Topical application of marine briarane-type diterpenes effectively inhibits 12-O-tetradecanoylphorbol-13-acetate-induced inflammation and dermatitis in murine skin
© Wei et al; licensee BioMed Central Ltd. 2011
Received: 17 November 2011
Accepted: 21 December 2011
Published: 21 December 2011
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.
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.
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.
Our results suggest that BrDs warrant further investigation as natural immunomodulatory agents for control of inflammatory skin diseases.
Skin is the largest organ in the body. As the primary interface between the body and environment, it serves as the first line of defense against microbial pathogens as well as physical and chemical stress or insults [1, 2]. The skin does not only serve as a physical and a chemical barrier, but is also an immune-competent organ that elicits effective innate and adaptive immune responses to protect the human body. The cutaneous immune system maintains a balance between restricting excessive inflammation following tissue damage or injury and preserving the ability to rapidly respond to pathogen infection . It is clear that inadequate or misdirected immune response is involved in the pathogenesis of a variety of acquired inflammatory skin disorders . Therefore, systematic investigation of the mechanisms of action of immunomodulatory agents on the skin's immune system is necessary for the development of therapies for skin disorders.
Acute inflammation is the initial immune response to harmful stimuli. Acute inflammation in the skin often involves an increase in the vascular permeability of skin tissues, resulting in an accumulation of fluid at the inflamed site (edema). The release of mediator molecules such as nitric oxide and prostaglandins also elicits vascular permeability, thus permitting the efficient migration of leukocytes, mainly neutrophils, to the inflamed tissue site. Matrix metalloproteinase-9 (MMP-9) has been reported to be a crucial player in such neutrophil migration by degrading some major cellular components of the epidermis and dermis . In addition, it is well-known that secretions of cytokines such as TNF-α, IL-1α and IL-6 by keratinocytes or antigen-specific cells can play a key role in mediating the cutaneous inflammatory response [2, 5, 6]. These mediators were employed as indicators of skin inflammation in this study.
Several novel approaches have been explored to manage risk factors for skin cancers, tissue damage from UVB exposure, and inflammatory skin disorders . Several phytochemicals and tissue extracts from medicinal plants have been reported to confer immunostimulatory activities and have potential clinical applications [8, 9]. Our laboratories previously reported that a small phytochemical from Lithospermum erythrorhizon (shikonin) can inhibit the transcriptional activation of human TNF-α promoter in vivo in mouse skin . We also showed that caffeic acid suppresses UVB radiation-induced expression of IL-10 and activation of MAPKs in mouse skin tissues . More recently, we demonstrated that ferulic acid, a phenolic phytochemical, can effectively inhibit UVB-induced matrix metalloproteinases in mouse skin via a posttranslational mechanism .
Natural products from plants and terrestrial microorganisms have traditionally provided good sources of lead compounds/agents for human medicines. However, due to the biological diversity of the marine environment and the discovery of marine compounds with certain unique structures and pharmacological activities, compounds from marine organisms are expected to be a major source of lead compounds for future generations of pharmaceuticals . A spectrum of different novel marine compounds have been identified, and their bioactivities evaluated for potential pharmaceutical application . Among these compounds, we isolated and identified a group of briarane-type diterpenes (BrDs) from Briareum excavatum, a Formosan gorgonian coral [14, 15].
In this study, we selected a group of specific BrDs from our chemical library of marine origin for systematic study and showed that BrD1 (excavatolide B) can drastically inhibit 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute inflammation in murine skins, as determined by its effect on vascular permeability, edema and key inflammatory mediators. We further determined a structure-activity relationship (SAR) of selected BrDs in terms of their regulation of cytokine expression. The possible molecular mechanisms for the mode of action of these BrDs were also studied.
Recombinant cytokines mIL-4 and mGM-CSF were purchased from PeproTech (Rocky Hill, NJ). TPA (12-O-tetradecanoyl-13-phorbol-acetate), Evans blue dye, N, N-dimethyl-formamide and LPS (Escherichia coli 055:B5) were purchased from Sigma-Aldrich (St. Louis, MO). BrDs were prepared from marine soft coral (Briareum excavatum) as previously described .
