Ⅱ. Materials and methods
2.8. Statistical analysis
Statistical analysis was performed with SPSS Statistics Software. The data were expressed as means ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by Tukey’s HSD test was used for comparing differences between multiple groups. Differences were considered significant at p < 0.05.
Ⅲ . Results
3.1. 7,8,4’-THIF alleviated DNCB-induced AD-like symptoms in NC/Nga mice
To investigate the effects of 7,8,4’-THIF on DNCB-induced AD-like symptoms in NC/Nga mice, I took the images of skin lesions, evaluated the dermatitis score, and measured the ear thickness. On day 21, the ear and the dorsal skin of DNCB-treated NC/Nga mice showed severe erythema, erosion, and dryness (Fig.
1A). Co-treatment of 7,8,4’-THIF or tacrolimus, showed less severe AD-like symptoms (Fig. 1A). The dermatitis score was gradually increased in DNCB-treated NC/Nga mice (Fig. 1B). On day 21, the dermatitis score was significantly high in DNCB-treated NC/Nga mice (6.78±0.79), compared with naïve control (0.22±0.16) (Fig.
1B). Treatment with 200 nmol 7,8,4’-THIF (4.78±0.58), 400 nmol 7,8,4’-THIF (4.78±0.58) or tacrolimus (4.78±0.29) significantly decreased the DNCB-induced increases in dermatitis score (Fig.
1B). The ear thickness was gradually increased in DNCB-treated
NC/Nga mice (Fig. 1C). On day 21, the ears of DNCB-treated NC/Nga mice (0.50±0.02 mm) were significantly thicker than those of the naïve control mice (0.21±0.00 mm) (Fig. 1C). Treatment with 200 nmol 7,8,4’-THIF (0.37±0.02 mm), 400 nmol 7,8,4’-THIF (0.39±0.02 mm) or tacrolimus (0.38±0.01 mm) markedly attenuated the DNCB-induced increase in ear thickness (Fig. 1C). On day 21, the ear skin of each mouse was prepared and observed. The ears of DNCB-treated NC/Nga mice became swollen and showed the epidermal hypertrophy (Fig. 1D). In contrast, the ears of 7,8,4’-THIF or tacrolimus-treated mice showed thinner and less severe epidermal hypertrophy than those of the DNCB-treated NC/Nga mice (Fig. 1D).
3.2. 7,8,4’-THIF alleviated DNCB-induced scratching behavior in NC/Nga mice
Analysis of spontaneous scratching behavior in NC/Nga mice is a possible approach to evaluate anti-pruritics for subjects with AD [28]. To investigate the effect of 7,8,4’-THIF on DNCB-induced scratching behavior in NC/Nga mice, the mice were
monitored and the time rubbing nose, ears and dorsal skin with the hind paws was measured as scratching time. On day 21, scratching time was markedly increased in DNCB-treated NC/Nga mice (156.33±25.47 sec) compared with naïve control mice (30.69±8.84 sec), whereas treatment with 200 nmol 7,8,4’-THIF (81.46±7.61 sec), 400 nmol 7,8,4’-THIF (68.34±15.38 sec), or tacrolimus (95.13±10.93 sec) reduced the DNCB-increased scratching time (156.33±25.47 sec) (Fig. 2).
3.3. 7,8,4’-THIF decreased DNCB-induced infiltration of eosinophils and mast cells into skin lesions in NC/Nga mice
To investigate the effect of 7,8,4’-THIF on DNCB-induced infiltration of eosinophils and mast cells into skin lesions in NC/Nga mice, tissue sections collected on the last day of the experiment (day 21), were stained with Congo red and toluidine blue to discriminate eosinophils and mast cells, respectively. The number of eosinophils stained with Congo red in mm2 skin lesions of DNCB-treated NC/Nga mice (564.44±49.64 cells) was significantly increased compared with that of naïve control mice
(62.22±15.07 cells) (Fig. 3A and B). In contrast, topical application of 200 nmol THIF (271.11±45.60 cells), 400 nmol 7,8,4’-THIF (222.22±36.58 cells per) or tacrolimus (293.33±37.11 cells) markedly lowered the number of eosinophils in dorsal skins of DNCB-treated NC/Nga mice (Fig. 3A and B). Furthermore, the number of mast cells stained with toluidine blue in mm2 skin lesions of DNCB-treated NC/Nga mice (862.22±72.45 cells) was significantly increased to that of naïve control mice (244.44±15.56 cells) (Fig. 3A and C). Treatment with 200 nmol 7,8,4’-THIF (471.11±49.79 cells), 400 nmol 7,8,4’-THIF (448.89±60.65 cells) or tacrolimus (555.56±25.34 cells) significantly reduced the number of infiltrated mast cells in dorsal skins of DNCB-treated NC/Nga mice (Fig. 3A and C).
