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Lab. Anim. Res. 2010: 26(1), 47-53 Inhibitory Effect of Luteolin Liposome Solution by Animal Model for Atopic Dermatitis in NC/Nga Mice Moon-Jae Choi 1,2, Young-Moo Lee 2, Byung-Suk Jin 3 and Bae-Hwan Kim 4 * 1 Nabion Corporation, Seoul, Korea 2 Department of Chemical Engineering, Hanyang University, Seoul, Korea 3 Department of Applied Chemistry, Dongduk Women s University, Seoul, Korea 4 Department of Public Health, Keimyung University, Daegu, Korea Atopic dermatitis is a common chronic inflammatory skin disease, associated with marked inflammatory cells (of mast cells and eosinophils) and severe itching, which leads to clinical problems in the pediatric population. This study was designed to investigate the inhibitory effects of luteolin liposome solution, that is entrapped the hydrophobic luteolin (one of the flavonoids) into ethosome to improve its stability, by using hapten-induced atopic dermatitis animal model (NC/Nga mice).the luteolin liposome treated mice showed anti-inflammatory effect as evidenced by the lowering of erythema and edema in clinical observation, reduction of inflammatory cell infiltration and epidermal thickness in histopathological examination, when compared with TNCB induced controls. Luteolin liposome solution also reduced the serum IgE level which played important roles in the atopic dermatitis model. These results suggest that luteolin liposome solution has some merit in this formulation showing inhibitory effects for the atopic dermatitis. Key words: Atopic dermatitis, animal model, TNCB, luteolin liposome (Received 21 January 2010; Revised version received 15 February 2010; Accepted 13 March 2010) mv v w w w w y xü» š, 20%, 1-3% ƒ wš, ƒwš y (Schultz-Larsen and Hanifin, 2002; McGirt and Beck, 2006). w w», w mv v e w w e ƒ š (Furue et al., 2004; Schuller et al., 2004; Wisselink and Willemse, 2009), w w v w» š. w z mv v e w w sƒ ã w š. mv v w NC/ Nga w (spontaneously developed *Corresponding author: Bae-Hwan Kim, Department of Public Health, Keimyung University, 2800 Dalgubeoldaero, Dalseo-Gu, Daegu, 704-701, Korea Tel: +82-53-580-5933 Fax: +82-53-580-5164 E-mail: kim9399@kmu.ac.kr dermatitis) wš, hapten sw š w š (Matsuda et al., 1997; Taniguchi et al., 2003; Yano et al., 2003). NC/Nga mv w w (Shiohara et al., 2004), ûš(50% w), r w» (Matsumoto et al., 1999; Shiohara et al., 2004), w» w hapten mite e e» w mv v x x ùkù w ƒ ü w š (Yun et al., 2008) l (luteolin; 3',4',5,7-tetrahydroxyflavone), w s r w w y, w, w z ƒ š š w w w t, t, y t y w ƒ w š (Chen et al., 2007; Ashokkumar and Sudhandiran, 2008; Kaefer and Milner, 2008). ù l w 47

48 Moon-Jae Choi et al. y w š w y û š w w w» w ethosome w s y w. Ethosome v n k w g carrier, k carrier š x w û š ƒ d v û w w š (Touitou et al., 1998; 2000). x NC/Nga hapten w w mv v, l s v sw mv v yw y w. lò Õ 7 f NC/Nga p (» û) l w, 7 k x w. x y 23±3 o C, 40-60%,» 12 w, x (u ùg ) ( ³ ) w œ w. x y 3R x z(institutional Animal Care and Use Committee, IACUC) e wš d š w ww. l FUIPTPNF Ethosome HPC (hydrogenated phosphatidyl choline) l (Wilshire Tech) 9:1 yw 1g k 1mL wì v j z š 60 o C ƒ w z, k k 1mL š 2 j y (hydrated liquid crystal) x g ( y ). z» k ƒw rv 60 ml 5mL/min w ƒw y ù j» carrier xk w ethosome. w 10 ƒ ww z v j bath type sonicator 10 q ƒw z 1 j þƒ g. l 7 z,» x 5 4 ù. x, x n ú w, 3 mv v šw (Taniguchi et al., 2003), 5% TNCB (1-Chloro-2,4,6-trinitrobenzene, Aldrich) 150 µl 1z sw z, 2 l 1% TNCB 1z 5 mv v w. TNCB (1%) sw z, T1 l s, T2 l PEG400 0.1% ƒ 150 µl 5z 2 v sw, TNCB NC( ) w. ²n x» 1z w. x w» w, e ú eg w 30 z w z w. w w w grooming w (Carstens and Kuraishi, 2004), w y ² i w (facial scratching) wš w mv v w (hindlimb scratching) w z d w. j TNCB e x v d e Tewameter (TM300, CK electronic GmbH, Germany) w TEWL (transepidermal water loss) d w, Corneometer (CM825, CK electronic GmbH, Germany) w w d w w. j j á x n w v v (, x,, y, ƒvkv ) ¾ w š, v w 10% neutral formalin š wš k k z paraffin s w r w. r H&E w tv v v w, tv Ì w. w toluidine blue mw s k w. l *H& )JTUBNJOF Th2 cytokine w B cell w x ü IgE level (Renz et al., 1994), s rw sƒw mv v x q w w tƒ d w. x ü IgE w Enzyme-Linked Immunosorbent (ELISA) Assay w x ƒ isoflurane w

