Fig. 5. Effects of Seirogan treatment on FITC- or oxazolone-induced CHS. The mice were killed 24 h after the FITC or oxazolone challenge, and colon specimens were frozen, cut into thin sections, treated with anti-IgA antibody and then stained with FITC-conjugated second antibody. The data show one typical experiment involving ten animals. Magnification, 200×. The data are presented as the means ± SD from ten animals. *p<0.05. to CHS by FITC or oxazolone. Microscopic observation of colon samples stained with hematoxylin and eosin revealed infiltration of large numbers of lymphocytes and neutrophils in response to CHS by FITTC or oxazolone (Fig. 4). The infiltration of smaller numbers of lymphocytes and neutrophils was observed in the colons from the Seirogan treated mice. Immunohistological changes in the Seirogan treated mice. The expression of IgA was observed in the colon from Seirogan treated animals. The colonic expression of IgA markedly increased after either the FITC or oxazolone challenges. Fig. 5 shows that the expression of IgA in the CHS groups decreased in the Seirogan treated mice. In addition, there was little expres- sion in the animals that smelled uncoated Seirogan in comparison to those that received the oral administration of coated Seirogan. In addition, all the results (Fig. 1–5) of the sham-stressed mice were equal to those of the control mice. Discussion Delayed-type allergic reactions, such as type IV allergies, are often accompanied by serious inflammation. The present study found that both FITC and oxazolone successfully increased ear and colon thickness at 24 h after challenge. Furthermore, histo- pathological observations of the ear and the colon sections revealed that edema and infiltration of inflammatory cells occurred simultaneously in these mice. This study reported that the overproduction of TNF-α is suppressed by administration of Seirogan. TNF-α is a pleiotropic cytokine, originally identified by its anti-tumor activity,(7) and is 94 believed to play a role in many immunological and inflammatory reactions.(8,9) In addition, blocking TNF-α inhibits allergic skin dermatitis. For example, treatment with an anti-TNF-α antibody can inhibit CHS,(10) arthus reaction(11) and type I allergies.(12) Mediators, such as TNF-α, are induced by acetylcholine, initiate and amplify inflammatory response.(12,13) Seirogan also inhibits spontaneous longitudinal contractions was induced by acetylcho- line.(3,14) It is therefore possible that oral administration of Seirogan can thus improve the inflammatory and allergic condi- tions induced by FITC or oxazolone. The current results demonstrated that allergic inflammation was completely suppressed by inhalation of the smell of Seirogan. This suggested that smell stimulation acted through the hypo- thalamic pituitary adrenal axis (HPA axis) because the plasma level of the ACTH and cortisol increased. The cortisol increases noninflammatory cytokines such as IL-10 or IL-4 and represses inflammatory cytokines such as TNF-α or interferon gamma (IFN-γ).(15,16) Furthermore, the smell of Seirogan affects the auto- nomic nervous system because it activates the HPA axis (stress axis). As a result, the acetylcholine level in plasma decreases,(17,18) and a reduction of TNF-α is induced,(19) because the para- sympathetic system is controlled. The two pathways that use this stress axis may act as the mechanism of the allergic restraint by the smell of Seirogan. Furthermore, when rats were burdened with stress, the excretion of feces aggravated the condition. In this situation of stress-induced feces excretion, corticotropin- releasing factor (CRF) participates as a neurotransmitter in the brain.(20) This CRF promotes the release of the ACTH, and as a result, increases the secretion of cortisol. In this study, it is doi: 10.3164/jcbn.11-82 ©2012 JCBN