3.4. 7,8,4’-THIF decreased DNCB-induced increase in serum IgE level in NC/Nga mice
To investigate the effect of 7,8,4’-THIF on DNCB-induced increase in serum IgE level in NC/Nga mice, blood samples were collected on the last day of the experiment (day 21). Repeated
topical application of DNCB significantly increased the serum IgE level in DNCB-treated NC/Nga mice (32695.3±8513.29 ng/ml) than naïve control mice (155.25±10.61 ng/ml) (Fig. 4). However, treatment with 200 nmol 7,8,4’-THIF (12055.2±3620.46 ng/ml), 400 nmol 7,8,4’-THIF (6947.13±1808.97 ng/ml) or tacrolimus (767±331.45 ng/ml) significantly decreased the level of serum IgE in DNCB-treated NC/Nga mice (Fig. 4).
3.5. 7,8,4’-THIF decreased DNCB-induced increase in chemokine TARC and Th2 and Th1 cytokines in NC/Nga mice
To elucidate the effect of 7,8,4’-THIF on DNCB-induced increase in chemokine TARC and Th2 and Th1 cytokines in NC/Nga mice, dorsal skins were collected on the last day of the experiment (day 21). I found that the level of chemokine TARC was markedly increased in skin lesions of DNCB-treated NC/Nga mice, compared to naïve control mice (Table 1). However, treatment with 200 nmol 7,8,4’-THIF, 400 nmol 7,8,4’-THIF, or tacrolimus significantly reduced the level of TARC in dorsal skins of DNCB-treated NC/Nga mice (Table 1).
On the other hand, the level of Th2 cytokines IL-4, IL-5, and IL-13 was significantly increased in skin lesions of DNCB-treated NC/Nga mice, compared with naïve control mice (Table 1).
The level of Th1 cytokines IL-12 and IFN-γ was also significantly increased in skin lesions of DNCB-treated NC/Nga mice, compared with naïve control mice (Table 1). However, treatment with 200 nmol 7,8,4’-THIF, 400 nmol 7,8,4’-THIF, or tacrolimus significantly decreased the level of Th2 and Th1 cytokines 4, IL-5, IL-13, IL-12, and IFN-γ in dorsal skins of DNCB-treated NC/Nga mice (Table 1).
3.6. 7,8,4’-THIF decreased DNCB-induced increase in TEWL in NC/Nga mice
To investigate the effect of 7,8,4’-THIF on DNCB-induced loss of water through epidermal layer in NC/Nga mice, the level of TEWL was measured on the last day of the experiment (day 21).
DNCB-treated NC/Nga mice (35.96±1.06 g/m2⋅h) showed an increase in TEWL compared to naïve control mice (8.31±0.40 g/m2⋅h) (Fig. 5). Treatment with 200 nmol 7,8,4’-THIF (28.60±0.99
g/m2⋅h), 400 nmol 7,8,4’-THIF (18.14±0.58 g/m2⋅h) or tacrolimus (12.30±0.73 g/m2⋅h) significantly reduced TEWL in DNCB-treated NC/Nga mice (Fig. 5).
