The prevention of 2,4-dinitrochlorobenzene-induced inflammation in atopic dermatitis-like skin lesions in BALB/c mice by Jawoongo

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: 2,4-dinitrochlorobenzene; Atopic dermatitis; Cytokine; Inflammation; Jawoongo.

Fig. 1

General schematic diagram for the study protocol

Fig. 2

Effects of Jawoongo on the skin of mice with DNCB-induced AD. Skin thickness in mice with DNCB-induced AD that were treated with Jawoongo (a). Jawoongo reduced the infiltration of inflammatory cells into the skin. Skin sections were stained with hematoxylin and eosin (b). Arrows indicate inflammatory cells. Jawoongo reduced the infiltration of mast cells into the skin. Skin sections were stained with toluidine blue (c). Arrows indicate inflammatory cells. The sections were evaluated under a microscope at an original magnification of 200×. The data were presented as mean ± SEMs (n = 8 mice/group). *P < 0.05, **P < 0.01 and ***P < 0.001 as compared to DNCB-stimulated group, respectively

Fig. 3

Jawoongo reduced leukocyte numbers in the blood. Blood samples were analyzed using a HEMAVET 950 hematological analyzer. The data were presented as mean ± SEMs (n = 8 mice/group). *P < 0.05, **P < 0.01 and ***P < 0.001 as compared to DNCB-stimulated group, respectively

Fig. 4

Jawoongo reduced cytokine levels in serum. Cytokine levels were measured by ELISA. The data were presented as mean ± SEMs (n = 8 mice/group). *P < 0.05, **P < 0.01 and ***P < 0.001 as compared to DNCB-stimulated group, respectively

Fig. 5

Effects of Jawoongo on cytokine mRNA expression in mouse skin tissue. IL-2, IL-4, IL-13 and TNF-α mRNA expression was measured by RT-PCR, shown in (a), (b), (c), and (d), respectively, in mouse skin tissue. The data were presented as mean ± SEMs (n = 8 mice/group). *P < 0.05, **P < 0.01 and ***P < 0.001 as compared to DNCB-stimulated group, respectively

Fig. 6

Distribution of CD4+ cells in the skin of mice with DNCB-induced AD. Skin sections were immunostained with CD4+ antibodies. CD4+ cells display a brown color. The sections were evaluated under a microscope at an original magnification of 200×

Fig. 7

Effects of Jawoongo on cytokine expression in HMC-1 cells. HMC-1 cells were stimulated with ionomycin (500 ng/ml) and PMA (5 ng/ml) and then treated with different concentrations of Jawoongo (50–200 μg/ml) for 24 h. IL-4, IL-13 and TSLP mRNA expression was measured by RT-PCR (a). Whole cell lysates were analyzed by Western blotting (b). The culture medium of the cells was harvested, and IL-4, IL-6 and IL-13 cytokine levels were measured by ELISA (c). The data were presented as mean ± SEMs (n = 8 mice/group). *P < 0.05, **P < 0.01 and ***P < 0.001 as compared to Ionomycin and PMA-stimulated group, respectively

Fig. 8

Effects of Jawoongo on NO production in RAW264.7 cells. RAW264.7 cells were stimulated with LPS (1 mg/ml) and then treated with different concentrations of Jawoongo (25–100 μg/ml) for 24 h. NO production was measured using the Griess reagent system (a). iNOS and TNF-α mRNA expression was measured by RT-PCR (b). Whole cell lysates were analyzed by Western blotting (c). The data were presented as mean ± SEMs (n = 8 mice/group). *P < 0.05, **P < 0.01 and ***P < 0.001 as compared to LPS-stimulated group, respectively

Fig. 9

Effects of Jawoongo on cytokine expression in splenocytes. Splenocytes were stimulated with LPS (1 μg/ml) and then treated with different concentrations of Jawoongo (25–200 μg/ml) for 24 h. IL-4, IL-6 and TNF-α mRNA expression was measured by RT-PCR (a). The culture medium of the cells was harvested, and IL-6 and TNF-α cytokine levels were measured by ELISA (b). Whole cell lysates were analyzed by Western blotting (c). The data were presented as mean ± SEMs (n = 8 mice/group). *P < 0.05, **P < 0.01 and ***P < 0.001 as compared to LPS-stimulated group, respectively

Fig. 10

LC-MS chromatogram. a Identification of decursin in Jawoongo. b Mass spectrum peak at 53.028 min

Fig. 11

Pathway diagram of the mechanism of histamine inhibition of ERK, JNK and NF-kB activation. RAW264.7 cells and Splenocyte activated with a LPS agonist activated the MEK/ERK MAPK signaling and NF-kB signaling cascade to induce production of TNF. Jawoongo inhibited MEK/ERK and NF-kB activation and suppressed production of TNF