Topical Application of Dieckol Ameliorates Atopic Dermatitis in NC/Nga Mice by Suppressing Thymic Stromal Lymphopoietin Production  Gabsik Yang, Jee-Woo Oh, Hye Eun Lee, Bong Ho Lee, Kyung-Min Lim, Joo Young Lee  Journal of Investigative Dermatology  Volume 136, Issue 5, Pages 1062-1066 (May 2016) DOI: 10.1016/j.jid.2015.12.046 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Dieckol suppresses TSLP production through the inhibition of NF-κB activation in keratinocytes. (a) Chemical structure of dieckol (4-[4-[6-(3,5-dihydroxyphenoxy)-4,7,9-trihydroxydibenzo-p-dioxin-2-yl]oxy-3,5-dihydroxyphenoxy]dibenzo-p-dioxin-1,3,6,8-tetrol; molar mass = 742.549 g/mol). (b, c) KCMH-1 cells were treated with dieckol or dexamethasone (10 μmol/L) for (b) 6 hours or (c) 24 hours. TSLP mRNA or protein levels were determined by (b) RT-real-time quantitative PCR (n = 3) or (c) ELISA (n = 6). mRNA levels of TSLP were normalized with GAPDH mRNA levels. (d) KCMH-1 cells were transfected with pNF-κB-luciferase plasmid with β-galactosidase expression plasmid. Dieckol was treated for 30 minutes. Luciferase activity was normalized by β-galactosidase activity (n = 6). (e) Pam212 cells were pretreated with dieckol (40 μmol/L) for 1 hour and stimulated with IL-1β (10 ng/ml) and subjected to immunoblotting. (f) Pam212 cells were transfected with pNF-κB-luciferase reporter plasmid with β-galactosidase expression plasmid. After cells were pretreated with dieckol for 2 hours, cells were treated with IL-1β (10 ng/ml) for 18 hours and dieckol for 30 minutes (n = 3). (g) After KCMH-1 cells were incubated with dieckol for 24 hours, the conditioned media was collected and added to mouse primary bone marrow-derived dendritic cells. OX40L mRNA levels were determined by RT-real-time quantitative PCR and normalized with GAPDH mRNA levels (n = 6). Data are shown as the mean ± SEM. ∗P < 0.05 vs. (b, c, d) vehicle alone, (f) IL-1β alone, or (g) vehicle with conditioned media by 1-way analysis of variance followed by Dunnett’s post hoc test. Dex, dexamethasone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IκBα, inhibitor of kappa B alpha; μM, μmol/L; OX40L, OX40 ligand; p-IκBα, phosphor-IκBα; RT, reverse transcription; SEM, standard error of the mean; TSLP, thymic stromal lymphopoietin; Veh, vehicle. Journal of Investigative Dermatology 2016 136, 1062-1066DOI: (10.1016/j.jid.2015.12.046) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Topical application of dieckol alleviates AD-like skin symptoms in NC/Nga mice stimulated with HDM/DNCB. (a) Representative pictures of mouse dorsal skin area. One week after the first boost with 1% DNCB (200 μl/mouse), HDM (Dermatophagoides farina body ointment, 100 mg/mouse) and DNCB were alternately applied to dorsal skin for a week. After 2 weeks of induction, dieckol (1% in 200 μl of a 3:1 mixture of acetone:olive oil) was topically applied every day for 4 weeks (n = 8/group). (b) Dermatitis scores were measured once a week for 6 weeks. The dermatitis score was the sum of scores graded as 0 (none), 1 (mild), 2 (moderate), or 3 (severe) for each of the 4 measured symptoms (erythema/hemorrhage, scarring/dryness, edema, and excoriation/erosion). (c) Representative picture of the histopathological features of skin lesions. Tissues were excised, fixed in 10% formaldehyde, embedded in paraffin, and sectioned. The sections were stained with hematoxylin and eosin and toluidine blue (original magnification ×100 and ×200, scale bar = 100 μm). (d) Serum IgE levels (n = 8) and (e, f) skin TSLP and IL-33 levels (n = 8) were measured by ELISA. (g, h) The mRNA expression levels of GATA-3 and T-bet were determined in skin lesions by RT-real-time quantitative PCR (n = 8). (i–l) The levels of IL-4, -5, -13, and interferon gamma in skin were determined by ELISA (n = 8). Data are presented as the mean ± SEM. #P < 0.05 vs. normal group; ∗P < 0.05 vs. HDM/DNCB alone by 1-way analysis of variance followed by Dunnett’s post hoc test. DNCB, 2,4-dinitrochlorobenzene; GATA3, GATA binding protein 3; H&E, hematoxylin and eosin; IFN-γ, interferon gamma; ND, not detected; RT, reverse transcription; SEM, standard error of the mean; Veh, vehicle. Journal of Investigative Dermatology 2016 136, 1062-1066DOI: (10.1016/j.jid.2015.12.046) Copyright © 2016 The Authors Terms and Conditions