Red ginseng inhibits scratching behavior associated with atopic dermatitis in experimental animal models

Pruritus is a severe symptom that is difficult to treat in atopic dermatitis patients. Red ginseng (RG), a natural medicine, has various biological activities such as anti-inflammatory effects. In this study, we examined the efficacy of RG extract (RGE) and its mechanism on experimental atopic dermatitis in mice. The effects of RGE on vascular permeability and itching were first evaluated. Histamine-induced permeability and itching were significantly inhibited by embrocation with RGE as well as diphenhydramine, an antihistamine drug. Next, we assessed the therapeutic effect of topical RGE in a mouse model of atopic dermatitis. Dermatitis was induced by repeated application of 2,4-dinitrofluorobenzene (DNFB) acetone solution to the mouse ear. The effects of tacrolimus (a calcineurin blocker), dexamethasone (a corticosteroid), and RGE on dermatitis and associated scratching behavior were compared. Repeated DNFB application caused frequent scratching behaviors and ear swelling. Topical treatment with tacrolimus, dexamethasone, and RGE for 8 days before the final challenge with DNFB significantly inhibited ear swelling. Tacrolimus and RGE significantly inhibited scratching behavior, whereas dexamethasone failed to do so. DNFB-induced nerve growth factor expression and nerve fiber extension were significantly attenuated by tacrolimus and RGE, but not by dexamethasone. RGE may have the potential for treatment of atopic dermatitis.     

Inhibitory effects of Saururus chinensis (LOUR.) BAILL on the development of atopic dermatitis-like skin lesions in NC/Nga mice

The present study was performed to examine whether the leaves of Saururus chinensis (LOUR.) BAILL (SC), an herb used for the management of various skin diseases including atopic dermatitis (AD) in Eastern countries, inhibited the development of AD-like skin lesions in NC/Nga mice which was induced by repeated application of picryl chloride (PiCl). The efficacy of SC was judged by measurement of skin severity, itching behavior, histological study, serum IgE levels, IL-4 and IFN-gamma in lymph nodes. Oral administration of SC extract to the PiCl-treated NC/Nga mice for 8 weeks (5 d per week) inhibited significantly the development of AD-like skin lesions macroscopically. Histologically, SC inhibited dermatitis changes like hypertrophy, hyperkeratosis, and infiltration of inflammatory cells into epidermis and dermis. The itching behavior and serum IgE level decreased significantly after SC administration. SC administration enhanced IFN-gamma mRNA expression but did not have an effect on IL-4 mRNA expression. These results suggest that SC could inhibit the development of AD-like skin lesions in NC/Nga mice possibly through modulating the Th1/Th2 imbalance by the promoting of Th1 cell response. Thus, SC may be an alternative substance for the management of AD patients.     

A novel animal model of pruritus induced by successive application of glucocorticoid to mouse skin

Topical glucocorticoids are commonly applied for treatment of atopic dermatitis, and are often administered over a long period. However, itching often occurs as a rebound phenomenon after cessation of long-term glucocorticoid application. The present study was an initial trial designed to establish an animal model of glucocorticoid-induced pruritus by topical application of dexamethasone over a long period in mice with contact dermatitis. BALB/c mice with chronic allergic contact dermatitis induced by 5 weeks of repeated application of 2,4,6-trinitro-1-chlorobenzene (TNCB) were treated topically with dexamethasone for 3 weeks from 2 weeks after the elicitation of dermatitis. The effects of dexamethasone on inflammation and pruritus were evaluated by measurement of ear-swelling and scratching behavior, respectively. Significant enhancement of pruritus was confirmed after chronic application of dexamethasone. The increased frequency of scratching behavior was reduced by withdrawal of dexamethasone. On the other hand, ear-swelling was markedly ameliorated by dexamethasone treatment, but rapidly relapsed after dexamethasone withdrawal. The level of interleukin (IL)-4 mRNA in ear skin and that of IgE in serum were increased in the mice with dermatitis and reduced by dexamethasone treatment. On the other hand, the level of nerve growth factor (NGF) mRNA was slightly increased by dexamethasone treatment and remained high even after its discontinuation. It is anticipated that this novel animal model of glucocorticoid-induced pruritus will be useful for clarifying the mechanisms of the rebound phenomenon induced by chronic treatment with topical glucocorticoids, and for developing a new form of therapy.     

