DNCB-treated Mice Research Articles

Jageum-Jung improves 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in mice and suppresses pro-inflammatory chemokine production by inhibiting TNF-α/IFN-γ-induced STAT-1 and NFκB signaling in HaCaT cells

Ethnopharmacological relevanceJageum-Jung (JGJ) is an oriental herbal formula comprising five herbs (Melaphis chinensis Bell, Cremastra variabilis Nakai, Knoxia valerianoides Thorel, Euphorbia lathyris L., and Moschus moschiferus L.). It has been used for detoxification and treating cancer and inflammatory diseases in China, Japan, and Korea. However, the mechanism of action of JGJ on keratinocyte inflammatory response is poorly understood. Aim of the studyIn the present study, we investigated the anti-inflammatory mechanism of JGJ and studied the effects of JGJ on atopic dermatitis-like skin lesions in mice. Materials and methodsWe elucidated the anti-inflammatory and anti-inflammatory effects of JGJ on tumor necrosis factor-α/interferon-γ (TNF-α/IFN-γ)-treated human keratinocyte cells, IgE-sensitized RBL-2H3 cells, and 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD)-like mice, respectively. ResultsThe results showed that JGJ suppressed the production and mRNA revels of IL-8, IL-6 and, conspicuously, both TARC and RANTES. JGJ inhibited nuclear translocation of the inflammatory transcription factors NFκB and STAT-1. Moreover, JGJ improved AD-like skin lesions in DNCB-treated mice and inhibited degranulation of mast cell. ConclusionsThe results of this study suggest that JGJ can be considered as a candidate agent for AD treatment.

A new flavonoid from Stellera chamaejasme L., stechamone, alleviated 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in a murine model

Stellera chamaejasme L. (family Thymelaeaceae), also known as ‘Langdu’, has been traditionally used to treat of skin-related diseases, such as, psoriasis and skin ulcers. The aim of this study was to identify the biologically active component of S. chamaejasme and evaluate its preventive effects on IL-4 and mast cell degranulation in RBL-2H3 cells and on the development of atopic dermatitis (AD) in 2,4-dinitrochlorobenzene (DNCB)-treated SKH-1 hairless mice. A novel flavonoid, genkwanin 5-O-xylosyl(1 → 2)glucoside (named stechamone), and three known compounds (umbelliferone, luteolin, and luteolin-7-O-glucoside) were isolated from the aerial parts of S. chamaejasme using chromatographic methods. Of these four compounds, stechamone most potently inhibited IL-4 production and mast cell degranulation in RBL-2H3 cells. Topical application of 0.5% stechamone improved atopic skin symptoms, including, erythema (redness), pruritus (itching), exudation (weeping), excoriation (peeling), and lichenification (skin thickening) in DNCB-treated AD mice by accelerating skin barrier recovery function and suppressing inflammatory cell infiltration. In addition, stechamone attenuated DNCB-induced increases in IL-4 (an inflammatory TH2 cytokine) expression and in serum IgE levels in our murine model of AD. DNCB induced AD-like skin lesions, but treatment with stechamone exhibited strong anti-atopic activity by regulating skin barrier function and reducing inflammatory responses. The results obtained suggest stechamone is a potential anti-atopic agent and treatment for skin inflammatory diseases.

Sea Buckthorn (Hippophaë rhamnoides L.) Oil Improves Atopic Dermatitis-Like Skin Lesions via Inhibition of NF-κB and STAT1 Activation

