The prevention of 2,4-dinitrochlorobenzene-induced inflammation in atopic dermatitis-like skin lesions in BALB/c mice by Jawoongo
Jawoongo is an herbal mixture used in traditional medicine to treat skin diseases. This study aimed to investigate whether Jawoongo ameliorates Atopic dermatitis (AD)-like pathology in mice and to understand its underlying cellular mechanisms.
AD was induced by 2, 4-Dinitrocholrlbenzene (DNCB) in BALB/c mice. Treatment with Jawoongo was assessed to study the effect of Jawoongo on AD in mice. Histological Analysis, blood analysis, RT-PCR, western blot analysis, ELISA assay and cell viability assay were performed to verify the inhibitory effect of Jawoongo on AD in mice.
We found that application of Jawoongo in an ointment form on AD-like skin lesions on DNCB-exposed BALB/c mice reduced skin thickness and ameliorated skin infiltration with inflammatory cells, mast cells and CD4+ cells. The ointment also reduced the mRNA levels of IL-2, IL-4, IL-13 and TNF-α in the sensitized skin. Leukocyte counts and the levels of IgE, IL-6, IL-10 and IL-12 were decreased in the blood of the DNCB-treated mice. Furthermore, studies on cultured cells demonstrated that Jawoongo exhibits anti-inflammatory activities, including the suppression of proinflammatory cytokine expression, nitric oxide (NO) production, and inflammation-associated molecule levels in numerous types of agonist-stimulated innate immune cell, including human mast cells (HMC-1), murine macrophage RAW264.7 cells, and splenocytes isolated from mice.
These findings indicate that Jawoongo alleviates DNCB-induced AD-like symptoms via the modulation of several inflammatory responses, indicating that Jawoongo might be a useful drug for the treatment of AD.
KeywordsAtopic dermatitis Jawoongo 2,4-dinitrochlorobenzene Cytokine Inflammation
Angelica gigas Nakai
Bovine serum albumin
Human mast cells
Inducible nitric oxide synthase
Optical cutting temperature
Tumor necrosis factor-α
Thymic stromal lymphopoietin
White blood cells
Atopic dermatitis (AD) is the most common chronic inflammatory and chronically relapsing skin disease. The prevalence of AD has increased continuously, and approximately 10 million people worldwide are currently affected. The disease leads to a significantly reduced quality of life [1, 2]. The pathogenesis of AD is not well understood but appears to be associated with the activation of innate immune responses, including inflammation.
Common features of AD include excessive infiltration of inflammatory cells and granulated mast cells into AD skin lesions and high immunoglobulin E (IgE) levels and leukocyte counts in blood . Notably, CD4+ T cells are critical for the development of allergic inflammatory diseases. CD4+ T cell activation induces the secretion of cytokines and chemokines and drives inflammation and allergic sensitization . Furthermore, the development of AD has been attributed to the activation of mast cells [5, 6] and T-helper 2 (Th2)-dependent cells [7, 8]. Mast cells are activated by IgE through the high-affinity IgE receptor (Fc휀R) [9, 10]. These cells are then recruited into AD skin lesions, where they promote skin hypersensitivity reactions by releasing histamine; prostaglandin D2 (PGD2); AD-related Th2 cytokines, including IL-4, IL-5, and IL-13; and proinflammatory cytokines, including IL-4 and IL-6.
Tacrolimus is an effective immunosuppressant that inhibits the production of various cytokines, such as IL-2, IL-4 and IL-5. Many studies have demonstrated that tacrolimus suppresses allergic cytokine production by T cells [11, 12] and is effective against AD in animal models [13, 14, 15]. Tacrolimus ointment is used for the treatment of AD in adults and children [16, 17, 18]. However, previous studies have shown that treatment with tacrolimus elevated total and specific IgE levels and caused transient burning and erythema in ~ 60% of patients [19, 20]. Consequently, the development of alternative remedies is necessary to reduce these side effects.