Oral administration of Astragalus membranaceus inhibits the development of DNFB-induced dermatitis in NC/Nga mice

Epicutaneously administered chemical antigens like 2,4-dinitrofluorobenzene (DNFB), evoke an atopic dermatitis (AD)-like dermatitis reaction in NC/Nga mice under specific pathogen free (SPF) conditions. Astragalus membranaceus (AM), is a popular herbal medicine used to treat allergic diseases in East Asia. In the present study, we examined whether AM suppress AD-like skin lesions in NC/Nga mice treated with DNFB under SPF conditions. Oral administration of AM to DNFB-treated NC/Nga mice was found to inhibit ear thickness increases and the skin lesions induced by DNFB. Moreover, IFN-gamma production by CD4(+) T cells from the lymph nodes of DNFB-treated NC/Nga mice was significantly inhibited by AM treatment, although levels of IL-4 and total IgE in serum were not. Study findings suggest that AM may suppress the development of AD-like dermatitis in DNFB-treated NC/Nga mice by reducing IFN-gamma production.     

Evaluation of antipruritic effects of several agents on scratching behavior by NC/Nga mice

We investigated the effects of several agents on the established itching model in NC/Nga mice, model of atopic dermatitis-like disease, to elucidate related characteristics. The number of spontaneous scratching behaviors (the duration time is over 1.5 s) by NC/Nga mice with severe skin lesions was measured before and after administration of agents for 24 h. The scratching behavior by NC/Nga mice was significantly suppressed by administration of dexamethasone or tacrolimus, but not by chlorpheniramine maleate or cyproheptadine hydrochloride. These results suggest that this method shows a good correlation with the effectiveness of drugs prescribed for itching in humans with atopic dermatitis, and histamine and serotonin do not play an important role in causing the scratching behavior seen by NC/Nga mice. The scratching behavior was also significantly suppressed by naloxone hydrochloride, dibucaine or capsaicin. These results suggest that the scratching behavior seen in this model is caused by itching signal transmission through neural system. Furthermore, we found that theophylline, pinacidil or limaprost had scratching suppression effects in this model.     

Oral chymase inhibitor SUN13834 ameliorates skin inflammation as well as pruritus in mouse model for atopic dermatitis

Chymase is a chymotrypsin-like serine protease exclusively stored in secretory granules of mast cells and has been thought to participate in allergic diseases. It has already been shown that chymase inhibitor SUN13834 improves dermatitis in NC/Nga mice that spontaneously develop dermatitis resembling atopic dermatitis. In the present study, effect of chymase inhibitor SUN13834 on itch, the major feature of atopic dermatitis, was examined using a mouse dermatitis model induced by repeated topical application of 2,4-dinitrofluorobenzene (DNFB). Oral administration of SUN13834 once a day for 5 weeks inhibited not only skin swelling but accumulation of inflammatory cells including mast cells and eosinophils in the skin of the mice. In addition, SUN13834 also decreased significantly at 10 and 50 mg/kg the amount of scratching behavior induced by the DNFB challenge. This result indicates for the first time that mast cell chymase may be involved in itch induction. In conclusion, SUN13834 is thought to be useful as therapeutic agent for atopic dermatitis.     

