Modulation of experimental atopic dermatitis by topical application of Gami-Cheongyeul-Sodok-Eum

Abstract

Background: Gami-Cheongyeul-Sodok-Eum (GCSE), an herbal formula of traditional Korean medicine, comprises nine herb components. GCSE has various biological activities such as anti-inflammatory, anti-bacterial and anti-viral activities. However, it is still unclear whether GCSE has any immunomodulatory effect on atopic dermatitis (AD).

Methods: GCSE was treated to primary B cells and CD4+ T cells isolated from atopic mice to compare its inhibitory effects on IgE secretion and cytokine expression. Experimental AD was established by alternative treatment of 2, 4-dinitrochlorobenzene (DNCB) and house dust mite extract to the ears of BALB/c mice. GCSE was topically applied to ears of atopic mice every day for 3 weeks. AD progression was analyzed by measuring ear thickness, serum IgE level, histological examination of ear tissue by H&E staining and cytokine profile of CD4+ T cells and CD19+ B cells by real time PCR and ELISA.

Results: Treatment of GCSE significantly reduced IgE production and expression of AD associated pathogenic cytokines such as IL-4, IL-5, IL-10, IL-13, IL-17, TNF-α, and IFN-γ by lymphocytes isolated from AD-induced mice. Topical application of GCSE on the ears of AD-induced mice significantly reduced ear thickness, clinical score and lymphocytes infiltration to ears as compared to control group. GCSE treatment also reduced serum IgE level and the levels of major pathogenic cytokines such as IL-4, IL-5, IL-10, IL-13 and IL-17. In addition, GCSE treatment significantly increased Foxp3 expression level.

Conclusions: The protective effect of GCSE in experimental AD is mediated by inhibition of IgE production, by reduction in the levels of pathogenic cytokines and by induction of Foxp3, all of which are suggesting the beneficial effect of GCSE on modulating atopic dermatitis.

Figure 1

HPLC chromatograms of marker substances in individual herbs of Gami-Cheongyeul-Sodok-Eum (GCSE). HPLC analysis was performed on YMC-Pack Pro C18 column (4.6 × 250 mm, 5 μm). The optimum mobile phase and wavelength for detection were acetonitrile and water for (A) nodakenin, (B) decursin and (C) decursinol angelate (gradient, 20% - 80% ACN, 330 nm); (D) acetonitrile and water (3.4 g of monobasic potassium phosphate and 1.7 g of sodium lauryl sulfate/1000 mL) for berberine (1:1, 345 nm); aqueous acetic acid and acetonitrile for (E) glycyrrhizin (3:2, 254 nm) and (F) liquiritigenin (7.5:2.5, 276 nm); aqueous acetic acid and acetonitrile : methanol (7:3) for (G) baicalin, (H) baicalein, and (I) wogonin (gradient, 25% - 52% ACN-MeOH, 277 nm). Data are representative of three independent experiments.

Figure 2

Effect of GCSE treatment on T cells and B cells isolated from AD-induced mice. (A) Mouse splenocytes were incubated with various concentrations of GCSE for 72 hrs. Cell viability was estimated with WST-1 assay. (B) Draining lymph node CD19+ B cells isolated from AD-induced mice were stimulated with LPS (10 μg/ml)/IL-4 (5 ng/ml) in the presence of various concentrations of GCSE dissolved in 70% alcohol for 72 hrs. Mouse IgE levels in the supernatant of B cell culture were measured by ELISA. Same volume of 70% alcohol was treated as control. (C) Draining lymph node CD4+ T cells from AD-induced mice were stimulated with PMA (50 ng/ml)/ionomycin (1 μM) in the presence of GCSE (0.25 mg/ml) for 4 hrs. Relative expression of cytokines of GCSE treated samples was compared with control samples by qRT-PCR. Expression level of HPRT was used as an internal control. (D) CD4+ T cells from AD-induced mice were stimulated with PMA /ionomycin for 72 hrs in the presence of GCSE (0.25 mg/ml), then protein level of each cytokine was analyzed by ELISA. Error bars indicate SD. One (*), two (**) and three (***) indicate p < 0.05, p < 0.01, and p < 0.001 respectively. Data are representative of three independent experiments.

Figure 3

Inhibition of AD progression by topical application of GCSE. (A) Representative ear pictures of control group or GCSE treated groups were shown. (B) AD progression was assessed by measuring ear thickness, 24 hrs after each DNCB or mite extract treatment. (C) Ears excised from each group were fixed with 4% paraformaldehyde for 24 hrs and embedded in paraffin. Paraffin embedded ears were sectioned into 6 μm and stained with hematoxylin and eosin (H&E). Infiltration by lymphocytes and thickness of epidermis were observed under the microscope (50X, 100X and 200X). (D) Total serum IgE levels were measured by ELISA, left. CD19+ B cells isolated from draining lymph node of each group were stimulated with LPS (10 μg/ml)/IL-4 (5 ng/ml) for 72 hrs then IgE levels in the culture supernatant were measured by ELISA, right. Error bars indicate SD. One (*), two (**) and three (***) indicate p < 0.05, p < 0.01 and p < 0.001 respectively. Data are representative of three independent experiments.

Figure 4

Down-regulation of pathogenic cytokines by GCSE treatment. (A) Draining lymph node CD4+ T cells from each treatment group were stimulated with PMA (50 ng/ml)/ionomycin (1 μM) for 4 hrs. Relative cytokine levels of GCSE treated samples were compared with control samples by qRT-PCR. The expression level of HPRT was used as an internal control. (B) Draining lymph node CD4+ T cells from each group were stimulated with PMA/ionomycin for 72 hrs, then, IL-4, IL-10, IL-17, and IFN-γ levels were measured by ELISA. (C) Draining lymph node CD19+ B cells from each treatment group were stimulated for 4 hrs. Relative cytokine levels of GCSE treated samples were compared with control samples by qRT-PCR. The expression level of HPRT was used as an internal control. Error bars indicate SD. One (*), two (**), and three (***) indicate p < 0.05, p < 0.01 and p < 0.001 respectively. Data are representative of three independent experiments.

Figure 5

Up-regulation of Foxp3 expression in iTreg by GCSE treatment. CD4+ T cells isolated from the spleen and lymph node of 8 week old Foxp3-GFP knock in mice were stimulated in a medium supplemented with anti-CD3 (1 μg/ml) / CD28 Ab (3 μg/ml), anti-IL-4 Ab (10 μg/ml), anti-INF-γ Ab (10 μg/ml) and TGF-β (5 ng/ml) at day 1 and additional 50 U/ml rhIL-2 at day 3. Then iTreg cells were stimulated with various concentrations of GCSE in the presence of PMA (50 ng/ml)/ ionomycin (1 μM) for 12 hrs. (A) Relative expression levels of Foxp3 of GCSE treated samples were compared with control sample by qRT-PCR. Expression level of HPRT was used as an internal control. (B) GFP signal was measured as protein level of Foxp3 by flow cytometry. Error bars indicate SD. One (*), two (**), and three (***) indicate p < 0.05, p < 0.01, and p < 0.001 respectively. Data are representative of three independent experiments.