Jawoongo is a traditional herbal medicine composed of Lithospermum root and Angelica gigas Nakai (AGN). AGN contains numerous active ingredients, including decursin. In previous studies, decursin exhibited anti-allergic effects in an asthma model and anti-metastatic effects in colon cancer [21, 22, 23]. Decursin has also been used for the treatment of various dermatitis-associated skin diseases, including eczema and chilblain. Recent studies have indicated that decursin is effective in driving artificial wound healing and ameliorating skin inflammation [24, 25, 26]. As known as, DNCB allergens elicited a systemic immune response, because increased cytokine levels in the serum of mice .
We investigated effect of jawoongo in DNCB induced AD model in Balb/c mice. The goal of this study was to explore the effects of Jawoongo on 2,4-dinitrochlorobenzene (DNCB)-induced AD-like symptoms in BALB/c mice and several types of immune cell.
Preparation of Jawoongo ointment and tacrolimus ointment
Jawoongo ointment was supplied by Han-poong Pharm Co., Ltd. (Jeonjoo, Republic of Korea). Jawoongo is made from Lithospermum root and Angelica gigas Nakai (AGN). The main compound in Lithospermum root is shikonin. Shikonin inhibits inflammation and imflammasomes [28, 29]. The main compounds in AGN are decursin and Nodakenin. Decursin and Nodakenin are known to inhibit inflammation. Additionally, decursin is known to inhibit the proliferation of ovarian cancer cells [30, 31, 32]. A 0.1% protopic tacrolimus ointment was also utilized (Astellas Pharma Tech, Japan). Tacrolimus is made from FK506, which has been used to treat dermatitis, as it suppresses the development of Th2 cells [33, 34].
Six-week-old male BALB/c mice were obtained from Orient Bio, Inc. (Seoul, Korea). The mice were maintained for 1 week under a controlled temperature (23 ± 3 °C) and humidity (55 ± 15%) with a 12 h light/12 h dark cycle before initiating the experiment. The body weights and food intake of the animals were measured once every 2 days. All procedures performed on the mice were approved by the animal care center of Kyung-Hee University (Kyung Hee University Study Proposal (SEOUL) – 12-014; Approval No. KHUASP(SE)-12–014). Upon completion of the experiment, the mice were anesthetized with a 1.2% avertin solution (0.5 g 2,2,2-tribromoethanol powder dissolved into 1 ml 2-methyl-2-butanol and 39 ml phosphate-buffered saline (PBS) at 55 °C) that was filtered through a Nalgene 0.22-μm filter (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and sacrificed via exsanguination [35, 36].
Induction of AD-like lesions and drug treatment
Skin samples (20 μm thick) were embedded in Tissue-Tek optical cutting temperature (OCT) compound (Leica, CA, Richmond, USA). The skin samples were stained with hematoxylin and eosin (H&E) to visualize inflammatory cells and with toluidine blue (TB) to visualize mast cells and then examined under a light microscope (Olympus). The mast cells and inflammatory cells were counted in 10 sections of high-power fields (HPFs) at 40×, 400× and 1000× magnification.
CD4+ lymphocytes were detected by immunohistochemical analysis using anti-CD4+ antibodies (Santa cruz biotechnology, Dallas, Texas, USA). After deparaffinization, the slides were rehydrated and antigen retrieval done by microwave treatment, they were treated with 3% hydrogen peroxide in PBS for 15 min to inhibit the endogenous peroxidase activity of blood cells. Following the hydrogen peroxide treatment, the sections were incubated with 5% bovine serum albumin (BSA) in PBS as a blocking reagent for 1 h at room temperature. The sections were then incubated with mouse monoclonal CD4+ antibodies (1:100 dilution) overnight at 4 °C. After washing with PBS, subsequently incubated with secondary biotinylated anti-rabbit IgG for 1 h at room temperature. The sections were treated with an avidin-biotin HRP complex (Vectastain ABC kit, Vector Labs, CA, USA) for 30 min at 4 °C and stained with diaminobenzidine (DAB) tetrachloride as a substrate. The slides were mounted in an aqueous mounting solution (DAKO, Glostrup, Denmark) and cover-slipped. All of the sections were analyzed using an Olympus microscope, and images were captured using a digital video camera.