Female C57BL/6JNarl mice (5-6 weeks old) were purchased from the National Laboratory Animal Breeding and Research Center, Taipei, Taiwan. All mice were maintained in a laminar airflow cabinet in a specific pathogen free (SPF) animal room kept at 24 ± 2°C and 40-70% humidity with a 12 h light/dark cycle under SPF conditions. All facilities were approved by the Institutional Animal Care and Utilization Committee of Academia Sinica, and animal experiments were all conducted according to institutional guidelines.
Preparation of 12-acyloxyl analogues of BrD1
An appropriate acyl chloride (0.02 mmol) was added to a solution of BrD1 (excavatolide B) (10 mg) in 5 ml of pyridine; the mixture was allowed to stand overnight at room temperature. Four milliliters of water was added to the reaction mixture followed by extraction with EtOAc (5 ml × 3). The EtOAc layers were combined, dried over anhydrous MgSO4 and evaporated. The afforded residue was purified by column chromatography on silica gel using EtOAc/hexane (1:8) as eluent to yield analogues of BrD1 (BrD1-5C, BrD1-6C, BrD1-7C and BrD1-10C). Yields varied from 61.6% to 78.3%.
Measurement of vascular permeability
TPA-induced vascular permeability assay was modified and performed as previously described . Shaved abdominal skins of female C57BL/6JNarl mice were topically treated with vehicle (acetone, 200 μl/site) or TPA (10 nmol in 200 μl acetone/site) for 6 h or treated with TPA for 10 min and subsequently treated for 6 h with the indicated concentrations of BrD1. Abdominal skins of untreated mice were used as the control group. One percent Evans blue dye (100 μl) was injected into mouse tail veins. After 20 min, anatomical appearances of mouse abdomens from the various treatments were photographically recorded. Abdominal skins representative of each test group were removed, turned over and photographed. Evans blue dye extravasated into the skins was extracted by incubation of the skin samples in 99% N, N-dimethyl-formamide overnight at 60°C and optical density was measured at 620 nm.
Generation of mouse bone marrow-derived dendritic cells
For mouse bone marrow-derived DCs (BMDCs), five to six-week-old female C57BL/6JNarl mice were purchased from the National Laboratory Animal Center, Taiwan and kept under SPF conditions. BMDCs were generated from bone marrow cells of C57BL/6 mice as described previously . In brief, bone marrow was isolated from femurs and tibiae which were then flushed with RPMI-1640 medium using a syringe with a 0.45-mm needle on Day 0. Red blood cells in suspension were lysed for 5 min with ACK lysing buffer (150 mM NH4Cl, 1.0 mM KHCO3, 0.1 mM EDTA). Bone marrow cells were suspended at a density of 1 × 107cells/30 ml in RPMI-1640 containing 10% FBS, 2 mM L-glutamine, 1% of nonessential amino acids, 100 U/mL penicillin and 100 μg/mL streptomycin supplemented with 20 ng/mL of mGM-CSF in 15-cm dishes at 37°C with 5% CO2. On day 2, two-thirds of the medium was removed and 30 mL fresh medium with mGM-CSF was added to the cells. On day 5, culture plates were gently swirled and the floating and loosely adherent cells were discarded. Aliquots of 75% culture media were replenished with 20 ng/mL mGM-CSF and 20 ng/mL mIL-4. On day 7, mouse BMDCs (95% pure CD11b and MHCII) were harvested and incubated in RPMI 1640 medium containing 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 U/ml penicillin, 100 μg/ml streptomycin, and supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) for 24 h at 37°C with or without test chemicals in the presence or absence of LPS (100 ng/ml).
Measurement of pro-inflammatory cytokines
BMDCs were treated with or without test chemicals in the presence or absence of LPS (100 ng/ml) for 24 h at 37°C. Aliquots of supernatants from DC cultures were assayed for IL-6 and TNF-α using commercial ELISA kits (R&D Systems, Minneapolis, MN) following the manufacturer's recommendations.
