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Lab. Anim. Res. 2010: 26(1), 7-13 Anti-inflammatory Effect of Quercetin on Picryl Chloride-induced Contact Dermatitis in BALB/c Mice Hyeong-Jin Kim 1, Jin Kim 1, So-Jung Kim 1, Seung-Ho Lee 1, Young-Seok Park 1, Byung-Kwon Park 1, Byeong-Soo Kim 1, Sang-Ki Kim 1, Sung-Dae Cho 2, Ji-Won Jung 3, Jeong-Seok Nam 4, Changsun Choi 5 and Ji-Youn Jung 1 * 1 Department of Companion and Laboratory Animal Science, Kongju National University, Yesan, Korea 2 Department of Oral Pathology, School of Dentistry, Chonbuk National University, Jeonju, Korea 3 Division of Intractable diseases, Center for Biomedical Sciences, National Institute of Health, Seoul, Korea 4 Laboratory of Tumor Suppressor, Lee Gil Ya Cancer and Diabetes Institute, Gachon University of Medicine and Science, Incheon, Korea 5 Department of Food and Nutrition, College of Human Ecology, Chung-Ang University, Anseong, Korea In this study, we investigated that anti-inflammatory effect of quercetin on picryl chloride(pcl)-induced contact dermatitis in BALB/c Mice. Experimental animals were divided into three groups and comprising five animals. All groups of oral administration was begun on the first day of PCL treatment and ceased on day 5. For the induction of contact dermatitis, BALB/c mice were sensitized with 40 µl of 1.5% picryl choloride (PCL) to the left and right ear, respectively. Ear swelling responses were much weaker in highdose group (100 mg/kg) than control group (0 mg/kg). Total serum IgE levels and histamine levels were measured by sandwich ELISA method using mouse IgE, histamine measuring Kit. Both total serum IgE and histamine levels were significantly decreased in high-dose group (100 mg/kg) than other groups. Degranulation of mast cells were also confirmed by Toluidine Blue (TB) staining method. In high-dose group (100 mg/kg), the number of mast cells were significantly decreased and there are many mast cells were shown degranulation in control group (0 mg/kg). All of these results demonstrate that the pharmacological actions of quercetin indicate their potential activity for allergic inflammatory diseases through the down-regulation of mast cell activation. Key words: Quercetin, picryl chloride, contact dermatitis, IgE, histamine (Received 8 October 2009; Revised version received 5 March 2010; Accepted 10 March 2010) v w w y w ù (Kim et al., 1998), w w ƒ w ƒ ƒ l w w y š»¾ w xk ùkü (Lee et al., 2002),» w»¾ ƒ» ƒ wš y wù f š (Park et al., 1998). *Corresponding author: Ji-Youn Jung, Department of Companion and Laboratory Animal Science, Kongju National University, Yesan Chungnam, 340-702, Korea Tel: +82-41-330-1526 Fax: +82-41-330-1529 E-mail: wangza@kongju.ac.kr v p w allergen w. w v T v s w s x, w 72» x (delayed type hypersensitivity) š (Yin et al., 2008). v w s T v (helper T lymphocytes), s y (activation) (proliferation) w w p s T v (antigen-specific cytotoxic T lymphocytes) w p» y w w p ù. T v y v w v w w w œ s(antigen presenting cell) tvü w s (Langerhans cells) v mw w w ( 600 M.W. w hapten) v 7