Background and Objectives: The objective of this study was to evaluate the topical effects of sea buckthorn (SBT) oil on atopic dermatitis (AD)-like lesions in a mouse model generated by repeated topical administration of DNCB in BALB/c mice. Methods: DNCB was applied repeatedly on the dorsal skin of mice to induce AD-like lesions. Following AD induction, SBT oil was applied daily on the dorsal skin for 4 weeks. The severity of skin lesions was examined macroscopically and histologically. We further measured the production of MDC/CCL22 and TARC/CCL17 in IFN-γ/TNF-α activated HaCaT cells. Results: Topically applied SBT oil in DNCB-treated mice ameliorated the severity score of dermatitis, decreased epidermal thickness, reduced spleen and lymph node weights, and prevented mast cell infiltration. In addition, SBT oil suppressed the Th2 chemokines TARC and MDC via dose-dependent inhibition of NF-κB, JAK2/STAT1, and p38-MAPK signaling pathways in IFN-γ/TNF-α-activated HaCaT cells. Conclusion: These results suggest that SBT oil had a beneficial effect on AD-like skin lesions, partially via inhibition of the Th2 chemokines TARC and MDC in inflamed skin.

Fluoxetine Ameliorates Atopic Dermatitis-Like Skin Lesions in BALB/c Mice through Reducing Psychological Stress and Inflammatory Response.

Atopic dermatitis (AD) is a common chronic inflammatory skin disorder, and patients with AD suffer from severe psychological stress, which markedly increases the prevalence rate of depression and anxiety disorders in later life. Fluoxetine, a selective serotonin reuptake inhibitor, has recently been reported to exert anti-inflammatory and immunosuppressive effects. However, it is unclear whether fluoxetine is effective in the treatment of AD through reducing psychological stress and inflammatory reaction. Here, we reported that a BALB/c mouse model of AD was induced by application of 2,4-dinitrochlorobenzene (DNCB) onto hairless dorsal skin. Chronic fluoxetine treatment (10 mg/kg per day, i.p.) significantly attenuated AD-like symptoms, as reflected by a dramatic decrease in scratching bouts, as well as a decrease in anxiety- and depressive-like behaviors. Furthermore, these behavioral changes were accompanied by a significant decrease in epidermal thickness, the number of mast cells in skin tissue, mRNA levels of interleukin-4 (IL-4) and IL-13 in the spleen, as well as serum immunoglobulin E (IgE) in the DNCB-treated mice by treatment with fluoxetine. Taken together, these results indicate that fluoxetine may suppress psychological stress and inflammatory response during AD development, and subsequently ameliorate AD symptoms, suggesting that fluoxetine may be a potential therapeutic agent against AD in clinic.

Evaluation of 5-methylcytosine and 5-hydroxymethylcytosine as potential biomarkers for characterisation of chemical allergens

Epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune responses. Chemical allergens form two categories: skin sensitizing chemicals associated with allergic contact dermatitis, and chemicals that cause sensitization of the respiratory tract and occupational asthma. In mice these are characterized by different T helper (Th) cell responses. Changes in DNA methylation in particular have been implicated in the in vivo responses to chemical allergy. As such it was hypothesised that differentially methylated regions (DMR) may provide candidates biomarkers of chemical allergy To examine this, mice were exposed to 2,4-dinitrochlorobenzene (DNCB; a contact allergen) or trimellitic anhydride (TMA; a respiratory allergen). DNA from draining lymph nodes was processed for methylated (5mC) and hydroxymethylated (5hmC) DNA immunoprecipitation (MeDIP/hMeDIP) then selected DMR analysed by qPCR. We describe a number of DMRs which, by combined analysis of 5mC and 5hmC, differentiate between responses induced by DNCB and those by TMA. Furthermore, these changes in methylation are specific to the draining lymph node. The Gmpr DMR is suggested as a possible biomarker for contact allergen-induced immune responses; it is characterised by divergent levels of 5mC and 5hmC DNCB-treated mice only. In contrast, the Nwc DMR was characterised by divergent 5mC and 5hmC specifically in response to TMA, highlighting its possible utility as a biomarker for responses induced by chemical respiratory allergens. These data not only represent novel analysis of 5hmC in response to chemical allergy in vivo, but with further investigation, may also provide a possible basis for differentiation between classes of chemical allergens.

