Kampo Medicines for Mite Antigen-Induced Allergic Dermatitis in NC/Nga Mice

Abstract

We have established an allergic dermatitis model in NC/Nga mice by repeated local exposure of mite antigen for analyzing atopic dermatitis. We examined how four Kampo medicines, Juzen-taiho-to, Hochu-ekki-to, Shofu-san and Oren-gedoku-to, on the dermatitis model to obtain basic information on their usefulness for treating atopic dermatitis. Mite antigen (Dermatophagoides farinae crude extract) solution at a concentration of 10 mg/ml was painted on the ear of NC/Nga mice after tape stripping. The procedure was repeated five times, at 7 day intervals. An apparent biphasic ear swelling was caused after the fourth and fifth antigen exposures with elevated serum IgE levels and accumulation of inflammatory cells. In the cervical lymph nodes and ear lobes, the five procedures of antigen exposure induced interleukin-4 mRNA expression but reduced interferon-gamma mRNA expression. Oral administration of all four Kampo medicines inhibited the formation of ear swelling and inflammatory cell accumulation. Juzen-taiho-to and Hochu-ekki-to apparently prevented the elevation of serum IgE level. Furthermore, the four Kampo medicines showed a tendency to prevent not only the increase in interleukin-4 mRNA expression but also the decrease in interferon-gamma mRNA expression. The present results indicate that Juzen-taiho-to, Hochu-ekki-to, Shofu-san and Oren-gedoku-to may correct the Th1/Th2 balance skewed to Th2, and this activity helps inhibit dermatitis in NC/Nga mice. The ability of the Kampo medicines to correct the Th1/Th2 balance seems to underlie their effectiveness in treating of atopic dermatitis.

Figure 1

HPLC profile of Juzen-taiho-to. Ingredients of Juzen-taiho-to (1.0 g of preparation) were extracted with 20 ml of methanol for 30 min under sonication and 30 μl of the extract was used for the HPLC analysis. Column, TSK-GEL 80_TS (4.6 mm × 250mm, Tosoh, Japan); pump, LC-10AD_VP (Shimadzu, Japan); detector, SPD-M10A_VP (Shimadzu). Mobile phase: (A) 50 mM AcOH–AcONH₄ buffer, (B) CH₃CN, from 90% A + 10% B to 100% B in 60 min (linear gradient). Column temperature, 40°C; flow rate, 1.0 ml/min; wavelength range, 200–400 nm.

Figure 2

Varied doses of Juzen-taiho-to, Hochu-ekki-to, Shofu-san, Oren-gedoku-to and prednisolone influence ear thickness from repeated painting with mite antigen solution in NC/Nga mice. Ear thickness was measured immediately before, and 1, 4 and 24 h after each antigen application. Results after the first and the second antigen applications were omitted because increased ear thickness and drug effects were negligible. Each value represents the mean ± SEM of 5–8 mice. ^*P < 0.05, ^**P < 0.01 for Kampo medicine-administered groups by multiple comparison test, ^#P < 0.05, ^##P < 0.01 for prednisolone-administered group by t-test.

Figure 3

Mixed results of Juzen-taiho-to, Hochu-ekki-to, Shofu-san, Oren-gedoku-to and prednisolone administration on serum total IgE levels in NC/Nga mice repeatedly treated with mite antigen. Serum samples were obtained 24 h after each antigen application and serum IgE was quantified by enzyme-linked immunosorbent assay. Each value indicates the mean ± SEM of 5–8 mice. ^*P < 0.05, ^**P < 0.01 for Kampo medicine-administered groups by multiple comparison test; ^##P < 0.01 for prednisolone-administered group by t-test.

Figure 4

Application of Juzen-taiho-to, Hochu-ekki-to, Shofu-san, Oren-gedoku-to and prednisolone influence the expression of IL-4 and IFN-γ mRNA in the cervical lymph nodes and ears of NC/Nga mice treated repeatedly with mite antigen. Cervical lymph nodes and ears were obtained 4 h after the fifth antigen application. Expression of mRNA was examined using RT–PCR. Resultant electrophoretic bands were semi-quantitatively evaluated using NIH Image software. Experiments were repeated at least twice and a representative experiment is indicated. Results are expressed as a ratio of (A) IL-4 and (B) IFN-γ to β-actin mRNA expression. Non, no antigen; Cont, control; TJ-48, Juzen-taiho-to; TJ-41, Hochu-ekki-to; TJ-22, Shofu-san; TJ-15, Oren-gedoku-to; Pred, prednisolone; ND, not detected.

Figure 5

Application of Juzen-taiho-to, Hochu-ekki-to, Shofu-san, Oren-gedoku-to and prednisolone initiates histological changes in ear tissues of NC/Nga mice treated repeatedly with mite antigen. Mouse ears were separated 24 h after the fifth antigen application. Tissue sections were stained with hematoxylin–eosin. (A) no antigen; (B) control; (C) Juzen-taiho-to 100 mg/kg; (D) Juzen-taiho-to 300 mg/kg; (E) Hochu-ekki-to 100 mg/kg; (F) Hochu-ekki-to 300 mg/kg; (G) Shofu-san 100 mg/kg; (H) Shofu-san 300 mg/kg; (I) Oren-gedoku-to 100 mg/kg; (J) Oren-gedoku-to 300 mg/kg; (K) prednisolone 3 mg/kg.