Primer design and RT-PCR
YES: Yesp Shaven abdominal skins of female C57BL/6JNarl mice were untreated or topically treated with vehicle (acetone, 200 ml/site) or TPA (10 nmol in 200 ml acetone/site) for 6 h or treated first with TPA for 10 min and then with the indicated concentrations of BrD1 for 6 h. Total RNA was extracted from treated abdominal skins of female mice using TRIzol reagent (Invitrogen Corp., Carlsbad, CA). RT-PCR reactions using AccessQuick RT-PCR system (Promega, Madison, WI) were carried out as described previously . The primers contained the following sequences: mouse MMP-9 sense primer 5'-CTAGTGAGAGACTCTACACGGAG-3', and anti-sense primer 5'-GAGCCACGACCATACAGATACTG-3'; mouse GAPDH sense primer 5'-CATCACTGCCACCCAGAAGACTGTGGA-3', and anti-sense primer 5'-TACTCCTTGGAGGCCATGTAGGCCATG-3'. Gel images were scanned and densitometry analysis of the captured image was performed using Gene Tools software (Syngene, Cambridge, UK).
Mice were treated or not treated topically on their shaven abdomens with acetone (vehicle only) or TPA (10 nmol in 200 μl acetone/site) for 6 h or treated first with TPA for 10 min, then with the indicated concentrations of BrD1 for 6 h. Mice were killed by cervical dislocation. Abdominal skin tissues were collected, fixed with formalin buffer and embedded in paraffin. Tissue sections (5 mm) were cut and laid onto silanized glass slides and deparaffinized three times with xylene for 5 min each prior to rehydration using a graded alcohol bath. For hematoxyline and eosin staining, the sections were stained with hematoxyline and eosin staining. For immunohistochemical staining, the deparaffinized sections were boiled in 10 mM citrate buffer (pH 6.0) for 10 min for antigen retrieval and rinsed with PBS containing 0.05% Tween-20 buffer for 5 min. The sections were treated with 3% hydrogen peroxide in methanol for 15 min to decrease non-specific binding. They were subsequently washed with blocking solution (PBS containing 1% BSA) for 30 min and then PBST twice for 5 min. All slides were incubated first with 2% goat serum in blocking solution for 30 min, then with a 1:200 dilution of polyclonal iNOS antibody (eBioscience, San Diego, CA) at room temperature for 1 h. The slides were further developed using HPR EnVisionTM system (Dako, Glostrup, Denmark). Subsequently, peroxidase-binding sites were determined by staining with 3,3'-diaminobenzidine tetrahydrochloride (Dako). Eventually, Mayer's hematoxyline was used for counterstaining.
Western blotting analysis
Shaven abdominal skins of female C57BL/6JNarl mice were not treated or topically treated with vehicle (acetone, 200 μl/site) or TPA (10 nmol in 200 μl acetone/site) at indicated time points or treated first with TPA for 10 min, then with the indicated concentrations of BrD1 at the indicated time points. Abdominal skin samples were collected, homogenated and lysed to prepare total proteins. Protein samples were subsequently resolved by SDS-PAGE using a gradient gel. The resolved proteins were transferred to a PVDF Immobilon-P membrane (Millipore, Bedford, CA.), and the membrane was blocked with 5% non-fat dry milk in PBST buffer (phosphate-buffered saline (PBS) containing 0.1% Tween 20) for 60 minutes at room temperature. The membranes were incubated overnight at 4°C with commercially available antibodies (1:1000 dilutions). Loading of equal amounts of protein was assessed using mouse β-actin. The blots were rinsed three times with PBST buffer for 5 minutes. Washed blots were incubated with HRP-conjugated secondary antibody (1:100,000 dilution), then washed again three times with PBST buffer. The transferred proteins were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). Quantification of bands was performed using Image J software.
Statistical analyses were performed with GraphPad Prism software, version 5. Data are presented as mean ± SD and statistical significance was determined by a one-way ANOVA followed by Tukey multiple comparison tests. Means were considered significantly different if the P value was less than 0.05.
Excavatolide B (BrD1) effectively inhibits TPA-induced vascular permeability in mouse skin
BrD1 inhibits TPA-induced edema
BrD1 suppresses TPA-induced COX-2 and iNOS expression in mouse skin
BrD1 inhibits TPA-induced MMP-9 expression in mouse skin
BrD1 inhibits TPA-stimulated NF-κB and Akt activation in mouse skin
BrD1 inhibits LPS-induced IL-6 and TNF-α expression in mouse bone marrow-derived dendritic cells
Structure-activity relationship of BrDs that underlies inhibition of LPS-induced IL-6 expression
Inhibitory effect of briarane-type diterpenes from marine coral Briareum excavatumon on LPS-induced IL-6 in mouse BMDCs.
