Ameliorative effects of green tea extract from tannase digests on house dust mite antigen-induced atopic dermatitis-like lesions in NC/Nga mice

Abstract

Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases, which is affected by several factors. Anti-histamines, steroids, and immunosuppressive agents have been used for the treatment of AD. However, many studies have reported that long-term use and abuse of these drugs causes many side effects. This study was performed to evaluate the ameliorative effect of green tea extract on AD-like lesions in NC/Nga mice. Green tea extract from tannase digest (GTT), beta-hexosaminidase, and histamine were measured in IgE-antigen complex-stimulated RBL-2H3 cells. Dorsal skin application of house dust mite-ointment induced AD-like symptoms in NC/Nga mice. Dermatitis scores, skin moisture, transepidermal waterloss (TEWL), thickness of skin and ear, T-cell proliferation, levels of immunoglobulins and cytokines, and infiltration of mast cell were measured to assess the degree of AD induction. Skin moisture and TEWL were measured using probes, and ELISA was performed to measure the immunoglobulin and cytokine levels in blood. GTT was selected based on its ability to inhibit the release of beta-hexosaminidase and histamine in IgE-antigen complex-stimulated RBL-2H3 cells. Oral administration of GTT significantly suppressed the skin inflammation and symptoms of AD-like skin lesions in NC/Nga mice. GTT may have a potential therapeutic effect in the treatment of AD.

Keywords: Atopic dermatitis (AD); Green tea extract; House dust mite; NC/Nga; Tannase.

Fig. 1

HPLC analysis of green tea extract from tannase digest (GTT)

Fig. 2

DPPH and SOD radical-scavenging activities of green tea extracts for screening. a DPPH radical-scavenging activity increased significantly compared with the control in a dose-dependent manner. b Superoxide-radical-scavenging activity was significantly increased compared with the control in a dose-dependent manner. Experiments were performed three times. Data are shown as mean ± SD of the changes in DPPH and superoxide-radical-scavenging activity. ***p < 0.001 compared with the control

Fig. 3

Cytotoxicity of green tea extracts and inhibitory effects on the release of β-hexosaminidase and histamine by green tea extracts on RBL-2H3 cells for screening. a Cell viability of green tea extracts on RBL-2H3 cells. Green tea extracts had no significant cytotoxicity on RBL-2H3 cells. b Inhibitory effects of green tea extracts on β-hexosaminidase from IgE-antigen complex-stimulated RBL-2H3 cells. Sample no. 7, the green tea GTT, had a significant inhibitory effect at all concentrations. Therefore, GTT was selected for measuring histamine release. c Inhibitory effect of GTT on histamine release from IgE-antigen complex-stimulated RBL-2H3 cells. The levels of β-hexosaminidase and histamine were significantly decreased by GTT administration in a dose-dependent manner. Experiments were performed three times. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control

Fig. 4

Experimental procedure and changes in the severity of atopic dermatitis-like skin conditions a For the induction of AD-like symptoms, house dust mite (HDM) was topically applied to the skin of NC/Nga mice twice weekly for 4 weeks. Mice were fed daily with GTT for 4 weeks. b Weekly dermatitis scores were evaluated during the treatment period. c Ear thickness was measured with a vernier caliper. d Moisture capacity of the skin was measured weekly with a CM825 probe for 2 weeks. e Transepidermal waterloss (TEWL) is the evaporation of skin moisture. It was weekly measured with a TM300 probe for 4 weeks. GTT resulted in a significant recovery from atopic dermatitis-like skin condition. Sample size was n = 7 per group. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control

Fig. 5

Changes in splenocyte numbers and splenomegaly in atopic dermatitis-like lesions. a Spleen weight was measured on day 28. B Proliferation of splenocytes obtained from NC/Nga mice was measured by MTT assay on day 28. LPS stimulates B-cell proliferation and ConA stimulates T-cell proliferation. ConA-induced splenocytes were significantly enhanced by the induction of AD. Splenomegaly and the proliferation of T cells were significantly decreased by GTT in a dose-dependent manner. Sample size was n = 7 per group. Data are shown as mean ± SD. *p < 0.05 and ***p < 0.001 compared with control

Fig. 6

Effect of consecutive administration of GTT on the concentrations of immunoglobulins, cytokines, and histamine in atopic dermatitis-like lesions. a Blood samples were collected weekly during the experimental period. On day 28, 400 mg/kg GTT significantly reduced the increased IgE levels in blood. b Level of IgG2a was measured using ELISA on day 28. The decreased IgG2a level was increased by the administration of GTT. c–e The levels of cytokines and histamine in the blood were not significantly changed by the induction of atopic dermatitis. Sample size was n = 7 per group. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control

Fig. 7

Effect of GTT on skin thickness and local infiltration of mast cells. a, c Hematoxylin and eosin stained the dorsal skin sections of NC/Nga mice (× 200). Atopic dermatitis and administration of GTT significantly changed the epidermal thickness. b, d Toluidine blue-stained violet mast cells in the skin sections (× 200). GTT inhibited the infiltration of mast cells into the skin. Sample size was n = 7 per group. Data are shown as mean ± SD. ***p < 0.001 compared with the control