Cells and reagents
Human skin keratinocytes (HaCaT cells) were obtained from the American Type Culture Collection (USA) and maintained in Dulbecco’s modified essential medium containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 100 units/mL penicillin, and 100 µg/mL streptomycin. N-acetyl-l-cysteine (NAC), z-DEVD-fmk, and cell proliferation kit I (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA); FAK inhibitor (FAKi; PF-562,271) was obtained from MedKoo (Chapel Hill, NC, USA); and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) was purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against FAK (Millipore, MA, USA), pY397-FAK (Invitrogen), extracellular signal-regulated kinase (ERK), p-ERK, c-Jun N-terminal kinase (JNK), p-JNK, p38, p-p38, IKKα, p-IKKα, procaspase-3, cleaved caspase-3, PARP (Cell Signaling, Danvers, MA, USA), and GAPDH (Millipore, MA, USA) were obtained as indicated.
Preparation of Cyanidin-3-O-glucoside (C3OG) and cyanidin 3-O-rutinoside from cherry fruits (
Prunus serrulata L. var. tomentella Nakai)
Cyanidin-3-O-glucoside (C3OG) and cyanidin-3-O-rutinoside (C3OR) were extracted from cherry fruits collected in June 2019 from trees in Jeungpyeong Gun, Chungbuk, South Korea (Fig. 1 A). 1.0 L of 95% ethanol was added to 0.20 kg of the fresh cherry fruits, grinded and squeezed to produce a purple juice, which was filtered through a vacuum filtration device to yield crude extract that was used for further preparation of anthocyanin-enriched extracts. The crude extract was purified by column chromatography through a Dowex ECR-S resin. The preparation thus obtained was washed with acidified water (0.1% HCl) to remove naturally-occurring sugars and acids contained in the fruits. Thereafter, the resulting preparation was eluted with acidified methanol (0.1% HCl), concentrated, and freeze-dried to yield an anthocyanin-rich powder [44]. The two target anthocyanins, C3OG and C3OR, were isolated by semi-preparative HPLC using a YMC Triart C18 column (250 × 10 mm, 5 μm, 12 nm), yielding 8 and 35 mg of pure anthocyanin compound, respectively, from 1 L of cherry juice (200 g of fresh cherry fruits). We included anthocyanins content of extracts in Additional file data (Additional file 1: Fig. S2).
High performance liquid chromatography (HPLC-DAD)
All HPLC analyses were performed using a Youngrin YL9100 HPLC system equipped with a photodiode array (PDA) detector. Samples were analyzed using a YMC ODS-A C18 column (250 × 4.6 mm, 5 μm, 12 nm), for which we used a gradient mobile phase comprising solvent A (H2O containing 0.1% trifluoroacetic acid) and solvent B (acetonitrile). The gradient elution program used was as follows: an initial A to B ratio of 90:10, followed by an increase in B from 10 to 15% in 40 min at a flow rate of 1.0 mL/min, and a subsequent increase in B from 15 to 80% in 10 min. The retention time of compound 1 was 27.4 min, whereas compound 2 was eluted at 30.0 min. Based on comparing the chromatogram peaks obtained for isolated compounds and those of standard compounds, compounds 1 and 2 were identified as C3OG and C3OR, respectively (Fig. 8B and D). The results of full-wave scanning (Fig. 8 C and E) revealed that both C3OG and C3OR have two maximum absorption peaks at approximately 281 and 518 nm and 279 and 515 nm, respectively, which is consistent with the previously reported characteristics of these anthocyanins. We also further confirmed the chemical structures of two major anthocyanin compounds using NMR spectroscopy (Additional file 1: Figs. S3, S4) [45, 46].
Cell viability assay
Cell viability was determined using the thiazolyl blue tetrazolium bromide (MTT) assay. HaCaT cells were cultured in 96-well plates (clear-bottomed, dark-sided 96-well microplate; Thermo Fisher Scientific, Rochester, NY, USA) at a density of 1 × 104 cells/well and pretreated with or without NAC (10 mM) for 1 h, and C3OG (10–200 µM) or C3OR (10–200 µM) for 24 h prior to exposure to 2,500–20,000 lx blue LED light for 1 h. After 24 h, the cells were incubated with MTT (0.25 mg/mL) at 37 °C in a CO2 incubator for 4 h. The resulting MTT formazan products were dissolved in DMSO, and absorbances were measured at 570 nm using a microplate reader (Bio-Tek Instruments Inc., Santa Clara, CA, USA).
