Original Article EjtJ01KOchfoC980byreu18.iBnof3grS20n1tNi-onea1605,cal-40iJ8lo/e08acj2Aftcp96ybCrattfniloci.n1rl ei1oFc-ra8el2feBRioatcdhihceaml ieRstersyesanrcmdhNJuapteraitnilonl of Seirogan, a wood creosote, on dermal and intestinal mucosal immunity and allergic inflammation Keiichi Hiramoto,1,2,* Yurika Yamate,3 Hiromi Kobayashi,1 Masamitsu Ishii,1 Takanori Miura,4 Eisuke F. Sato2 and Masayasu Inoue2 1Department of Dermatology and 3Department of Physiology, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abeno, Osaka 545-8585, Japan 2Brain Science Institute, Osaka City University Medical School, 1-4-3 Asahimachi, Abeno, Osaka 545-8585, Japan 4Taikoh Pharmaceutical Co., 3-34-14 Uchihonmachi, Suita, Osaka 564-0032, Japan (Re9ceived 22 June, 2011; Accepted 25 October, 2011; Published online 6 July, 2012) svCSteirdi2TCeirecao0hodtt1egipsvdt2ayhenireusi,gsoaCaehnro,itgwmod©ipionmse2aotnorl0dinw1basc2ucorcterJiAekoCosntssiBto,sraiNtapbrentru,iodctihploeranersdplyirbLsotecirdceiituebnecnudtsut.ieosd,enduwinahdsiaecnrhaytnhpmeaenteremdtriidmiutismasro,ruhfpnetrhtoeiec- drug in Asian countries including Japan for many years. This antidiarrhetic has recently been used as a sugar-coated pill because Seirogan has a strong smell. The strong smell of the uncoated form of Seirogan may modulate the defense systems of animals because the sense of smell is important for the detection of toxic metabolites in foods contaminated with pathogens. This study examined the effect of the sugar-coated and uncoated forms of this antidiarrhetic on the immunological response and inflammatory reactions in mice that had been sensitized with either fluorescein isothiocyanate or oxazolone. The sensitization of mice with either FITC or oxazolone markedly increased the plasma levels of tumor necrosis factor-α and mucosal IgA and elicited severe inflammation in the colon by a mechanism that could be suppressed by exposure of animals to the smell of uncoated Seirogan as effectively as the oral administration of the agent. Dermal inflammation in the FITC- and oxazolone-sensitized mice was also suppressed effectively either by the exposure to the smell or oral administration of the agent. Biochemical and histochemical analyses revealed that the elevated levels of plasma tumor necrosis factor-α and mucosal IgA were significantly decreased by exposure to the smell of uncoated Seirogan as well as by oral administration of the agent. Exposure of mice to the smell of Seirogan but not oral administration of the agent selectively increased plasma levels of adrenocorticotropic hormone and cortisol, particularly in the sensitized animals. These observa- tions suggest that exposing the animals to the smell of Seirogan per se activated the hypothalamo-pituitary-adrenal axis and systemically modulated immunological reactions to suppress the allergic reactions. Key Words: wood creosote, TNF-α, FITC, oxazolone, inflammatory bowel disease Wboth acute and chronic digestive disorders associated with WIonotdrocdreuocstoitoen(Seirogan) has long been used in Asia to treat diarrhea. Seirogan is extracted by distillation from the beechwood tree and is a mixture of simple phenolic compounds, including guaiacol, creosol and related compounds, and is chemically distinct from, and should not be confused with, coal tar creosote. In addition, the binding medicine that wood creosote is a main ingredient is not seen in Southeast Asia and China. Seirogan appears to possess antimotility activity in both the small intestine and colon. Studies in small animals have shown that Seirogan inhibits spontaneous and electrically stimulated contractility in isolated segments of guinea pig small intestine, doi: 10.3164/jcbn.11-82 ©2012 JCBN colon, and rat ileum in smooth muscle baths.(1–3) Seirogan also retards the expulsion of a rectal bead in the rat, suggesting the inhibition of propulsive patterns of colonic motility.(4) Kuge et al.(5) revealed that administration of Seirogan by oral gavage inhibited stress-induced secretion and normalized basal short-circuit current in the jejunum and colon. In addition, peptide hormones such as corticotrophin-releasing factor participate in these suppressive effects.(6) This suggests that Seirogan may modulate the mucosal neuro-immune network. The effect Seirogan on the type IV allergic reactions in the skin and intestine was investigated to test this hypothesis. In addition, the suppres- sive effect of exposure to the smell was examined because Seirogan has unique smell. Materials and Methods Animals. Specific pathogen-free male ICR mice were pur- chased from SLC (Hamamatsu, Japan). The mice were 8 weeks old at the beginning of each experiment and were housed in filter- protected cages. Ambient light was controlled automatically to produce a 12-h light/12-h dark cycle, and sterile water was provided ad libitum. The animals were subjected to experiments according to the animal care regulations of Osaka City University Medical School. Chemicals. 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone) was obtained from Sigma-Aldrich Chemical (Milwaukee, WI), fluorescein isothiocyanate (FITC) isomer-I was supplied by Dojindo (Kumamoto, Japan). Oxazolone solution was dissolved in 1:1 v/v ethanol:distilled water and FITC solution was dissolved in 1:1 v/v acetone:dibutyl phthalate. Solutions were prepared freshly immediately prior to dosing. Contact sensitization. The skin on the back of each mouse was shaved with electric clippers and used as a sensitizing area. The mice were sensitized by applying 50 μl of 0.5% FITC or 0.5% oxazolone solution. The mice were challenged for 5 days after sensitization by applying the FITC or oxazolone solution (4 μl) to both the dorsal and ventral surfaces of the right ear. Ear thickness was measured with a micrometer, and contact hypersensitivity (CHS) was measured as the difference between the ear thickness measured before challenge and that measured 24 h after challenge. Either FITC or oxazolone solution was administered to the colon through the rectum via a 3.5 F catheter equipped with a 1 ml syringe. The catheter was inserted so that the tip was 4 cm proximal *To whom correspondence should be addressed. E-mail: hiramoto@msic.med.osaka-cu.ac.jp J. Clin. Biochem. Nutr. | September 2012 | vol. 51 | no. 2 | 91–95