Figure 3
12-HHT reduces UVB-induced IL-6 synthesis via inhibition of the p38 MAPK/NF-κB pathway. (A) HaCaT cells were starved for 12 h and irradiated with UVB (5 mJ/cm2) for various times (0, 30, 60 and 120 min). The cell lysates were examined by western blotting to analyze p-p38, p38, p-JNK, JNK, p-ERK and ERK. (B) Starved HaCaT cells were pretreated with SB203580 (20 µM), PD98059 (20 µM), SP600125 (20 µM) or Bay11-7082 (20 µM) for 60 min and then irradiated with UVB (5 mJ/cm2) for 24 h. The level of IL-6 synthesis was measured by ELISA. (C) Upon UVB irradiation (5 mJ/cm2), the HaCaT cells were immediately incubated with ethanol (control) or 12-HHT (150 nM) for 60 min. The cell lysates were prepared and examined by western blotting using antibodies specifically recognizing p38 MAPK and p-p38 MAPK. The p-p38 MAPK signals are presented as the fold induction relative to the control samples and are shown with densitometry values expressed as the means ± S.D. of three independent experiments. (D) HaCaT cells were co-transfected with both NF-κB-dependent luciferase construct and pSV40-β-galactosidase construct for 24 h and serum-starved for an additional 6 h. These transfected cells were either sham-irradiated or stimulated with UVB (5 mJ/cm2) and treated with ethanol (control) or 12-HHT (150 nM) for 1 h. The relative fold increase of luciferase activity was calculated, as described in the Methods. Data indicate the means ± S.D. of three independent experiments (*P < 0.05 and **P < 0.01).
