells were observed under a fluorescent microscope (Olympus IX70-S1F2; Olympus Corporation, Tokyo, Japan), and the other half of the cells were collected and analyzed with an Epics Altra Flow Cytometer, as described previously (11).

Cells (2×10⁶) were collected at 72 h post-irradiation. Total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and cDNA was synthesized from 2 µg total RNA using 200 U of reverse transcriptase (MMLV-RT) and 20 pM oligo dT (Promega Corporation, Madison, WI, USA) at 42°C for 1 h. The expression of pro-collagen I was determined by qPCR using SYBR Green PCR Master mix (Applied Biosystems, Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 45 sec, using the Strata Gene Mx3000p (Agilent Technologies, Inc., Santa Clara, CA, USA). Expression was quantified using the 2^−∆∆Cq method (12). The primers employed were as follows: Pro-collagen I, forward: 5′-CTCGAGGTGGACACCACCCT-3′ and reverse: 5′-CAGCTGGATGGCCACATCGG-3′. All amplifications were run in triplicate and the results were normalized to the housekeeping gene GAPDH, the primers of which were as follows: Forward: 5′-CAAAAGGGTCATCATCTCTG-3′ and reverse: 5′-CCTGCTTCACCACCTTCTTG-3′. Three independent experiments were performed.

Cells (2×10⁶) were collected at 72 h post-irradiation. Total protein was extracted for western blot analysis and the expression of collagen type 1 (Col-1) and glutathione peroxidase 1 (GPX-1) was measured. Proteins were harvested and collected with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined with a bicinchoninic acid protein assay. Subsequently, proteins (20 µg/lane) were separated by 12% SDS-PAGE. Following electrophoresis at 100 V for 2 h, proteins were transferred to polyvinylidene difluoride membranes at 350 mA for 90 min. The membranes were blocked with 5% non-fat milk in Tris-buffered saline and Tween 20 (TBST) at room temperature for 2 h, followed by incubation with the following primary antibodies overnight at 4°C: Anti-Col-1 (cat. no. ab6308; 1:1,000; Abcam, Cambridge, UK), anti-GPX-1 (cat. no. ab108427; 1:2,000; Abcam) and β-actin (cat. no. 4970S; 1:2,000; Cell Signaling Technology, Inc.). The membranes were washed three times with TBST and subsequently incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (cat. no. 115-035-062; 1:2,000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at room temperature for 2 h. Protein expression levels were analyzed by visualizing the bands with enhanced chemiluminescence (Pierce, Rockford, USA). ImageJ 1.50i software (National Institutes of Health USA) was used for densitometry.

Data are expressed as the mean ± standard deviation. Unpaired t-tests were used for direct comparisons between the UVB and the UVB + crocin groups. For multiple comparisons, one-way analysis of variance followed by Tukey's post hoc test was performed. All analyses were performed using SPSS v.13.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To test the potential cytotoxicity of crocin on dermal fibroblasts, cells were treated with various concentrations (0, 12.5, 50 and 100 µM) of crocin for 72 h prior to measuring the levels of proliferation using a CCK-8 assay. As shown in Fig. 1, crocin did not inhibit the proliferation of cells at the concentrations tested, up to 100 µM, indicating that crocin at this dose range may not be toxic to fibroblasts.

To determine the optimal dose of UVB irradiation, fibroblasts were exposed to a range of UVB doses (0, 25, 50 and 100 mJ/cm²) and a CCK-8 kit was used to measure cell proliferation following 72 h. UVB irradiation significantly inhibited cell proliferation at the dose of 100 mJ/cm² (Fig. 2A). Therefore, 100 mJ/cm² was selected for the subsequent experiments. Following pre-incubation with crocin (12.5, 50 and 100 µM) for 24 h, cells were irradiated by UVB and cell proliferation was measured following 72 h. High doses (50 and 100 µM) of crocin significantly rescued the cell proliferation inhibited by UVB irradiation (P<0.05), and the rate was similar to that following Vit C treatment (Fig. 2B and C).

Flow cytometry analyses of the cell cycle are shown in Fig. 3. Cell populations in the G₂/M phases were significantly increased following UVB irradiation, indicating that cells were arrested at the G₂/M phase. As a positive control, Vit C treatment rescued cell cycle arrest as expected. High doses (50 and 100 µM) of crocin restored cell cycle arrest to levels similar to those of the control group (without UVB irradiation), indicating that crocin could rescue UVB-induced cell cycle arrest.

To determine whether crocin could prevent cells from UVB-induced photoaging, SA-β-gal staining was performed following UVB irradiation (Fig. 4A). A significant increase in SA-β-gal-positive cells was observed following UVB irradiation (Fig. 4). A decrease in SA-β-gal-positive cells was observed in the Vit C-treated group as well as in the high dose (50 and 100 µM) crocin-treated groups (Fig. 4). Statistical analysis confirmed that the percentage of SA-β-gal-positive cells was increased in the UVB group, and decreased following Vit C or crocin treatment (Fig. 4B).

An increase in ROS is one of the most important mechanisms in photoaging (4). In the present study, UVB irradiation significantly increased intracellular ROS when compared with the control group, while the ROS levels were reduced by Vit C and crocin treatment (Fig. 5A). These results were confirmed by flow cytometric analysis of the fluorescent intensity following DCFH₂-DA staining (Fig. 5B). To verify the possible underlying mechanisms, free radical scavenging activity of crocin was measured. Crocin inhibited DPPH radial activity in a dose-dependent manner (Fig. 5C), indicating that it could partially neutralize ROS. In addition, the expression of the antioxidant protein GPX-1 was measured by western blot analysis. An increase in GPX-1 expression was observed in the Vit C and crocin-treated groups (Fig. 5D and E), indicating that crocin may reduce UVB-induced ROS, partially via the enhanced expression of antioxidant protein GPX-1.

Secretion of ECM is one of the major functions of dermal fibroblasts. A decrease in collagen secretion is commonly observed during skin aging (13). As shown in Fig. 6, a significant decrease in Col-1 expression was observed at the mRNA and protein level following UVB irradiation. In addition, Col-1 expression was upregulated by Vit C and crocin treatment at the mRNA and protein levels, suggesting that cell functions were also restored following Vit C and crocin treatment.