ROS measurement
HaCaT cells were cultured in 96-well plates (clear-bottomed, dark-sided) at a density of 1 × 104 cells/well and pretreated with or without NAC (10 mM) for 1 h and C3OG (10–200 µM) or C3OR (10–200 µM) for 24 h prior to exposure to 20,000 lx blue LED light for 1 h. Thereafter, the cells were stained with 10 µM H2DCFDA for 30 min at 37 °C and subsequently washed with phosphate-buffered saline prior to analysis using a fluorescence microplate reader or visualized under a fluorescence microscope.
LED light exposure
Direct sunlight can reach a light intensity of up to 100,000 lx and 25,000 lx in full daylight. Comparatively, indoor light intensities are considerably lower, with standard office lighting typically not exceeding 500 lx. During the day, light levels are determined by the presence of clouds and haze. They can vary to differing degrees and durations, depending on factors such as the prevailing cloud cover and atmospheric turbidity. Consequently, in the present study, we simulated overcast (2500 lx) and full-daylight (20,000 lx) conditions for 1 h, as was used in a previous study [8].
As a source of illumination, we used LED (GT-P25G6: blue, 460–470 nm, 9 W, GT-P25WB; Shenzhen Getian Opto-Electronics Co., Ltd., Shenzhen, China), which were attached to a fan and a heat sink to reduce the transfer of heat to samples. To eliminate unwanted thermal effects caused by the LED light, we monitored that the temperature of the incubator and cell culture dish was maintained at 37 °C in all experiments performed. HaCaT cells were seeded into 96-well cell culture plates (for MTT assays or ROS measurement) or six-well cell culture plates (for western blotting or adhesion assays). After 24 h, the cells were exposed to blue (450 nm) LED light at a unified illuminance of 2,500–20,000 lx for 1 h in an incubator equipped with an LED box. Illuminance was measured and adjusted using a Hioki 3423 lx HiTester lux meter (Hioki E. E. Corporation, Japan) on the sample surface. As a control, a similar plate of cells was incubated in an unilluminated incubator. To avoid any potential bias, the cells used for the control and light irradiation treatments cells were derived from the same stock.
Immunoblotting
To observe cellular canonical signaling, HaCaT cells in six-well plates were pre-treated with NAC (10 mM) and PF-271 (1 µM) for 1 h or C3OG (10–200 µM) or C3OR (10–200 µM) for 24 h prior to exposure to blue LED illumination (20,000 lx) for 30 min, and thereafter immediately lysed (Fig. 4). Similarly, to determine the activation of caspase-3, HaCaT cells in six-well plates were pre-treated with NAC (10 mM) and DEVD-fmk (200 µM) for 1 h or C3OG (200 µM) or C3OR (200 µM) for 24 h prior to exposure to blue LED illumination (20,000 lx) for 1 h, followed by cell lysis (Fig. 6). Clarified lysates were run on 4–12% NuPAGE Tris-Bis gels (Invitrogen). The separated proteins were transferred to polyvinylidene fluoride membranes, blocked with 3% bovine serum albumin, and incubated overnight with primary AT antibodies at 4 °C. The following day, the membranes were washed with Tris-buffered saline containing 0.1% Tween® 20 detergent and then incubated with horseradish peroxidase-conjugated secondary antibodies. Bound antibodies were visualized using Enhanced chemiluminescence in conjunction with a ChemiDoc MP Imaging System (Bio-rad, Hercules, CA, USA).
Cell detachment assay
HaCaT cells in six-well plates were pretreated with NAC (10 mM) and DEVD-fmk (200 µM) for 1 h or C3OG (200 µM) or C3OR (200 µM) for 24 h prior to being exposed to blue LED illumination (20,000 lx) for 1 h. After 24 h, floating (detached cells in the supernatant) and adherent cells (obtained by trypsinization) were harvested separately. Cell counts were performed using a hemocytometer and the trypan blue exclusion method, with all viable (unstained) and dead (stained) cells being counted. Using the count data thus obtained, we calculated the percentages of cell adhesion and detachment using the following equations 8:
Percentage
adherent cells = (number of adherent cells/total number of cells) × 100%
Percentage detached cells = (number of floating
cells/total number of cells) × 100%
Statistical analysis
Statistical significance between experimental groups was determined using the Student’s t-test or two-way analysis of variance (ANOVA) in conjunction with the Sidak multiple comparisons test (Prism software, v7.0d; GraphPad Software, La Jolla, CA, USA).