A Herbal Formula, Atofreellage, Ameliorates Atopic Dermatitis-Like Skin Lesions in an NC/Nga Mouse Model

Abstract

We evaluated the anti-atopic dermatitis (AD) effect of Atofreellage (AF), a herbal formula composed of 10 medicinal plants. AD was induced on the dorsal skin areas of NC/Nga mice (male, seven weeks old) by daily application of 2,4-dinitrochlorobenzene (DNCB) for five weeks. After three weeks of DNCB application, 200 μL of AF (0, 25, 50 or 100 mg/mL) was applied to the skin lesions. Histological findings, blood cell populations, serum levels of immunoglobulin E (IgE), histamine, pro-inflammatory cytokines, and inflammatory signaling in the skin tissue, and T-helper cell type 2 (Th₂)-related cytokines in splenocytes were analyzed. Histopathological findings showed AF treatment notably attenuated the thickness of dorsal skin, and eosinophil infiltration. AF treatment (especially 100 mg/mL) also demonstrably ameliorated the blood cell population abnormalities, as the notable elevation of serum concentrations of IgE, histamine, TNF-α, IL-6 and IL-1β were remarkably normalized by AF treatment. Western blot analysis evidenced the apparent normalization of inflammatory signals (ERK, p38 MAP kinase, JNK, and NF-κB) in the skin tissue. Additionally, AF treatment notably attenuated the activation of Th₂-dominant cytokines (IL-13, IL-4, and IL-5) in Con A-treated splenocytes in an ex vivo assay. In conclusion, this study provides experimental evidence for the clinical relevance of Atofreellage.

Keywords: NC/Nga murine model; atopic dermatitis; herbal medicine; inflammation.

Figure 1

Fingerprint of Atofreellage. Atofreellage and its reference compounds were subjected to HPLC analysis. Histograms of Atofreellage (A,C) and reference compounds (B) and the quantitative analysis of Atofreellage (D) are presented.

Figure 2

Histopathological findings. Dorsal skin lesions of NC/Nga mice were stained with H & E (A) and toluidine blue staining (B). All images were analyzed under 100× magnification. The skin thicknesses of epidermal (C) and dermal (D) tissues, and infiltration mast cells (E) were analyzed. The data are expressed as the mean ± SD (n = 8). Arrows indicated the infiltration of basophils. ^## p < 0.01 and ^### p < 0.001, compared with the normal group; * p < 0.05 and *** p < 0.001, compared with the control group.

Figure 3

IgE and histamine in serum. Serum levels of IgE (A) and histamine (B) were measured using ELISA kit. The data are expressed as the mean ± SD (n = 8). ^### p < 0.001, compared with the normal group; ** p < 0.01 and *** p < 0.001, compared with the control group.

Figure 4

Pro-inflammatory cytokines in the serum. Serum levels of pro-inflammatory cytokines, including TNF-α (A); IL-6 (B); and IL-1β (C), were measured using ELISA kits. The data are expressed as the mean ± SD (n = 8). ^### p < 0.001, compared with the normal group; * p < 0.05 and *** p < 0.001, compared with the control group.

Figure 5

Western blot analysis of inflammatory signaling pathway components. Protein levels of NF-κB and MAP kinase sub-family members were determined by western blotting (A). Protein levels were quantified using ImageJ software (B). The data are expressed as the mean ± SD (n = 8). ^### p < 0.001 compared with the normal group; * p < 0.05, ** p < 0.01, and *** p < 0.001, compared with the control group.

Figure 6

Th₂-related cytokines in cultured splenocytes. The levels of IL-13 (A); IL-4 (B) and IL-5 (C) in culture media were measured using ELISA kits. The data are expressed the mean ± SD (n = 8). ^### p < 0.001, compared with the normal group; * p < 0.05 and *** p < 0.001, compared with the control group.