8 Hyeong-Jin Kim et al. w z w œ s (Boerrigter et al., 1988; Rees et al., 1990). š w k w w œ s v mw v w naive T v w w (sensitization) j (Cumberbatch et al., 1990), 1 T v { (thoracic duct) mw y ƒ recirculating lymphocyte pool w w ù p v» ew. w z w w» T v (memory T lymphocytes) recirculating lymphocytes pool postcapillary venules mw ü w.» T v ƒ w w p w üv s (endothelial homing receptors) k w. p w» T v t v y v w sù v s (dermal dendritic cell) l w š w 2 w v j š š š. v v ü ù s w w w œ s w ü y(internalization) e z w œ w w v paracortex¾ (migration)w, œ w v ü T v T cell receptor (TCR) w autocrine interleukin 2 (IL-2) w cytokine mw y ù. cytokine w y s T v, w p» T v (TCR + CD4 IL-2R DR T lymphocyte) x mw w (homing)w s k (Baadsgaard et al., 1991; Cooper et al., 1994; Li-weber et al., 2002; Acuto et al., 2003). s ù w w, w, w e, x,» w, immunoglobulin E (IgE) w (Suh et al., 2004). compound 48/80 s O 2 w š š š (Lee et al., 1992), s w y w w w w (Peden and Dailey, 1995). v e v y w k ƒ š ù w e» n w ƒ š š w e v w, p w k š (Tasaka, 1986). Quercetin flavonoid wù flavonol m w, ù, p q (Formical et al., 1995). w s³ 25 mg w (Lamson et al., 1991), t w quercetin flavonoid t š (Park et al., 2006). Quercetin z in vivo in vitro model w ³, y (Cavallini et al., 1978) y (Edenhader et al., 1996), x w, x y (Daniel et al., 2003) š w³z (Kimura et al., 1984). Flavonoid w y y» ƒ (Middleton et al., 1993; Kim et al., 2004), p w flavones/flavonoids C-ring2,3- double bond ƒ ü mitogen-induced lymphoblastosis p T-cell y j, B-cell w š š (Mookerjee et al., 1986; Hirano et al., 1989; Namgoong et al., 1994). quercetin v e w» w v j y w picryl chloride(pcl) female BALB/c mice g j jš, PCL ú l w quercetin 1z 5 5z n w w quercetin v z. lò x 7 f BALB/c (30±5g) p (» û) l w w š, x œ w p w x y» e x w. x x šx ( kg g,» ) gš, t 23±2 o C, 50±10%, y»z 10~12z/hr, 12 (06:00AM~ 18:00PM), 150-300 lux œ w p w x x w. x œ w x z x z ³ w. j Picryl chloride (PCL, Fluka, MO, USA) olive oil

acetone 1:4 (OA) 1.5% w z w, v j» w PCL BALB/c mice ü tv ƒƒ 40 µl g jš, PCL ú l w quercetin 1z 5 5z n w w quercetin v z. x 30 w, PCL z Ì y d w» w IgE histamine ƒ ƒ x w» w ù ƒƒ 5 3 (0 mg/kg), (50 mg/kg), š (100 mg/kg) w x w w. x quercetin (Sigma, MO, USA) n ƒƒ 5% k xk g (0 mg/kg), (50 mg/ kg), š (100 mg/kg) n 0.2 ml/ kg w n w. Anti-inflammatory effect of quercetin 9 š, HRP-avidin solution 50 µl 1 g. chromogenic substrare (TMB) reagent 50 µl 20 k z reaction stopping solution 50 µl wš 450 nm q ELISA reader (Molecular Devices Emax, USA) w Ÿ d w. )JTUBNJOF-FWFM Quercetin n Histamine y d w» w þ ( 70 o C)w x w w Histamine EIA kit (SPI-BIO, Bretonneux, France) d w. polypropylene tube standard x ƒƒ 200 µl š derivatization buffer derivatization reagent 50 µl, 20 µl ƒw vortex mixing w. Antibodyƒ gq well standard serum 100 µl š histamine-ache tracer 100 µl 4 o C 24 g. óù Ellmans reagent 200 µl š w z 1 w ELISA (Molecular Devices Emax, USA) w 405 nm q Ÿ d w. &BSTXFMMJOH 1.5% PCL 40 µl BALB/c mice ü tv g v wš 0, 3, 6, 9, 12, 18, 24 ƒ e r (vernier calipers) w Ì d w Ì y y w. Ì PCL Ì z ƒ Ì sƒw q w. l ƒ še w x» w PCL z Ì y d w PCL z 3 ̃ še w y w. PCL z 3 w, k l x w 1 š k z, (3,000 rpm, 15, 26 o C) w x þ ( 70 o C)w. *H&-FWFM þ ( 70 o C) x d w w w, IgE ELISA Kit (SHIBAYAGI, Gunma, Japan) w IgE d w. Antibodyƒ gq well IgE standard solution sample ƒƒ 50 µl 2 k z biotin-conjugated anti-ige antibody solution 50 µl 2 j j á Quercetin n w mast cell v e w w w PCL z ̃ še w 3 w, w 10% s 7 š wš»(shandon, Tokyo, Japan) k q v en e z, q v s» (Shandon, Tokyo, Japan) s w. 5 µm Ì r w š, Hematoxylin & Eosin (H&E) Toluidine blue (TB) w v Ÿ wx w tv v s k y w. x e Mean±SE t w š, one way ANOVA w P<0.05 w, ùkú Duncan's multiple range test w. &BSTXFMMJOH m PCL v quercetin n ƒ e w» w PCL z quercetin» w (0 mg/kg), (50 mg/kg), š (100 mg/kg) n