Atopic inhibitory effect of Luteolin liposome 49 w z l x wš» 3,000 rpm 10 w x w, 20 o C w w. IgE d sandwich ELISA w w (SHIBAYAGI ELISA kit, Japan). m SPSS 17.0 for windows (SPSS Inc., USA) w one-way ANOVA w š ƒ w» w Duncan's multiple range test w z w š, m P<0.05 w. ²n x» x» w y y w (data not shown). v z (NC) w TNCB ƒ ƒ š(p<0.05), x e w w (Table 1). j TEWL e x e T1 (Luteolin Liposome solution) T2 (Luteolin) e (0 week) w 2 z (P<0.01), TNCB w (P<0.01) z w j e q, x d TNCB w x e T1 T2 w ù w w (Table 2). Corneometer w w d, T1 (Luteolin Liposome solution) T2 (Luteolin) (NC) w û (P<0.05), TNCB w (Table 3). j j á e TNCB y, ƒvkv w x ¾ mv v w v ùkü š, T1 (Luteolin Liposome solution) z, T2 (Luteolin) û (Figure 1). t tv Ì(epidermal hyperplasia, hyperkeratosis) TNCB w ƒƒ š, T1 (Luteolin Liposome solution) T2 (Luteolin) TNCB w v ü š. w se TNCB w T1 T2 z (Table 4, Figure 2). v ü s(mast cell) TNCB (NC) w ƒƒ š, TNCB w x e T1 (Luteolin Liposome solution) T2 (Luteolin) Table 1. The frequency of scratching in NC/Nga mice 0 0.8±0.8 a 6.0±2.6 b 5.2±2.6 b 5.2±2.2 b 2 0.6±0.5 a 4.4±2.1 b 2.6±1.5 ab 2.4±1.7 ab Unit: times in 30 minutes NC: Normal control, TNCB: TNCB induced control, T1: Luteolin liposome solution, T2: Luteolin Values with different superscripts (a,b) in the same row are significantly different (P<0.05) by ANOVA and Duncan s multiple range test. Table 2. Changes inf transepidermal water loss (TEWL) of NC/Nga mice 0 12.08±2.93 a 75.04±13.89 b 80.28±15.58 b 76.44±27.05 b 2 12.34±3.78 a 41.68±12.78 b 38.44±20.27 b 0 38.90±22.51 ab Unit: g/m²/h Values with different superscripts (a,b) in the same row are significantly different (P<0.05) by ANOVA and Duncan s multiple range test. Table 3. Changes in water capacity of NC/Nga mice by Corneometer 0 37.74±25.79 b 16.86±2.93 a 14.01±7.27 a 16.74±5.04 a 2 30.85±4.32 b 0 019.06±11.22 a 19.86±4.37 a 20.23±7.19 a Unit: Arbitrary unit Values with different superscripts (a,b) in the same row are significantly different (P<0.05) by ANOVA and Duncan's multiple range test.

50 Moon-Jae Choi et al. Figure 1. Gross lesions of NC/Nga mouse skin. A: Normal control, B: TNCB induced control, C: Luteolin liposome solution, D: Luteolin. ƒ. k w (Table 5, Figure 3). l *H& x mv v t w t x ü IgE d w TNCB w m v ƒƒ, x e T1 (Luteolin Liposome solution) T2 (Luteolin) x ü IgE level TNCB w (Table 6). mv v w z y sƒw» w sƒ kw w. z sƒ mv v in vivo x» x ³ w w, x w. p š NC/Nga v x, NC/Nga hapten e w j mv v x ƒÿ š (Shiohara et al., 2004). mv v e z sƒ ƒ hapten w z sƒw. hapten DNCB (2,4-dinitrochlorobenzene) (Lee et al., 2006)ù TNCB (Taniguchi et al., 2003)ƒ š, sƒ TNCB w. hapten v v w, n z, š n Th1 w v w ù mv v p Th2» w ƒ š (Plitnick et al., 2002; Taniguchi et al., 2003; Goutet et al., 2005; Cumberbatch et al., 2005). Table 4. Epidermal thickness of NC/Nga mice 2 27.54±6.02 a 105.63±43.37 c 34.87±10.91 ab 52.74±17.76 b Unit: µm Epidermal thickness was determined by the average of three sites (short, medium, long-size each) in same fields of microscope. Values with different superscripts (a,b,c) in the same row are significantly different (P<0.05) by ANOVA and Duncan s multiple range test.