Analysis of mouse blood
Whole blood samples were collected by cardiac puncture and placed in Vacutainer TM tubes containing EDTA (BD Biosciences, USA) to prevent clotting. Anti-coagulated blood was processed to determine leukocyte counts, including lymphocytes, monocytes, eosinophils, basophils and neutrophils, using a HEMAVET 950 hematological analyzer (Drew Scientific, Inc., Oxford, USA).
PCR primer sequences
F: 5’-GCA GCT GTT GAT GGA CCT AC-3′
R: 5’-TCC ACC ACA GTT GCT GAC TC-3′
F: 5’-TCG GCA TTT TGA ACG AGG TC-3′
R: 5′-GAA AAG CCC GAA AGA GTC TC-3′
F: 5’-CGG CAG CAT GGT ATG GAG TG-3′
R: 5′-ATT GCA ATT GGA GAT GTT GGT CAG-3′
F: 5′-AAT GGC AAC ATC AGG TCG GCC ATC ACT-3′
R: 5’-GCT GTG TGT CAC AGA AGT CTC GAA CTC-3′
F: 5′-ATG AGC ACA GAA AGC ATG ATC-3′
R: 5’-TAC AGG CTT GTC ACT GGA ATT-3’
F: 5′-GAG GGG CCA TCC ACA GTC TTC-3’
R: 5’-CAT CAC CAT CTT CCA GGA GCG-3’
F: 5’-TGC CTC CAA GAA CAC AAC TG-3’
R: 5’-CTC TGG TTG GCT TCC TTC AC-3’
F: 5’-AAC CTT CCA AAG ATG GCT GAA-3’
R: 5’-CAG GAA CTG GAT CAG GAC TTT-3’
F: 5′-GGT CAA CAT CAC CCA GAA CC-3’
R: 5′-TTT ACA AAC TGG GCC ACC TC-3’
F: 5′-TAT GAG TGG GAC CAA AAG TAC CG-3’
R: 5′-GGG ATT GAA GGT TAG GCT CTG G-3’
F: 5’-CGT CTT CAC CAC CAT GGA GA-3’
R: 5’-CGG CCA TCA CGC CAC AGT TT-3’
Enzyme-linked immunosorbent assay
Levels of IgE, IL-4, IL-6, IL-10, IL-12 and IL-13 were assessed using a Duoset enzyme-linked immunosorbent assay (ELISA) system (BD Biosciences, USA) according to the manufacturer’s instructions. In brief, to assess the level of IgE, IL-4, IL-6, IL-10, IL-12 and IL-13 in the mice serum treated with Tacrolimus and Jawoongo, 96-well plates were coated with capture antibody in ELISA coating buffer and incubated overnight at 4 °C. The plates were then washed with PBS with 0.05% Tween 20 (PBS-T) and subsequently blocked with 10% FBS in PBS for 1 h at 20 °C. Serial dilutions of standard antigen or sample in dilution buffer (10% FBS in PBS) were added to the plates, and the plates were incubated for 2 h at 20 °C. After the plates were washed, biotin-conjugated anti-mouse IgE and streptavidin-conjugated horseradish peroxidase (SAv-HRP) were added to the plates, and the plates were incubated for 1 h at 20 °C. Finally, the tetramethylbenzidine (TMB) substrate was added to the plates, and after 15 min of incubation in the dark, 2 N H2SO4 was added to stop the reaction. The optical density was measured at 450 nm on an automated ELISA reader. (Versa Max, Molecular Devices, CA, USA).