Protective effect of diet supplemented with rice prolamin extract against DNCB-induced atopic dermatitis in BALB/c mice

BackgroundRice prolamin has been reported to possess antioxidative, anti-inflammatory and immune-promoting properties. This study is aimed to examine the protective effects of dietary rice prolamin extract (RPE) against dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD)-like skin lesions in mice.MethodsBALB/c mice were fed diet supplemented with 0–0.1 % RPE for 6 weeks. For the last 2 weeks, 1 % or 0.2 % DNCB was applied repeatedly to the back skin of mice to induce AD-like lesions. Following AD induction, the severity of skin lesions was examined macroscopically and histologically. In addition, the serum levels of IgE, IgG1 and IgG2a were determined by ELISA, and the mRNA expression of IL-4 and IFN-γ in the skin was determined by real-time PCR.ResultsDietary RPE suppressed the clinical symptoms of DNCB-induced dermatitis as well as its associated histopathological changes such as epidermal hyperplasia and infiltration of mast cells and eosinophils in the dermis. RPE treatment also suppressed the DNCB-induced increase in transepidermal water loss. Dietary RPE inhibited the DNCB-induced enhancement of serum IgE and IgG1 levels, whereas it increased the serum IgG2a level in DNCB-treated mice. In addition, dietary RPE upregulated the IFN-γ mRNA expression and downregulated the IL-4 mRNA expression in the skin of DNCB-treated mice.ConclusionsThe above results suggest that dietary RPE exerts a protective effect against DNCB-induced AD in mice via upregulation of Th1 immunity and that RPE may be useful for the treatment of AD.

High-pressure processing of milk alleviates atopic dermatitis in DNCB-induced Balb/c mice

High pressure (HP) is recognized as the alternative process technology of heat treatment in food production. Although milk was the first food product to be treated with HP, the allergenicity of HP-treated milk remains to be elucidated. Thus, this study was conducted to investigate the effects of HP treatment on the alleviation of atopic dermatitis (AD) caused by milk in a mouse model. To investigate the effect of HP treatment on the alleviation of AD, Balb/c mice were fed HP-treated milk orally for 7 days following induction of AD with 1-chloro-2,4-dinitro-benzene (DNCB). Mice fed with HP-treated milk exhibited markedly lower serum IgE level than DNCB-treated mice. These results suggest that HP may be applicable as an alternative process for the development of hypoallergenic milk products.

Estimation of the environmental effect of natural volatile organic compounds from Chamaecyparis obtusa and their effect on atopic dermatitis-like skin lesions in mice.

Aromatherapy has been suggested as an alternative therapeutic method for the treatment of atopic dermatitis (AD), eczema and other skin diseases. In the current study, the anti-atopic properties of the volatile organic compounds of Chamaecyparis obtusa (VOCCo) were examined to determine whether they are amenable for use as a pharmaceutical candidate. The alterations in histological features, serum IgE levels and mast cell infiltration following exposure to VOCCo were determined in a 2,4-dinitrochlorobenzene (DNCB)-induced AD-like mouse model. The results of these experiments demonstrated that VOCCo inhibited the development of AD-like skin lesions by reducing the serum IgE level and mast cell infiltration into the dermal and subcutaneous layers. This was supported by screening of immune cytokine mRNAs, including interleukin (IL)-1β and IL-6 from the skin of DNCB-treated mice. The expression of IL-1β and IL-6 in the skin lesions of mice was dose-dependently inhibited by treatment with VOCCo. Furthermore, treatment with VOCCo resulted in the recovery of histopathological features in AD-like skin lesions. These results suggest that VOCCo may have therapeutic and preventive effects for the development of AD.