Development of numerous nerve fibers in the epidermis of hairless mice with atopic dermatitis-like pruritic skin inflammation

Itching is the most important symptom in atopic dermatitis because the persistent scratching in response to itching aggravates the disease. However, the etiologic mechanisms of itching in atopic dermatitis remain uncertain. HR-1 hairless mice fed a special diet, HR-AD, develop atopic dermatitis-like symptoms with prolonged scratching episodes. The purpose of this study was to examine whether skin nerve fiber changes were involved in the prolonged scratching seen in this mouse model. On day 56 after the start of feeding, prolonged scratching, as well as atopic dermatitis-like skin changes, were clearly observed in HR-AD-fed mice, while no abnormal changes were observed in mice fed a normal diet. Immunohistochemical analyses of the skin using antibody to protein gene product 9.5 showed the development of numerous immunoreactive nerve fibers in the epidermis of HR-AD-fed mice. Furthermore, after cessation of HR-AD feeding, the reduction in intraepidermal nerve fibers coincided with decreased scratching. Neither the prolongation of scratching nor the increase in intraepidermal nerve fibers was affected by dexamethasone treatment. Thus, the increased number of intraepidermal nerve fibers could be involved in the aggravation of itch-related scratching observed in this model.     

Expression of IL-31 gene transcripts in NC/Nga mice with atopic dermatitis

To search for the pruritogen of atopic dermatitis, a characteristic symptom in atopic dermatitis patients, we examined interleukin-31 (IL-31) mRNA expression in NC/Nga mice as an animal model of atopic dermatitis. The expression of IL-31 mRNA in the skin of NC/Nga mice with scratching behavior was significantly higher than that in NC/Nga mice without scratching behavior. Our findings suggest that IL-31 may participate in the cause of itch sensation and promote scratching behavior in NC/Nga mice with atopic dermatitis.     

Pharmacological characterization of a chronic pruritus model induced by multiple application of 2,4,6-trinitrochlorobenzene in NC mice

Female NC/Jic mice were sensitized and challenged repeatedly at 48 h intervals for 10 and 30 days by painting 1% 2,4,6-trinitrochlorobenzene (TNCB) on both ears. Mice challenged with TNCB for 30 days developed an inflammatory dermatitis with high immunoglobulin E (IgE) titer. Histological analysis with acidic Toluidine Blue staining revealed that dermal mast cells markedly differentiated and intensely degranulated, consistent with a dramatic increase in scratching behavior. A significant increase in total scratching events could be observed in mice treated with TNCB for a short period of 10 days. Extending the term of TNCB application to 30 days, the IgE titer and number of mast cells elevated significantly, and thus various drugs were evaluated pharmacologically by using the mice treated with TNCB for 30 days. Terfenadine and cyproheptadine attenuated the chronic scratching behavior. Tacrolimus and dexamethasone were less effective and cromolyn showed no effect. In addition, terfenadine and tacrolimus suppressed the degranulation of mast cells. The present chronic scratching model could be suitable to evaluate drugs effective for suppression of mast cell differentiation and degranulation by irritation, and may represent a promising tool to develop new drugs for inflammatory pruritus associated with, for example, atopic dermatitis.     

Epigallocatechin-3-gallate improves Dermatophagoides pteronissinus extract-induced atopic dermatitis-like skin lesions in NC/Nga mice by suppressing macrophage migration inhibitory factor

Epigallocatechin-3-gallate (EGCG) has been shown to exert anti-inflammatory effects on the inflammatory skin conditions. However, little is known about its effect on atopic dermatitis (AD). We first attempted to assess the anti-inflammatory effect of topical application of EGCG in vivo AD model using NC/Nga mice and to determine whether EGCG exerts the anti-inflammatory effect by inhibiting macrophage migration inhibitory factor (MIF) and other cytokines that are related to immune dysregulation in the pathogenesis of AD. Murine AD-like skin lesions were made by painting Dermatophagoides pteronissinus extract (DPE). The effects of EGCG treatment were assessed by total clinical severity score and ear thickness, and by histological grading. In addition, the mRNA and protein expression of the cytokines including MIF were measured by real-time RT-PCR and immunohistochemistry. The serum levels of MIF and IgE were measured by ELISA. In the AD mouse model, EGCG significantly reduced the total clinical severity score and ear thickness (p<0.05). The histological grading was also markedly improved. The mRNA expression of MIF, TNF-alpha, IFN-gamma, IL-2 and IL-12 p40, but not of IL-4, IL-5 and IL-13 in the lesions was significantly reduced by EGCG (p<0.05). On the immunohistochemistry, EGCG also markedly diminished the expression of MIF, TNF-alpha and IFN-gamma. The serum MIF and IgE production was significantly reduced by EGCG (p<0.05). These results demonstrate that topical application of EGCG may improve the AD-like skin lesions by suppressing MIF and T helper 1 cytokines. Taken together, it is suggested that EGCG may be a potential therapeutic modality for AD.     