IL-6 (% of LPS control)
Name (20 μM)
% (Mean ± SD)
2.4 ± 0.93
22.5 ± 4.5
26.2 ± 4.0
34.7 ± 8.6
37.0 ± 9.6
56.6 ± 2.1
61.3 ± 9.5
81.3 ± 1.9
Steric hindrance of 12-acyloxyl substituents reduces the inhibitory bioactivity of briarane-type diterpenes
Our results suggest that steric hindrance of the 12-acyloxyl substituents of BrDs decreases their inhibitory activity. In order to confirm this SAR, we semi-synthesized several analogues of BrD1 by replacing the hydroxyl group at C-12 with longer acyloxyl groups (C5, C6, C7 and C10) (Figure 7B). We then examined the inhibitory effect of BrD1 and these BrD1 analogues (BrD1-5C, BrD1-6C, BrD1-7C and BrD1-10C) on cytokine secretion activity in LPS-stimulated mature DCs. BrD1 significantly inhibited the expression of cytokine IL-6 in LPS-stimulated, mature DCs (Figure 7C). This inhibitory activity gradually decreased when the hydroxyl group at C-12 was replaced by longer acyloxyl groups (C5 to C10), confirming that steric hindrance of 12-acyloxyl substituents can effectively suppress the capacity of BrDs to inhibit LPS-induced IL-6 expression. These results thus strongly suggest that the 8, 17-epoxide and 12-hydroxyl groups of BrD1 are instrumental in conferring the BrD-mediated inhibitory activity in mouse bone marrow-derived dendritic cells.
The immune response in skin tissues is systemic and sophisticated. A variety of vaccination strategies are administered through the skin to produce antigen-specific immune responses and protect the host [22–25]. The skin is also a useful organ for drug delivery, for example, certain pharmaceuticals can be delivered via the use of a transdermal patch. On the other hand, excessive inflammation or inadequate immune response in skin can lead to various cutaneous diseases such as allergic reactions, psoriasis and even skin cancers. Our laboratories have previously demonstrated that a number of phytochemicals derived from medicinal plants or fruits exhibit strong anti-inflammatory effects either on TPA-induced inflammatory mediators or UVB radiation-induced cytokine and MMP expression in mouse skin tissues [7, 11, 26]. In the present study, we showed that a group of marine briarane-type diterpenes, particularly BrD1, can strongly suppress TPA-induced inflammation and dermatitis in a mouse skin model. In combination with our previous studies [7, 10, 11, 26], the current study leads us to suggest that a specific combination of several skin model/system test sets  may warrant a systematic evaluation for use as the first in vivo platform for drug screening or efficacy verifications.
Epidermis tissue contains keratinocytes, Langerhans cells and other cells types. When the epidermis is exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA), keratinocytes and Langerhans cells are stimulated through activation of protein kinase C (PKC) resulting in activation of PI3K/Akt, Erk and NF-κB signaling transduction pathway [28–30]. This activation of Akt and NF-κB activity via TPA stimulation was faithfully observed in the present investigation (Figure 5A), providing a solid baseline from which to investigate the effect of BrD1. Akt and NF-κB play crucial roles in the activation of key inflammatory enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) [31–33]. Interestingly, our results showed that the expression of COX-2 and iNOS was detected mainly in the epidermis after TPA stimulation, indicating that COX-2 and iNOS were mainly released from epidermal cells (likely mainly from keratinocytes) (Figure 3A and 3B). In addition, we also observed considerable sporadic expression of iNOS in the dermal layer after TPA treatment, and BrD1 strongly inhibited this iNOS expression in both the epidermal and dermal layers (Figure 3B). The specific cell types in these two tissue layers that are responsive to BrD1 inhibition effect need to be further investigated. The major dermis cell types are fibroblasts, macrophages and fabric basophils (mast cells). During inflammation, immune cells, mainly neutrophils, can effectively infiltrate into the dermal layer. Keratinocytes, mast cells and infiltrated neutrophils in skin have been reported to secrete MMP-9 for tissue remodeling in response to inflammation [34–36]. MMP-9 has further been reported to be directly mechanistically involved in the increases of vascular permeability [37–39]. Our results (Figure 4A and 4B) show that BrD1 can effectively inhibit TPA-induced MMP-9 expression. We suggest that this may be partly due to the inhibitory effect of BrD1 on vascular permeability and edema. Together, our present results suggest that BrD1 may suppress the inflammatory responses mediated by different types of cells present in different tissue layers of the skin. Our data (Figure 5A and 5B) also indicate that the molecular mechanisms responsible for the anti-inflammatory effects of BrD1 may involve the Akt and NF-κB signaling transduction pathway.