Detection of nitric oxide
Nitric oxide (NO) production from RAW264.7 cells in culture was measured using Griess reagent (Welgene, Korea). Briefly, 150 μL of cell culture supernatant was mixed with 150 μL of Griess solution and incubated for 30 min at room temperature. The optical density was determined at 570 nm using a microplate reader.
Cell viability assay
An MTS assay was performed to determine cell viability. To accomplish this, cells (HMC-1, RAW264.7 and Splenocyte cells) were seeded into a 96-well plate at a density of 3 × 103 cells per well and treated 24 h later with varying concentrations of Jawoongo (5–500 μg/mL) for an additional 24 h. Ten microliters of WST solution was added to each well of the plate, which was incubated in the dark at 37 °C for another 2 h. Optical density was measured at 450 nm using an ELISA plate reader.
Western blot analysis
Cells (HMC-1, RAW264.7 and Splenocyte cells) were lysed with cell lysis buffer (50 mM Tris-Cl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and protease inhibitor). Twenty micrograms of protein was separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Protran nitrocellulose membrane, Whatman, UK). The membrane was blocked with 5% nonfat milk, probed with specific primary antibodies, incubated with HRP-conjugated secondary IgG antibodies (Calbiochem, San Diego, CA, USA), and visualized using an enhanced chemiluminescence detection system (Amersham ECL kit, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). The antibodies against COX-2, p-JNK, total JNK and iNos were obtained from Cell Signaling (Danvers, MA, USA). The antibodies against p-Erk, total Erk, phospho-NF-κB p65 (Ser536), total NF-κB and Actin were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). Tubulin antibody was obtained from Sigma-Aldrich (St. Louis, MO, USA).
Isolation of Splenocytes
Spleen suspensions from normal mice were prepared under aseptic conditions by homogenization in RPMI-1640 medium (containing 10% FBS, 1% antibiotics, and 0.05 mM β-mercaptoethanol). Red blood cell (RBC) lysis buffer (Sigma, St. Louis, MO, USA) was added to the cell suspension to remove RBCs. The spleen cells were centrifuged, suspended in complete RPMI-1640, and maintained at 37 °C in a humidified incubator with 5% CO2.
Liquid chromatography-mass spectrometry analysis.
An Agilent 1100 series liquid chromatography-mass spectroscopy (LC-MS) with an atmospheric pressure chemical ionization interface was used in negative and positive ionization modes. Data were collected using Chemstation software version A.09.03. A Shiseido capcell-pak C18 column (4.6 mm × 150 mm, 5 μm) was used with an injection volume of 10 μL for the HPLC separation. The mobile phases consisted of (A) Acetonitrile, (B) 0.1% Acetic acid and (C) Methanol at a flow rate of 1.0 mL/min. The gradient of the mobile phases (A: B: C) for separation was 0–90 min (35: 65: 0 to 0: 0: 100). Decursin was used as standard. Mass spectrometry was operated with an electrospray ionization source and positive mode.
All experiment results were expressed as the means ± SEM of at least three separate tests. Statistical significance at P < 0.05 < 0.01 and < 0.001 has been given respective symbols in the figures. Statistical analyses (ANOVA) were performed using PRISM software (GraphPad Software Inc., La Jolla, CA, USA,).
Effects of Jawoongo on a DNCB-induced mouse model of AD
Jawoongo treatment lowered the number of WBCs in the blood of mice
Application of DNCB increased both the total number of white blood cells (WBCs) and the number of each WBC subtype, including neutrophils, basophils, eosinophils, monocytes, and lymphocytes, in the serum of the mice. Importantly, treatment with Jawoongo or tacrolimus lowered the increased number of WBCs, indicating that Jawoongo and tacrolimus suppress inflammation by decreasing the number of WBCs in the blood (Fig. 3a, b, c, d, e and f).