Tacrolimus suppressed the production of cytokines involved in atopic dermatitis by direct stimulation of human PBMC system. (Comparison with steroids).

Tacrolimus (FK506) ointment showed remarkable efficacy against atopic dermatitis in animal models and clinical trials. The suppressive effect of tacrolimus on the production of the cytokines involved in atopic dermatitis (IL-2, IL-3, IL-4, IL-5, IFN-gamma and GM-CSF) from human peripheral blood mononuclear cells (PBMC) was investigated. We constructed a new cytokine production system in which T cells are activated by direct stimulation in vitro with anti-CD3/CD2 or anti-CD3/CD28 antibody combination. Tacrolimus inhibited the production of these cytokines by both stimulations. In a comparative study with steroids (alclometasone dipropionate and betamethason valerate) in anti-CD3/CD2 system, tacrolimus and both steroids inhibited Th1 cytokines (IL-2, IFN-gamma), Th2 cytokines (IL-4, IL-5) and IL-3, GM-CSF (produced by both Th1 and Th2). The suppressive effect of tacrolimus on cytokine production was stronger than that of alclometasone dipropionate and equal to or stronger than that of betamethason valerate. The effective dose of tacrolimus (IC50, 0.02-0.11 ng/ml) is almost the same as for Th1 and Th2 cytokines, and 1 ng/ml of tacrolimus suppressed all cytokines completely. These results suggest that tacrolimus suppresses the allergic cytokines from T cells, and that tacrolimus ointment is effective against atopic dermatitis through the inhibition of cytokine production.     

Development of new atopic dermatitis models characterized by not only itching but also inflammatory skin in mice

The present study was performed to develop a new atopic dermatitis model characterized by not only itching but also inflammatory skin using BALB/c mice. From 18 days after the first systemic immunization, daily epicutaneous application of ovalbumin was performed for 2 weeks. Antigen challenge (ovalbumin) resulted in a significant increase of scratching behavior from day 23 to day 32. Moreover, skin symptoms such as erythema/hemorrhage, edema, excoriation/erosion and dryness/desquamation were also observed from day 19 to day 32. The frequency of scratching in the first stage (from day 24 to day 26 after the systemic first immunization) was decreased by chlorpheniramine and epinastine; however, in the last stage (from day 27 to day 30 after the systemic first immunization), both drugs showed no inhibition of scratching behavior. Therefore, an endogenous mediator other than histamine may be responsible for provoking the itching sensation in the last stage. Naloxone dose-dependently reduced the frequency of scratching in the last stage. Moreover, repeated local application of dexametasone significantly inhibited both scratching behavior and skin symptoms from day 24 to day 30. From these findings, it may be concluded that this model is essentially similar to atopic dermatitis in humans and could be used to elucidate the pathogenic mechanisms of atopic dermatitis and to develop appropriate new drugs for therapy.     

