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).