Jawoongo treatment reduced the serum levels of IgE, IL-6, IL-10 and IL-12 in mice
Jawoongo treatment down-regulated mRNA expression of IL-2, IL-4, IL-13, and TNF-α in mouse skin
Jawoongo treatment reduced the number of CD4+ cells in skin
Jawoongo inhibited agonist-induced cytokine production in HMC-1 cells
Jawoongo inhibited LPS-induced inflammatory responses in RAW264.7 cells
Jawoongo inhibited LPS-induced inflammatory responses in isolated Splenocytes
Decursin is an Indicator molecule for Jawoongo
AD is a common pruritic and chronically relapsing inflammatory skin disease. Affecting approximately 10–20% of children and 1–3% of adults worldwide , AD is a major global public health problem. Additionally, the incidence of AD has steadily increased every year .
Several mouse models have been developed to evaluate drugs for the treatment of AD. A DNCB-patch model using BALB/c mice has been proposed as a suitable representative of human AD because mice treated with DNCB show symptoms similar to human AD, including epidermal hyperplasia, dermal mast cell infiltration, and elevated serum IgE levels . Activated mast cells release inflammatory mediators such as histamines, cytokines and chemokines [42, 43]. In this study, we investigated the anti-AD effects of Jawoongo using DNCB-treated BALB/c mice. We found that topical application of Jawoongo strongly suppressed DNCB-induced AD-like lesions and reduced skin thickness, CD4 levels and mast cell infiltration in sensitized skin. We observed that Jawoongo suppresses skin inflammation by inhibiting various DNCB-stimulated inflammatory responses.
Until now, the exact pathogenesis of AD has remained unclear. However, Th1 and Th2 cytokines play an important role in the etiology of AD. In particular, Th2 cytokines are important mediators of AD development . CD4+ T cells are key factors implicated in the pathogenesis of AD, and skin infiltration of CD4+ T cells is known to increase in severe AD cases .
Therefore, we investigated cytokines related to Th2 in an in vivo model. Jawoongo treatment reduced the increased serum levels of IgE, IL-6, IL-10 and IL-12 induced by DNCB treatment In fact, it was reported that IL-12 was increased by DNCB [46, 47], and this increase was suppressed by Jawoongo as expected. We also found that Jawoongo reduced DNCB-stimulated increases in eosinophil, neutrophil, monocyte, basophil, lymphocyte and WBC numbers and in IL-4, IL-13 and TNF-α mRNA expression. These results suggest that Jawoongo decreased the number of CD4+ cells entering the skin. Of note, Jawoongo and tacrolimus, which was used as a positive control in this study, showed similar effects on AD-like skin lesions, but Jawoongo exhibited more favorable effects than tacrolimus in some aspects, such as in decreased mast cell recruitment and serum IgE levels.
To improve our understanding of Jawoongo’s actions at the cellular level, we evaluated the effects of Jawoongo on several types of innate immune cell, including human mast cells (HMC-1), murine macrophage RAW264.7 cells, and splenocytes isolated from mice.
Our present study demonstrates that Jawoongo treatment suppresses DNCB-induced AD symptoms by downregulating serum IgE levels and the production of several inflammatory cytokines. In addition, our data indicate that Jawoongo treatment inhibits cytokine expression and activation of the NF-kB and MAPK pathways in several types of immune cell. Taken together, our results suggest that Jawoongo might be a useful candidate drug for the treatment of AD.
We thank our colleagues from the Laboratory of Prevention medicine for technique assistance and useful discussions.
This research was supported by a grant from the Korean Medicine R&D Project of the Ministry of Health and Welfare (HI12C1889 and HI13C0530).
Availability of data and materials
All data and materials are contained and described within the manuscript.
JMK carried out the experiment and drafting of manuscript. SHH, SRK, HC, HIK, DUK and SMO revised the research and manuscript and assisted in the research work. HSS, TYK and YCS guided the research, revised and submitted the manuscript. CC and SGK designed, supervised the experiments and corrected the manuscript. All the authors read and approved the final manuscript.
Ethics approval and consent to participate
Animal experiments were approved by Kyung Hee university institutional animal care of use committee (Approval No. KHUASP(SE)-12–014).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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