The pro-inflammatory cytokines TNF-α and IL-6, released by dendritic cells, are key mediators of inflammatory and immune responses. It is known that expression of these cytokines is controlled by TLR4-stimulated activation of NF-κB via regulation of the transcriptional activities of these cytokines in monocytes and dendritic cells. The results from this study show that BrD1 can inhibit the activation of NF-κB in TPA-induced mouse dermatitis (Figure 5B) and in LPS-stimulated mouse BMDCs (data not shown). However, BrD1 exhibited a differential inhibitory effect on TNF-α and IL-6 in LPS-induced mouse BMDCs. As seen in Figure 6B, BrD1 strongly inhibited LPS-induced IL-6 expression but only partially inhibited LPS-induced TNF-α expression. In order to distinguish between TNF-α and IL-6 inhibition, a time course study of LPS-induced TNF-α and IL-6 expression was performed. After LPS stimulation, a relatively high level of expression of TNF-α was detected between 1 h and 2 h post-treatment, and an elevated level of IL-6 expression was detected between 2 h and 4 h post-treatment (Figure 6A). Our previous findings and those of others have shown evidence of post-transcriptional regulation of TNF-α pre-mRNA in resting T-cells, B-cells and monocytes [40–42]. LPS-induced mRNA splicing can lead to massive and rapid expression of TNF-α mRNA in monocytes . The differential effect on expression of TNF-α and IL-6 in LPS-activated BMDCs seen in this study may have implications for the future use of BrD1 in pharmaceutical applications. The detailed molecular mechanisms responsible for this differential effect warrant further investigation.
In summary, we conclude that marine briarane-type diterpene (BrD1) can not only effectively suppress TPA-induced vascular permeability and edema, but can also decrease the expression of COX-2, iNOS and MMP-9, and reduce the activation of NF-κB and Akt in test mouse skin tissues. In addition, BrD1 can also strongly inhibit IL-6 expression in LPS-stimulated BMDCs, a key immunoregulatory cell type. The 8, 17-epoxide of the BrDs apparently contribute stereochemically to the inhibition of IL-6 expression, and steric hindrance of the 12-acyloxyl substituents was found to effectively reduce the inhibitory bioactivity of BrD1. In view of the unique structure and specific active functional group of BrD1, we suggest this marine natural product should be further evaluated for development as an immunotherapeutic agent for control of inflammation and skin diseases, and for other healthcare applications.
We thank Dr. Heiko Kuhn of the Academia Sinica Institute of Molecular Biology and Agricultural Biotechnology Research Center Joint Editors' Office, and Ms. Ruth Giordano of Dr. Ning-Sun Yang's laboratory for editing the manuscript. We also thank Dr. Chia-Chung Hou of the Academia Sinica Agricultural Biotechnology Research Center and Ms. Hui-Ming Chen of the Academia Sinica Agricultural Biotechnology Research Center for valuable suggestions and thank Ms. Chia-Chi Wu of the Academia Sinica Agricultural Biotechnology Research Center and Ms. Li-Ting Huang of the Academia Sinica Agricultural Biotechnology Research Center for performing some tests. This work was supported by a grant (NSC98-2113-M-110-002-MY3) from the National Science Council, Executive Yuan, Taiwan, ROC; and by grants (CCMP 99-RD-067 and CCMP100-RD-019) from the Committee on Chinese Medicine and Pharmacy, Department of Health, Executive Yuan, Taiwan, ROC
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