Preventive effects of a novel herbal mixture on atopic dermatitis-like skin lesions in BALB/C mice

Abstract

Background: A combination of parts of Cornus officinalis, Rosa multiflora, Lespedeza bicolor, Platycladus orientalis, and Castanea crenata is commonly used for alleviating inflammatory skin disorders. Therefore, this study was carried out to evaluate the in vitro and in vivo preventive effects of a novel herbal formula made from the five plants (C2RLP) against atopic dermatitis in BALB/C mice.

Methods: Mice were allocated into five groups (n = 8) including, control (Normal, petrolatum, and betamethasone treated) and treatment groups (treated with 2.5 and 5% C2RLP ointment). Atopic lesion was induced by applying 1-Chloro-2, 4-dinitrobenzene to the dorsal thoracic area of mice. Macroscopical and histological evaluations were performed to determine the effects of treatment on the progress of the skin lesions. The effects of treatment on the production and release of interleukins, interferon -ϒ, nitrite, prostaglandin E2, thymus and activation-receptor chemokine, and β-hexosaminidase were evaluated and comparisons were made between groups. In addition, the chemical compounds present in C2RLP were identified by Liquid Chromatography-Mass Spectrometry.

Results: Topical application of C2RLP reduced the dermatitis score and suppressed histopathological changes in mice. Treatment significantly reduced (P < 0.05) plasma IL-4 level, the production of nitrite, prostaglandin E2, and thymus and activation-receptor chemokine production. The lipopolysaccharide-induced iNOS-mRNA expression in RAW 264.7 cells was also suppressed by high concentrations of C2RLP. In addition, C2RLP showed an inhibitory effect against DPPH free radical (IC₅₀ = 147.5 μg/ml) and β-hexosaminidase release (IC₅₀ = 179.5 μg/ml). Liquid Chromatography-Mass Spectrometry analysis revealed the presence of various compounds, including loganin, ellagic acid, and kaempferol 3-glucoside.

Conclusion: Down-regulation of T- helper 2 cellular responses and suppression of inflammatory mediators contributed to the protective effects of C2RLP from atopic dermatitis in BALB/C mice.

Keywords: Atopic dermatitis; Inflammation; Mice; Skin.

Fig. 1

Experimental schedule for induction of atopic dermatitis and treatment with C2RLP in BALB/C mice

Fig. 2

Effects of C2RLP on viability (a), NO production (b), iNOS-mRNA expression (c), and PGE2 production (d) of LPS stimulated RAW 264.7 cells. The data shown represent the means of three independent experiments and bars with different letters indicate significant difference (P < 0.05)

Fig. 3

Effects of C2RLP on TARC production (a) and viability (b) in HaCa-T cells. The data shown represent the means of three independent experiments and bars with different letters indicate significant difference (P < 0.05)

Fig. 4

The effects of C2RLP on β-hexosaminidase release (a) and viability (b) in IgE-DNP antibody sensitized RBL-2H3 cells. The β-hexosaminidase inhibitory activity (IC₅₀ value) of C2RLP was also determined (c). The data shown represent the means of three independent experiments and bars with different letters indicate significant difference (P < 0.05)

Fig. 5

DPPH free radical scavenging activity of C2RLP (IC₅₀) following incubation of the C2RLP and DPPH solution at 37 °C for 30 min

Fig. 6

Representative photographs of mouse dorsal skin showing various degrees of alopecia, skin dryness, and erythematous lesions after 2 days of treatment indicated no difference between treatment and control groups (A). Photographs of mice treated with petrolatum (a), 2.5% C2RLP (b), 5% C2RLP (c), and Betamethasone (d) indicated treatment with the test substance enhanced the recovery of mice from AD-like lesions compared with petrolatum. Dermatitis score at the end of treatment (day 33) showing a significant difference with respect to DNCB treated control mice (e). The data shown in Fig. E represent mean + SD (n = 8) and bars with different letters indicate significant difference (P < 0.05)

Fig. 7

HE (a) and TB (b) stained skin sections (200x magnification) indicating the effect of C2RLP treatment on the degree of inflammatory cell infiltration and epidermal hyperplasia. Epidermal thickness (c) and, the number of mast cells (d) was determined in the HE and TB-stained sections, respectively (n = 6). The data shown represent the means of three independent experiments and bars with different letters indicate significant difference (P < 0.05)

Fig. 8

Effects of topical C2RLP treatment on serum levels of IL-4 (a) and IFN-γ (b) of DNCB treated mice. The data shown represent the mean ± SD (n = 8) and bars with different letters indicate significant difference (P < 0.05)