Topical application of marine briarane-type diterpenes effectively inhibits 12-O-tetradecanoylphorbol-13-acetate-induced inflammation and dermatitis in murine skin

Abstract

Background: Skin is the largest organ in the body, and is directly exposed to extrinsic assaults. As such, the skin plays a central role in host defense and the cutaneous immune system is able to elicit specific local inflammatory and systemic immune responses against harmful stimuli. 12-O-tetradecanoylphorbol-13-acetate (TPA) can stimulate acute and chronic inflammation and tumor promotion in skin. TPA-induced dermatitis is thus a useful in vivo pharmacological platform for drug discovery. In this study, the inhibitory effect of briarane-type diterpenes (BrDs) from marine coral Briareum excavatum on TPA-induced dermatitis and dendritic cell (DC) function was explored.

Methods: Evans blue dye exudation was used to determine vascular permeability. H&E-stained skin section was used to determine the formation of edema in mouse abdominal skin. We also used immunohistochemistry staining and western blot assays to evaluate the activation of specific inflammation makers and key mediators of signaling pathway in the mouse skin. Furthermore, mouse bone marrow DCs were used to determine the relationship between the chemical structure of BrDs and their regulation of DC function.

Results: BrD1 remarkably suppressed TPA-induced vascular permeability and edema in skin. At the biochemical level, BrD1 inhibited TPA-induced expression of cyclooxygenase-2, inducible nitric oxide synthase and matrix metalloproteinase-9, the key indicators of cutaneous inflammation. This inhibition was apparently mediated by interference with the Akt/NF-κB-mediated signaling network. BrD1 also inhibited TNF-α and IL-6 expression in LPS-stimulated BMDCs. The 8, 17-epoxide of BrDs played a crucial role in the inhibition of IL-6 expression, and replacement of the C-12 hydroxyl group with longer esters in BrDs gradually decreased this inhibitory activity.

Conclusions: Our results suggest that BrDs warrant further investigation as natural immunomodulatory agents for control of inflammatory skin diseases.

Figure 1

Briarane-type diterpene 1 (BrD1) inhibits TPA-induced vascular permeability in mouse skin. Abdominal skins of female C57BL/6 mice were treated topically with TPA (10 nmol) or acetone (vehicle control) for 6 h, or treated with TPA for 10 min and then treated for 6 h with the indicated concentrations of BrD1. One percent Evans blue dye (100 μl) was injected into mouse tail veins for 20 min. (A) Photograph of mouse abdominal skins subjected to various treatments and vascular permeability test. (B) Photographs of the dermal (internal) sides of representative abdominal skins subjected to the above treatment and test. (C) Evans blue extravasation in test skins was determined by assay of optical density at 620 nm. *, P < 0.05, and **, P < 0.01 versus LPS control. Data are representative of two independent experiments.

Figure 2

BrD1 inhibits TPA-induced edema. Abdominal skins of female C57BL/6 mice were topically treated as described above in Figure 1. Skin biopsies of abdominal skins were collected and stained with hematoxylin and eosin. a, b, c and d indicate the tissue layers: epidermis, dermis, hypodermis and peritoneum, respectively. Data are representative of two independent experiments.

Figure 3

BrD1 suppresses TPA-induced iNOS expression in mouse skin. Abdominal skins of female C57BL/6 mice were topically treated as described in Figure 1. Longitudinal tissue sections of abdominal skins were immunostained for COX-2 (A) and iNOS (B) proteins and counter-stained with hematoxylin, as described in Materials and Methods. Positive staining for COX-2 and iNOS are visualized as brownish cells in the dermis and epidermis (arrow). Data are representative of two independent experiments.

Figure 4

BrD1 inhibits TPA-induced MMP-9 expression in mouse skin. (A) Abdominal skins of female C57BL/6 mice were treated topically with TPA (10 nmol) or acetone (vehicle control) for 24 h, or treated with TPA for 10 min and then treated for 24 h with indicated concentrations of BrD1. Skin samples were collected, processed and analyzed for MMP-9 protein expression using western blot analysis. Mouse β-actin was used as a control. (B) Abdominal skins of female C57BL/6 mice were topically treated as described in Figure 1. MMP-9 mRNA expression was determined using RT-PCR assay. The ratio is presented as the value relative to the intensity of TPA-treated control skin. Data are representative of two independent experiments.

Figure 5

BrD1 inhibits TPA-induced NF-kB and Akt activation in mouse skin. (A) Mouse skin was untreated or TPA-treated for indicated time points, and test skin samples were collected and analyzed for phosphorylation levels of Akt and Erk1/2. (B) Abdominal skins of female C57BL/6 mice were treated topically with TPA (10 nmol) or acetone for 2 h or treated with TPA for 10 min and then treated for 2 h with the indicated concentrations of BrD1 for 2 h. Skin samples were collected, processed and analyzed for the phosphorylation levels of Akt, NF-κB, IκBα and Erk1/2 by Western blot analysis. Mouse β-actin was used as a control. The ratio is presented as the value relative to the intensity of TPA-treated control skin. Data are representative of two independent experiments.

Figure 6

BrD1 inhibits LPS-induced IL-6 and TNF-α expression in mouse BMDCs. (A) BMDCs from C57BL/6 mice were treated with LPS (100 ng/ml) for 1 to 24 h. (B) BMDCs from C57BL/6 mice were treated with LPS (100 ng/ml) for 24 h or LPS plus BrD1 at different concentrations for 24 h. Levels of IL-6 and TNF-α proteins in supernatants of conditioned media were analyzed by ELISA. *, P < 0.05, and ***, P < 0.001 versus LPS control. Data are representative of two independent experiments.

Figure 7

Structure-activity relationship of briarane-type diterpenes that confer the inhibition of LPS-induced IL-6 expression in mouse BMDCs. (A) Chemical structures of briarane-type diterpenes 1 to 8. The numbers labeling BrD1 indicate the carbon atoms. (B) The chemical structure of BrD1, and its analogs including BrD1-5C, BrD1-6C, BrD1-7C and BrD1-10C. (C) The chemical structure of BrD1, and its analogs including BrD1-5C, BrD1-6C, BrD1-7C and BrD1-10C. Immature DCs were co-treated with LPS and BrDs at 20 μM. The level of IL-6 proteins in supernatants was determined using ELISA. ***, P < 0.001 versus LPS+BrD1 treatment. Data are representative of two independent experiments.