Figure 1.
UV-absorbing properties and phototoxicity of afzelin.
A. Chemical structure of afzelin. B. The UV absorbance spectra for DMSO and afzelin (dotted line). Spectra were acquired on a BioTek UV-Vis spectrometer. C. Phototoxicity data for afzelin and chlorpromazine (CPZ), the positive control, in the 3T3 NRU phototoxicity test. Balb/c 3T3 cells were treated with different concentrations of the tested compounds and irradiated with UVA (5 J/cm2). Dotted line indicates the response in the 3T3-NRU assay in the absence of UVA irradiation and the solid line indicates the response in the presence of UVA (5 J/cm2) irradiation. Representative data, n = 3 (B–C).
Figure 2.
UVB-induced DNA damage is attenuated by the UV-absorbing activity of afzelin itself.
Cells were treated with different concentrations of afzelin (40–200 µg/ml) for 1 h before being exposed to UVB (20 mJ/cm2). A. At 30 min after UVB irradiation, the percentage of cellular DNA damage was detected by the comet assay after rewinding the DNA with alkaline buffer. Slides were stained with SYBR and viewed under a fluorescence microscope. Representative data, n = 3 B. Tail lengths (µm) of a minimum of 50 comets in each sample were analyzed using software image analysis. C. Enzyme-linked immunosorbent assay (ELISA) analysis of the percentage (%) of cyclobutane pyrimidine dimers (CPD) remaining in cells pretreated with vehicle or different concentrations of afzelin (40–200 µg/ml) at 30 min before UVB irradiation (20 mJ/cm2). D. Immunofluorescence analysis of phosphoactive histone H2A.X (γH2A.X; green) and phospho-p53 (serin 15) (green). Staining with propidium iodide (PI; red) was performed to observe cell nuclei. The number of Alexa Fluor 488-positive cells per 100 PI-positive cells was determined in two individual high-power fields per experiment by two independent assessors. Images were analyzed with GE IN Cell Analyzer1000 Workstation software. Data are presented as means ± SD, n = 3 (B-D). §P<0.01 compared with the vehicle-treated group, ¶P<0.01 compared with the UVB treated group (B–D).
Figure 3.
Radical scavenging effect of afzelin which was treated after UV irradiation.
A–D. Effects of afzelin on UVB-induced reactive oxygen species (ROS) production in HaCaT cells. CM-H2DCFDA (5 µM) was added, and the cells were incubated for 20 min in the dark. The cells were then washed and irradiated with 20 mJ/cm2 UVB. The cells were treated with afzelin for 1 h. Subsequently, A. DCF fluorescence was visualized by fluorescence microscopy. The average fluorescence intensity values were calculated using Image J software. Representative data, n = 3 B. ROS-induced DCF formation was measured using a spectrophotometer. C. Cell viability was measured 1 h after UVB irradiation. D. Effects of afzelin on UVB-mediated lipid peroxidation in HaCaT cells. HaCaT cells were treated with afzelin (40–100 µg/ml) for 12 h after being exposed to UVB (20 mJ/cm2) radiation. At 12 h after UVB irradiation, the cells were processed for lipid peroxidation. E. Effect of afzelin on ROS production in H2O2-treated HaCaT cells. The cells were treated as described in A but incubated for 30 min with 1 mM H2O2 instead of receiving UVB irradiation. Data are means ± SD, n = 3 (B-E). §P<0.01 compared with the vehicle-treated group, ¶P<0.01 compared with the UVB or H2O2-treated group (B–E).
Figure 4.
Afzelin treatment protects HaCaT cells against UVA, UVB, and H2O2-mediated decreased cell viability.
HaCaT cells were treated with different concentrations of afzelin (40–200 µg/ml) for 12 h after being exposed to oxidative stress (UVA, UVB, and H2O2). A. The percentage of viable cells was assessed using the MTT assay at 12 h after UVB irradiation. B. Phase-contrast microscopy. HaCaT cells were irradiated with UVB (20 mJ/cm2) and then incubated without or with afzelin (40–200 µg/ml) for 12 h. A representative picture from three independent experiments with similar results is shown. C. The cells were treated as described above but irradiated with UVA (5–10 J/cm2) instead of UVB irradiation. D. The cells were treated as described above but incubated for 12 h with 1 mM H2O2 instead of UVB irradiation. Data are presented as means ± SD, n = 5 (A,C and D). §P<0.01 compared with the vehicle-treated group, ¶P<0.01 compared with the UVB or H2O2-treated group (A,C and D).
Figure 5.
Inhibitory effect of afzelin on UVB-induced apoptosis.