The Inhibitory Effect of Premature Citrus unshiu Extract on Atopic Dermatitis In Vitro and In Vivo

Atopic dermatitis (AD) is a chronic, recurrent inflammatory skin disease that is associated with Th2 cellmediated allergy. The process that leads to infiltration of inflammatory cells into an AD lesion is remarkably dependent on various chemokines, especially TARC (thymus and activation-regulated chemokine/CCL17) and MDC (macrophage-derived chemokine/CCL22). Serum levels of these chemokines are over-expressed in AD patients. Citrus unshiu, which is known as Satsuma mandarin, has anti-oxidative, anti-inflammation, and anti-microviral activity. Therefore, we investigated the effect of EtOH extract of premature C. unshiu on AD. We did this using a DNCB-induced AD mouse model. We also tried to confirm an inhibitory effect for premature C. unshiu on the expression of inflammatory chemokines in IFN-γ and TNF-α stimulated HaCaT human keratinocytes. We found that extract of premature C. unshiu reduced DNCB-induced symptoms such as hyperkeratosis, increased skin thickness, and infiltrated mast cells, in our AD-like animal model. The extract decreased levels of IFN-γ and IL-4 in ConA-stimulated splenocytes isolated from DNCB-treated mice. Also, extract of premature C. unshiu inhibited mRNA expression and protein production of TARC and MDC through the inhibition of STAT1 phosphorylation. These results suggest that C. unshiu has anti-atopic activity by regulating inflammatory chemokines such as TARC and MDC.

Alternative therapeutic advantages of catfish bile on atopic dermatitis: protection of T cell-mediated skin disease via antioxidant activities

In the present study, we aimed to examine the anti-atopic properties of bile from the cat fish, Silurus asotus, to determine its possible use as a pharmaceutical product. The anti-atopic activities of cat fish bile were examined in a non-cell antioxidant, in-vitro assay (splenocytes and mast cells) and a 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis-like mouse model. The results of these experiments revealed that Silurus asotus bile (SAB) scavenges radicals and protects proteins from superoxide attacks, suggesting that SAB suppresses the T helper (Th) type 2-skewed immune response. Th1/Th2 mRNA cytokines (interleukin (IL)-2, interferon (IFN)-γ and IL-4) from mouse splenocytes were effectively inhibited, and the release of β-hexosaminidase in RBL-2H3 mast cells was significantly suppressed by SAB. These results were supported by screening the Th1/Th2 cytokine mRNAs (IL-2, IFN-γ and IL-4) from lymph nodes in DNCB-treated mice. More dramatic results were observed in the histological changes at higher SAB concentrations (5%) compared to the therapeutic control, visualized using hematoxylin-eosin (H&E) staining. The results presented in this study suggest that SAB may provide functional advantages with regard to treating atopic dermatitis because of its antioxidant and immune-suppressive effects.

Comparative Studies of Lymph Node Cell Subpopulations and Cytokine Expression in Murine Model for Testing the Potentials of Chemicals to Induce Respiratory Sensitization

To investigate immunological changes in lymph nodes based on expression of cell-specific receptors and cytokine expression profile and accompanying inflammatory reactions in lungs of mice treated with chemicals of known potentials to induce respiratory sensitization and those in which activity in this regard is unclear. On day 1 and 7, Balb/c mice received toluene-2,4-diisocyanate (TDI), trimellitic anhydride (TMA), 1-chloro-2,4-dinitrobenzene (DNCB), glutaraldehyde (GA), formaldehyde (FA), benzalkonium chloride (ChB) or vehicle. On day 14, they received a single intranasal instillation with the same chemical or vehicle. On day 15, auricular lymph nodes (LN) were excised and used for analyzes of T-, B-cells, expression of CD44 and for the estimation of IL-4 and IFN-gamma production after in vitro stimulation with concanavalin A (ConA) and also for IL-4 and IFN-gamma mRNA expression analyses using Real-Time PCR. Inflammatory changes in lungs were observed by estimation of TNF-alpha and MIP-2 concentrations and cell numbers and their type in BAL. There were no significant changes in cell subpopulations of T helper cells in LN. The percent of B cells was significantly increased after treatment with DNCB, TDI, and GA. Increased expression of CD44 on T cells was also observed. Both IL-4 and IFN-gamma were found increased in TDI- and FA-treated mice, while only IL-4 was increased in TMA-treated mice. Real-Time PCR analyses, however, showed increased IL-4 mRNA expression for TDI- and TMA-, and IFN-gamma mRNA expression for DNCB-treated mice. We haven't observed significant changes in inflammatory reactions in the lungs of exposed animals. Studying immunological changes with first determining the activation status of T cells followed by analyzes of expression of mRNA for Th1 and Th2 cytokines in murine model could be a useful method for assessment of the potentials of chemicals to induce respiratory sensitization but is not sufficient. Addition of ventilatory measurements, but not necessarily inflammatory reactions, could complete the model.