Atopic inhibitory effect of Luteolin liposome 51 Figure 2. Histological observation of NC/Nga mouse skin by H&E staining ( 100) (red bar=50 µm). A: Normal control, B: TNCB induced control, C: Luteolin liposome solution, D: Luteolin. mv v hapten n, z, Th1 y Th2 e e š š (Mueller et al., 1989; Goutet et al., 2005; Friedman, 2006), TNCB n z» y w mv w w š w. mv v v v y w, w epidermal hyperplasia v d ü s e š, s v ƒ y w. p» (Renz et al., 1994) w mv ƒ w t B s w x IgE e ƒ Th2 w x w mv v z sƒ ww x š w. mv v ƒ x IgE e Table 5. Number of mast cells and ratio of degranulation by Toluidine blue staining x (Luteolin Liposome solution Luteolin) s w w. mv ƒ x ü IgE v ü s(mast cell) k g histamine w w (Falus Merétey, 1992; Malaviya et al., 1996; Menzies-Gow et al., 2002; Ying et al., 1999) x IgE e k» wš. ƒ w vü s w» q. x tv (TEWL) v ü w d w, v sƒw t (Elias and Menon, 1991; Hruza and Pentland, 1993). x ƒ w TNCB w NC TNCB T1 T2 Total No of mast cell 79.2±11.5 a 176.6±17.8 c 134.0±26.1 b 125.3±37.8 b Mean No of degranulation.59±8.6 a 0.117±12.9 b 101.6±22.9 b 00.94±28.4 b Deganulation/total No. 74.5 66.1 75.8 75.2 The number of mast cells was determined by the average of five different fields with an eyepiece of 100 magnification. Values with different superscripts (a,b,c) in the same row are significantly different (P<0.05) by ANOVA and Duncan s multiple range test.

52 Moon-Jae Choi et al. Figure 3. Histological observation of NC/Nga mouse skin by Toluidine blue staining ( 100) (red bar=50 µm). A: Normal control, B: TNCB induced control, C: Luteolin liposome solution, D: Luteolin. There are many mast cells (arrow) in TNCB control (B). w ù j. l (luteolin) w w y, w, w ƒ s r (Chen et al., 2007; Ashokkumar and Sudhandiran, 2008; Kaefer and Milner, 2008) mv e z» w q š û x ethosome w s y mw z w. x s y l» l mv v t x ü IgE w w w š, t { œ q. x Luteolin Liposome solution TNCB mv TNCB w t v w w w v q š, p x ü IgE level v vü s w mv v z ƒ. k Ashokkumar, P. and Sudhandiran, G. (2008) Protective role of luteolin on the status of lipid peroxidation and antioxidant defense against azoxymethane-induced experimental colon carcinogenesis. Biomed. & Pharmacotherapy 62, 590-597. Carstens, E. and Kuraishi, Y. (2004) Animal models of itch: scratching away at the problem. In Itch Basic Mechanisms and Therapy (Yosipovitch, G. ed.), pp.35-50, Informa Healthcare, USA. Chen, C.Y., Peng, W.H., Tsai, K.D. and Hsu, S.L. (2007) Luteolin suppresses inflammation-associated gene expression by blocking NF-κB and AP-1 activation pathway in mouse alveolar macrophages. Life Sci. 81, 1602Æ1614. Cumberbatch, M., Clelland, K., Dearman, R.J. and Kimber, I. (2005) Impact of cutaneous IL-10 on resident epidermal Langerhans' cells and the development of polarized immune responses. J. Immunol. 175(1), 43-50. Elias, P.M. and Menon, G.X. (1991) Structural and lipid biochemical correlation of the epidermal permeability barrier. Table 6. Plasma IgE levels of NC/Nga mice 2 734.0±364.6 a 4,303.3±523.0 c 3101.1±780.5 b 2,503.2±1,041.5 b Unit: ng/ml Values with different superscripts (a,b,c) in the same row are significantly different (P<0.05) by ANOVA and Duncan s multiple range test.

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