Histamine H(4) receptor antagonist reduces dermal inflammation and pruritus in a hapten-induced experimental model

Effects of the histamine H(4) receptor antagonist 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120) were examined for 99 days in a long-term experimental model of pruritic dermatitis induced by repeated challenge with 2,4,6-trinitrochlorobenzene (TNCB) in HR-1 mice. Repeated application of TNCB to the back skin of mice elicited frequent scratching behavior and skin lesions at 24 h after challenge and beyond. JNJ7777120 (10 and 30 mg/kg) reduced this scratching behavior and ameliorated the skin lesions in a dose-dependent manner, whereas the histamine H(1) receptor antagonist fexofenadine had no such effect and did not reduce the inflammation score, even though dexamethasone reduced the scratching bouts. Each of the three agents reduced the increase in the serum IgE concentration induced by TNCB, but only JNJ7777120 reduced the number of mast cells in the skin lesions elicited by repeated application of TNCB. These results indicate that treatment with a H(4) receptor antagonist may be effective for amelioration of both skin inflammation and pruritus in patients with allergic dermatitis such as atopic dermatitis.     

Anti-IL-31 receptor antibody is shown to be a potential therapeutic option for treating itch and dermatitis in mice

Background and Purpose

IL-31, which is described as a pruritogenic cytokine, is linked to the itching that is associated with allergic and non-allergic eczema, but the precise pruritogenic mechanism of IL-31 and its potential as a therapeutic target for atopic dermatitis (AD) have not been determined.

Experimental Approach

We investigated the effects of existing drugs on the scratching behaviour induced by an i.v. injection of IL-31 to clarify whether IL-31 induced pruritus indirectly. In addition, we studied the effects of an anti-IL-31 receptor α subunit (anti-IL-31 receptor α) neutralizing antibody on chronic pruritus-inducing dermatitis in an AD-like model to determine whether IL-31 not only induces scratching behaviour, but is also the causative factor in an AD phenotype.

Key Results

The scratching behaviour induced by an i.v. injection of IL-31 was inhibited by pretreatment with an anti-IL-31 receptor α-neutralizing antibody. In contrast, it was not inhibited significantly by a non-sedative antihistamine (terfenadine), immunosuppressants (dexamethasone and tacrolimus), or a μ-opioid receptor antagonist (naloxone). The anti-IL-31 receptor α-neutralizing antibody reduced the ear swelling and dermatitis score in a chronic pruritus-inducing AD-like model. Moreover, treatment with the anti-IL-31 receptor α-neutralizing antibody showed therapeutic effects on the dermatitis even if it was injected after the disease had developed.

Conclusions and Implications

Anti-IL-31 receptor α is a potential novel therapeutic approach for escaping from the itch–scratch cycle and also a treatment for dermatitis in AD.     

The anti-pruritic efficacy of TS-022, a prostanoid DP1 receptor agonist, is dependent on the endogenous prostaglandin D2 level in the skin of NC/Nga mice

TS-022 is a prostanoid DP(1) receptor agonist, originally developed as a novel anti-pruritic drug for atopic dermatitis. The drug has been shown to suppress scratching and improve the skin inflammation in the NC/Nga (NC) mouse, a model of atopic dermatitis. Corticosteroids are commonly used as effective agents for the treatment of atopic dermatitis. We examined the anti-pruritic efficacy of TS-022 in NC mice cohabited with skin-lesioned NC mice, which showed spontaneous scratching without skin lesions in the early phase and chronic itching with severe dermatitis in the late phase, in comparison with that of dexamethasone. We have previously reported that prostaglandin D(2) might have a physiological role in the inhibition of pruritus. While after 2 weeks of cohabitation with skin-lesioned NC mice (early phase of dermatitis, characterized by the appearance of spontaneous scratching), topically applied TS-022 exhibited a weak anti-pruritic effect in the NC mice, after 6 weeks of cohabitation (late phase, characterized by both chronic scratching and dermatitis), the drug exerted potent anti-pruritic activity. In contrast, dexamethasone exerted potent anti-pruritic effect in both the early and late phases. Indomethacin aggravated the scratching in the early phase, but had no effect in the late phase. The skin prostaglandin D(2) level was significantly increased in the early phase, to subsequently declined and return to the basal level in the late phase. The cutaneous ability for prostaglandin D(2) production following topical application of arachidonic acid or mechanical scratching was decreased in the late phase. Moreover, the expression level of the prostanoid DP(1) receptor in the skin was increased in the late phase. These findings suggest that the potent anti-pruritic activity of TS-022 in the late phase might be attributable to the decrease of endogenous prostaglandin D(2) production and increase of prostanoid DP(1) receptor expression.     