Validation of a mouse model of chemical-induced asthma using trimellitic anhydride, a respiratory sensitizer, and dinitrochlorobenzene, a dermal sensitizer

Occupational asthma can be caused by chemicals. Previously, we established a murine model of immunologically mediated chemical-induced asthma using toluene diisocyanate. We sought to verify this model using trimellitic anhydride (TMA), a respiratory sensitizer, and 1-chloro-2,4-dinitrobenzene (DNCB), a dermal sensitizer. BALB/c mice received dermal applications (vehicle or chemical) on days 1 and 7. On day 10, they received an intranasal instillation (vehicle or chemical). Whole-body plethysmography (enhanced pause) was used to monitor changes in ventilatory function and methacholine reactivity. Pulmonary inflammation was assessed by using bronchoalveolar lavage (cells, TNF-alpha levels, and macrophage inflammatory protein 2 levels). Immunologic parameters included total serum IgE levels, lymphocyte distribution in auricular and cervical lymph nodes, and IL-4 and IFN-gamma levels in supernatants of lymph node cells incubated with or without concanavalin A. Mice dermally treated and intranasally challenged with TMA experienced markedly increased enhanced pause immediately after intranasal challenge and increased methacholine reactivity (24 hours later). Mice similarly treated with DNCB did not show any ventilatory changes. Neutrophil influx and increased macrophage inflammatory protein 2 and TNF-alpha levels were found in bronchoalveolar lavage fluid in both TMA- and DNCB-treated mice. The proportion of CD19+ B cells was increased in auricular and cervical lymph nodes of TMA-treated mice. IL-4 and IFN-gamma levels were increased in supernatants of concanavalin A-stimulated auricular and cervical lymph node cells of TMA- or DNCB-treated mice; however, the relative proportions of IL-4 and IFN-gamma levels differed between TMA- and DNCB-treated mice. Serum total IgE levels were increased in TMA-treated mice only. Both compounds induce a mixed T(H)1-T(H)2 response, but only TMA induced ventilatory changes. In the workplace avoiding skin contact with chemical sensitizers might be advised to prevent chemical-induced asthma.

Identification of contact and respiratory sensitizers using flow cytometry

Identification of the chemicals responsible for respiratory and contact allergies in the industrial area is an important occupational safety issue. This study was conducted in mice to determine whether flow cytometry is an appropriate method to analyze and differentiate the specific immune responses to the respiratory sensitizer trimellitic anhydride (TMA) and to the contact sensitizer dinitrochlorobenzene (DNCB) used at concentrations with comparable immunogenic potential. Mice were exposed twice on the flanks (days 0, 5) to 10% TMA or 1% DNCB and challenged three times on the ears (days 10, 11, 12) with 2.5% TMA or 0.25% DNCB. Flow cytometry analyses were conducted on draining lymph node cells harvested on days 13 and 18. Comparing TMA and DNCB immune responses on day 13, we found obvious differences that persisted for most of them on day 18. An increased proportion of IgE+ cells correlated to total serum IgE level and an enhancement of MHC II molecule expression were observed in the lymph node B lymphocytes from TMA-treated mice. The percentage of IL-4-producing CD4+ lymphocytes and the IL-4 receptor expression were clearly higher following TMA exposure. In contrast, higher proportions of IL-2-producing cells were detected in CD4+ and CD8+ cells from DNCB-treated mice. Both chemicals induced a significant increase in the percentage of IFN-γ-producing cells among CD8+ lymphocytes but to a greater proportion following TMA treatment. In conclusion, this study encourages the use of flow cytometry to discriminate between contact and respiratory sensitizers by identifying divergent expression of immune response parameters.