10 Hyeong-Jin Kim et al. Figure 1. Ear swelling responses in BALB/c mice at 0, 3, 6, 9, 12, 18, 24 hr following the sensitization with PCL to the ear. Each value was expressed as Mean±SE of 5 BALB/c mice. *Significantly different from 0 mg/kg group at P<0.05. w z Ì y d w (Figure 1). PCL 3 z ƒ ƒ w ùkû, 5 e PCL PCL z Ì ƒs ƒw w š, y, ƒv w w v x ùkû. Quercetin z w x PCL z ̃ É y w, PCL v w ƒ k q. PCL 3 z Ì 24 z ̃ j s. 5 e PCL z 3 Ì 0.124 mm, 0.114 mm, š 0.068 mm š ƒ û Ì (Figure 1). PCL w t z 3 ùkù» wš, 6 z s e, y e v š š (Ikeda et al., 2000; Jung et al., 2004). x Ì y w PCL 3 x w ear swelling ùkû, ƒ ear swelling ƒ ùkù Ì w, 100 mg/kg quercetin n w ear swelling j y. v y quercetin ƒ z q. *H&MFWFM m PCL mw v» Figure 2. Total serum IgE levels in female BALB/c mice at 3 hr after application with PCL. Each value was expressed as Mean±SE of 5 BALB/c mice. *Significantly different from 0 mg/ kg group at P<0.05. quercetin 1z 5 5z 5 n w š, ̃ še x w x ü IgE d w (Figure 2), x ü IgE e (0 mg/kg) 212.59 ng/ml, (50 mg/kg) 196.70 ng/ml, š (100 mg/kg) 110.73 ng/ml quercetin š n w w û IgE e. IgE p w d yw t» š, w zv IgE p w ƒ š š š (Kim et al., 2003). IgE Fc ƒ s w w y w j, IgE y yw Th2 sƒ IL-4 w B v isotype switching w ƒ w (Dekruyff et al., 1989), x ü IgE ùkü w tƒ.» z x n IL-4 w Th2 s w IgE ƒ w wš, y (Park et al., 2007; Park et al., 2008). w quercetin nuclear factor-kappab w cytokine w (Mainardi et al., 2009). w cytokine x (50 mg/kg), š (100 mg/kg) IgE eƒ (0 mg/kg) w û š quercetin IgE w s l cytokine ww y ƒ ùkù.

Anti-inflammatory effect of quercetin 11 Figure 3. Total serum Histamine levels in female BALB/c mice at 3 hr after application with PCL. Each value was expressed as Mean±SE of 5 BALB/c mice. *Significantly different from 0 mg/ kg group at P<0.05. )JTUBNJOFMFWFM PCL mw v w z quercetin 5 5 n wš, Ì ƒ še w x w x ü histamine d w (Figure 3). Histamine e 100%» w quercetin 50 mg/ kg n w 95.69%, 100 mg/kgn w š 90.77% e ùkþ. y y y» x ü w s(effector cell) ƒƒ k major basic protein yw w w (Kim et al., 2001). s compound 48/80 w k w PEM381 w k ƒ š, k w w ùkû (Choi et al., 2001). quercetin n ƒ y IgE eƒ n w w š, s y ww, histamine g z j quercetin w w w q. j PCL mw v w z quercetin 1z 5 5z n wš, ̃ še w w. H&E TB w, wš q v Ÿwx w 200 w (Figure 4) quercetin n w ̃ quercetin š Figure 4. Ear skin of 0 mg/kg group (A, C) and 100 mg/kg quercetin group (B, D) in BALB/c mice at 3 hr after application with PCL. (A) Intradermal edema with prominent inflammatory cell infiltration. H&E, 200. (B) Less severe changes are seen compared with (A). H&E, 200. (C) There are many mast cells including those showing degranulation. TB 200. (D) Mast cells show less degranulation compared with (C). TB 200.

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