The effects of olopatadine hydrochloride on the number of scratching induced by repeated application of oxazolone in mice

It is suggested that atopic dermatitis is a skin disease associated with itching as subjective symptoms, and histamine H(1) receptor antagonists are used in order to prevent the itching, and the deterioration for scratch by itching. Histamine H(1) receptor selective anti-histamine olopatadine hydrochloride (olopatadine; Allelock shows consistent efficacy and safety in the treatment of allergic disorders. We investigated the possible efficacy of olopatadine on the number of scratching induced by repeated application of oxazolone in BALB/c mice. The repeated treatment of olopatadine significantly inhibited the ear swelling and the increased number of scratching. It significantly inhibited the increased production of interleukin (IL)-4, IL-1beta and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the lesioned ear. Moreover, it significantly inhibited the increased production of nerve growth factor (NGF) and substance P. On the other hand, loratadine, bepotastine and chlorpheniramine did not inhibit the ear swelling and the increased number of scratching. These results indicate that olopatadine inhibited not only the increased production of cytokines but also NGF and substance P unlike other histamine H(1) receptor antagonists. It was suggested that olopatadine suppressed the increased number of scratching by the anti-inflammatory effects. Therefore, olopatadine appears to exert additional biological effects besides its blockade of a histamine H(1) receptor.     

Topical application of aloperine improves 2,4-dinitrofluorobenzene-induced atopic dermatitis-like skin lesions in NC/Nga mice

Aloperine has been shown to inhibit 2,4-dinitrofluorobenzene (DNFB) induced allergic contact dermatitis in BALB/c mice. In the present study, we further investigated the effect of aloperine on DNFB-induced atopic dermatitis-like skin lesions in NC/Nga mice. NC/Nga mice elicited atopic dermatitis-like skin lesions after the topical application of DNFB. Aloperine treatment significantly inhibited dermatitis index and ear thickness in DNFB-treated NC/Nga mice in a dose-dependent manner. Eosinophils, mast cells infiltration into the ears and plasma level of immunoglobulin (Ig) E were also suppressed by aloperine treatment. Finally, cytokine (interleukin (IL)-1β, IL-4, IL-6, IL-10, IL-13, tumor necrosis factor (TNF)-α and interferon (IFN)-γ) productions in ear biopsies homogenates were significantly elevated after DNFB challenge. Topical application of aloperine increased the immunosuppressive cytokine IL-10 level, while it reduced other cytokines production in a dose-dependent manner. Taken together, these data suggest that aloperine may be one of the effective therapeutic agents for the treatment of atopic dermatitis.     

Inhibition of atopic dermatitis by topical application of silymarin in NC/Nga mice

Silymarin has been known to inhibit chemical-induced irritant contact dermatitis. In the present study, we report that topical application of silymarin suppresses dust mite extract (DPE)-induced atopic dermatitis (AD) in NC/Nga mice. Repeated topical application of ears with DPE caused AD-like skin lesions in NC/Nga mice. However, silymarin reduced AD-like skin lesions in these mice, resulting in decreased ear swelling and leukocyte infiltration into the ear. Moreover, our results showed that mast cell infiltration into the ear was suppressed by silymarin treatment in DPE-treated NC/Nga mice. Silymarin also reduced plasma level of IL-4 and IgE in these mice. Further study demonstrated that the mRNA expression of IL-4 was increased and that of IFN-gamma was decreased by DPE treatment in the ears of NC/Nga mice. However, DPE-induced changes in IL-4 and IFN-gamma mRNA expression were reversed by silymarin. DPE-induced increase in TNF-alpha mRNA expression was also suppressed by silymarin treatment. The results presented in this report suggest that silymarin might be beneficial for the treatment of AD.