HaCaT cells were treated with afzelin (40–200 µg/ml) for 12 h after irradiation with 20 mJ/cm2 UVB. A. Cell cycle analysis with cellular DNA content was examined by flow cytometry. The sub-G1 region (presented as “M1”) includes cells undergoing apoptosis. The number in each panel refers to the percentage of apoptotic cells. B. Agarose gel electrophoresis of HaCaT cell nuclear DNA fragments exposed to UVB (20 mJ/cm2) C. Immunoblot analysis of various apoptosis-related proteins in HaCaT cells unexposed or exposed to UVB (20 mJ/cm2). Cell extracts were subjected to 10–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with antibodies against cleaved caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), procaspase-8, and procaspase-9. β-Actin was included as an internal control. Immunoblot bands were quantified using Image J software. Representative data, n = 5 (A–C). D. Reconstructed skin was exposed to UVB (20 mJ/cm2) and then treated for 12 h with or without 100 µg/ml afzelin. Skin specimens were removed, fixed, paraffin-embedded, and processed for analysis by hematoxylin and eosin (H&E) staining and for an analysis of apoptotic cells by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Sunburn cells (arrowheads point to some examples) were calculated as the mean of five randomly selected fields (400×) from each skin sample. Data are means ± SD, n = 5 (C-D). §P<0.01 compared with the vehicle-treated group, ¶P<0.01 compared with the UVB treated group (C–D).
Figure 6.
Afzelin inhibits UVB-induced mitochondrial effects.
A. Effect on UVB mediated changes on bid, Bcl-xL, Bcl-2, and Bax protein expression. HaCaT cells were irradiated with UVB (20 mJ/cm2) and incubated for 12 h. Cell lysates were subjected to immunoblot analysis with antibodies against bid, Bcl-xL, and Bax. The Bcl-xL/Bax and Bcl-2/Bax ratios are presented in graphs. B. Mitochondrial membrane potential (Δψm) was measured by staining HaCaT cells with JC-1 followed by fluorescence microscopy analysis. HaCaT cells were incubated with the JC-1 probe for 30 min. Mean (red/green) fluorescence, expressed as a percentage of the control, indicates the ratio of high/low mitochondrial membrane potential. Representative data, n = 3 (A–B). C. DHR 123 was employed to detect mitochondrial hydrogen peroxide. Reactive oxygen species (ROS)-induced DHR 123 fluorescence was measured using a spectrophotometer. D. Enzyme-linked immunosorbent assay (ELISA) analysis of cytochrome c in cytosolic and mitochondrial fractions of HaCaT cells exposed to UVB (20 mJ/cm2). HaCaT cells were treated with afzelin (40–100 µg/ml) for 12 h after exposure to UVB (20 mJ/cm2) radiation. Then, the cells were processed for cytochrome c analysis. Data are means ± SD, n = 3 (A–D). §P<0.01 compared with the vehicle-treated group, ¶P<0.01 compared with the UVB -treated group (A–D).
Figure 7.
Afzelin treatment protects HaCaT cells against the UVB-induced decrease in cell viability by inhibiting p38 MAPK and JNK activity.
A. HaCaT cells were irradiated with 20 mJ/cm2 UVB and subsequently treated with afzelin (100 µg/ml) for the indicated times. Total cell lysates were prepared and subjected to immunoblot analysis. Bands for phospho-p38 MAPK, and phospho-SAPK/JNK were detected and normalized to β-actin. B and C. HaCaT cells were irradiated with 20 mJ/cm2 UVB and treated for 30 min with various concentration of afzelin (0–200 µg/ml). Total cell lysates were prepared at 30 min after irradiation and subjected to immunoblot analysis. The bands for phospho-p38 MAPK (B), and phospho-SAPK/JNK (C) were detected and normalized to their total forms. The relative density of each band after normalization is shown under each immunoblot as a fold-change compared with that of the UVB-exposed control, which was assigned an arbitrary unit of 1. Representative data, n = 3 (A–C). D. HaCaT cells were exposed to UVB (20 mJ/cm2) and treated with SB203580 (5 µM), SP600125 (20 µM), and afzelin (100 µg/ml) for 24 h. Cell viability was analyzed by the MTT assay. Data are means ± SD, n = 3 (D). §P<0.01 compared with the vehicle-treated group, ¶P<0.01 compared with the UVB treated group (D).
Figure 8.
Inhibition of p38 mitogen activated protein kinase (MAPK) suppresses UVB-mediated induction of inflammatory cytokine production in human HaCaT keratinocytes.
HaCaT cells were treated with the test substances (5 µM SB203580, 20 µM SP600125, or 40–200 µg/ml afzelin) after irradiation with 20 mJ/cm2 UVB. A. After 12 h, cyclooxygenase (COX)-2 protein expression was assessed by Western blotting. The histogram depicting relative COX-2 protein expression levels was normalized to β-actin expression level. Representative data, n = 3 (A). B. Prostaglandin-E2 (PGE2), C. interleukin-6 (IL-6), and D. tumor necrosis factor-α (TNF-α) concentrations in cell culture supernatants from the same experiments. Data are means ± SD, n = 5 (B–D). §P<0.01 compared with the vehicle-treated group, ¶P<0.01 compared with the UVB-treated group (B–D).
Figure 9.
Schemic summary of the complex skin-protecting properties of afzelin.
Afzelin protects the skin from the deleterious effects of UVB by exerting cellular activities (DNA protective, antioxidative, and anti-inflammatory) as well as absorbing UV.