3.7. 7,8,4’-THIF increased DNCB-induced decrease in filaggrin in NC/Nga mice
To investigate the effect of 7,8,4’-THIF on DNCB-induced defective skin barrier of NC/Nga mice, dorsal skins were collected on the last day of the experiment (day 21). The immunohistochemical analysis of filaggrin was performed. The level of filaggrin was reduced in dorsal skin of DNCB-treated NC/Nga mice, while treatment with 200 nmol 7,8,4’-THIF, 400 nmol 7,8,4’-THIF, or tacrolimus restored the level of filaggrin, in dorsal skin of DNCB-treated NC/Nga mice (Fig. 6).
Table 1
DNCB+vehicle 117.85±11.82b 148.04±13.19b DNCB+7,8,4’-THIF 200 85.34±9.84ac 101.58±8.96a DNCB+7,8,4’-THIF 400 62.66±3.64a 86.12±5.83aDNCB+tacrolimus 62.41±4.59c 83.15±9.10a
Table 1. Effects of 7,8,4’-THIF on DNCB-induced increase in chemokine TARC and Th2 and Th1 cytokines in NC/Nga mice.
The pg/ml levels of Th-derived chemokine TARC and cytokines in NC/Nga mice are shown. Dorsal skins were collected on the last day of the experiment (day 21). The level of TARC, Th2 cytokines IL-4, IL-5, and IL-13, and Th1 cytokines IL-12 and IFN-γ in dorsal skin was measured using an ELISA. Data are the means ± SEM (n = 6). Means with letters (a-c) are significantly different from each other at p < 0.05.
Figure 1
Figure 1. Effect of 7,8,4’-THIF on DNCB-induced AD-like symptoms in NC/Nga mice.
(A) Images of skin lesions from the groups of mice were taken on the last day of the experiment (day 21). (B) Dermatitis scores were evaluated weekly from day -5 to 21. (C) Ear thickness was measured from day -5 to 21. (D) Hematoxylin and eosin staining of ear skin on day 21 (100×). Arrows indicate the epidermal hypertrophy. Data represent the mean ± SEM (n=9). Means with letters (a-d) within a graph are significantly different from each other at p < 0.05.
Figure 2
Figure 2. Effect of 7,8,4’-THIF on DNCB-induced scratching incidence in NC/Nga mice.
Scratching time was evaluated on the last day of the experiment (day 21). Data represent the mean ± SEM (n=9). Means with letters (a-b) within a graph are significantly different from each other at p
< 0.05.
Figure 3
Figure 3. Effect of 7,8,4’-THIF on DNCB-induced infiltration of eosinophils and mast cells in skin lesions of NC/Nga mice.
(A) Representative images depicting the histological features of skin collected on day 21 are shown. Staining with Congo red (CR) and toluidine blue (TB) was used to identify eosinophils and mast cells, respectively. The arrows indicate the CR-stained eosinophils and the TB-stained mast cells. Cells were counted under a microscope at 400× magnification. Scale bar, 50 µm. The numbers of (B) eosinophils and (C) mast cells in 1 mm2 of skin were calculated. Data represent the mean ± SEM (n=9). Means with letters (a-b) within a graph are significantly different from each other at p < 0.05.
Figure 4
Figure 4. Effect of 7,8,4’-THIF on DNCB-induced increase in serum IgE level in NC/Nga mice.
Blood was collected on the last day of the experiment (day 21). The level of serum IgE was measured using an ELISA. Data are the means ± SEM (n = 6). Means with letters (a-c) within a graph are significantly different from each other at p < 0.05.
Figure 5
Figure 5. Effect of 7,8,4’-THIF on DNCB-induced infiltration of eosinophils and mast cells in skin lesions of NC/Nga mice.
Effect of 7,8,4’-THIF on DNCB-induced increase in TEWL in NC/Nga mice. The level of TEWL in dorsal skin of mice was measured using a skin evaporative water recorder on the last day of the experiment (day 21). Data are the means ± SEM (n = 9). Means with letters (a-e) within a graph are significantly different from each other at p < 0.05.
Figure 6
Figure 6. Effect of 7,8,4’-THIF on DNCB-induced decrease in filaggrin (FLG) in NC/Nga mice.
Dorsal skins were collected on the last day of the experiment (day 21). The level of filaggrin in dorsal skin was measured using immunohistochemical staining (400×).