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1 26 3 ( ) J Korean Oriental Med 2005;26(3): Anti-allergic and Anti-inflammatory Effects of Jacho (Lithospermum Erythrorhizon) Mi-Hwa Kwon, Jin-Yong Lee, Deog-Gon Kim Dept. of Pediatrics College of Oriental Medicine, Kyung Hee University, Seoul, Korea Objectives : This study was performed to examine the anti-allergic and anti-inflammatory effects of Jacho (Lithospermum erythrorhizon). Methods : Macrophage cells were pretreated for 1 hour with Jacho. After pretreatment, macrophages were incubated with lipopolysaccharide (LPS) 100ng/ml for 12 h (TNF-, IL-6) or 24 h (IL-1, IL-10) and media collected and TNF-, IL-6, IL-1, and IL-10 concentrations in supernatants were each measured by enzyme-linked immunosorbent assay. Concentrations of Jacho used were 50, 100, 250, 500, and 1000, and hydrocortisones used were 10-8, 10-7, 10-6, 10-5, and 10-4 M. Results : Jacho showed inhibitory effect on TNF- by LPS-stimulated macrophage The inhibitory effect was most significant in 250, and was not in a dose-dependent manner as in the hydrocortisone group. Jacho also showed inhibitory effect on IL-6 by LPS-stimulated macrophage The inhibitory effect was most significant in 1000, and increased in a roughly dose-dependent manner. Jacho and hydrocortisone showed contrary effect on IL-1. Jacho obviously increased the expression of IL-1 in all five concentrations, and at the lowest concentration (50 ) the level of IL-1 was highest. On the other hand, hydrocortisone was observed to have inhibitory effect on IL-1 in all five concentrations. IL-10 was obviously inhibited by Jacho and hydrocortisone respectively in a roughly dose-dependent manner. Conclusions : By the findings of this experiment, Jacho was observed to have anti-allergic and anti-inflammatory effects through inhibiting pro-inflammatory cytokine TNF- and IL-6, and might be one of the effective therapeutic regimens for allergic diseases. Key Words: Jacho, Lithospermum erythrorhizon, allergic disease, atopic dermatitis, inflammatory cytokine, TNF-, IL-6, IL-1 IL-10 : : : :. ( ) 1 (Tel: , Fax: , aokop@hanmail.net ) 1963 Gell Coombs 20~30 1,2,3,4) 20% 5,6,7), 110

2 (569) 8,9,10)., 4 11). 1 anaphylaxis type IgE,,., Th1 Th2, IgE cytokine,, cytokine chemokine 12) ).. Lithospermi Radix Borraginacese Lithospermum erythrorhizon Sieb. et Zucc..,,,,,,, 21,22,23)., 24,25,13,26,27), shikonin, 28,29) 27,30). 31,32,33). 13,14,15,19). Vanisree Staniforth 26) Shikonin in vivo tumor necrosis factor- (TNF- ). 13) B.. in vitro macrophage cell lipopolysaccharaid(lps, 100 ng/ml) TNF-, IL-6, IL-1, IL-10 enzyme-linked immuno-sorbent assay (ELISA),. 1). 250 g 3 L 2,500 ml 2, extract 24.7 g. macrophage cell 50, 100, 250, 500, 1000 /. 111

3 (570) 26 3 ( ) 2) Lipopolysaccharide(LPS) Hydrocortisone Sigma (St. Louis, MO, U.S.A.). 3) Macrophage cell dulbecco s minimum eagle s medium (DMEM, 10% fetal bovine serum (FBS), penicillin; 100 U/ml, streptomycin; 100 U/ml), macrophage cell 24 well plate /well 5% CO2, 37. 1) macrophage cell, LPS(100 ng/ml) stimulation, hydrocortisone LPS stimulation hydrocortisone, LPS stimulation. 50, 100, 250, 500, 1,000 /, hydrocortisone 10-8, 10-7, 10-6, 10-5, 10-4 M. 2) Macrophage cell well plate overnight incubation, medium. 1 LPS (100 ng/ml) stimulation. TNF-, IL-6 12, IL-1 IL medium 2,000 rpm, ) Cytokine TNF-, IL-6, IL-10 IL-1 enzyme-linked immuno-sorbent assay (ELISA). Plate (Nunc Maxisorp) capture antibody 25, overnight coating, plate washing buffer washing. 1% BSA, 5% sucrose, 0.05% NaN3 phosphate buffered saline(pbs) blocking, sample (sample 0.1% BSA, 0.05% Tween 20 PBS ). Plate washing detection Ab plate washing, streptavidin-horseradish peroxidase 20 washing. Tetramethylbenzidine (TMB) substrate 20 stop solution (2N H2SO4), 450nm O.D. mean S.E. Student s t-test p<0.05. Cytokine hydrocortisone. TNF-, IL-6, IL-10 IL-1, hydrocortisone TNF-, IL-6, IL-10, IL-1. Macrophage cell LPS (100 ng/ml) proinflammatory cytokine TNF- ELISA (Table )(Fig 1). Hydrocortisone hydrocortisone TNF-. Hydrocortisone 10-8 M, 10-7, 10-6, 10-5, 10-4 M p< (50, 100, 250, 500, 1000 / ) TNF-. TNF- 112

4 (571) Table 1. Effects of Jacho on LPS-induced TNF- in Macrophages Treatment of cells TNF- (pg/ml)(mean S.E) Normal LPS LPS + hydrocortisone (10-8 M) LPS + hydrocortisone (10-7 M) ** LPS + hydrocortisone (10-6 M) ** LPS + hydrocortisone (10-5 M) ** LPS + hydrocortisone (10-4 M) ** LPS + Jacho (50 / ) ** LPS + Jacho (100 / ) ** LPS + Jacho (250 / ) * LPS + Jacho (500 / ) * LPS + Jacho (1000 / ) ** Fig. 1. Effects of Jacho on TNF- by LPS-stimulated in macrophages Cells were pretreated for 1 hour with drug. At the end of pretreatment, macrophage were incubated with LPS 100 for 12 h and media collected and analyzed as described under material and methods. Drugs were used 50, 100, 250, 500, Hydrocortisones were used 10-8, 10-7, 10-6, 10-5, 10-4 M. Data are presented as means standard error. *p<0.05 and ** p<0.01 indicate statistically significant differences from the LPS-treated group., 250 /. TNF- hydrocortisone 10-8 M 10-7 M. 2. IL-6 Macrophage cell LPS (100 ng/ml) proinflammatory cytokine IL-6 ELISA (Table )(Fig 2). Hydrocortisone 10-8 M IL M 10-6, 10-5, 10-4 M (p<0.01). Hydrocortisone hydrocortisone 10-4 M. 5 (50, 100, 250, 500, 1000 / ) IL-6 113

5 (572) 26 3 ( ) Table 2. Effects of Jacho on LPS-induced IL-6 in Macrophages Treatment of cells IL-6 (pg/ml) Normal LPS LPS + hydrocortisone (10-8 M) LPS + hydrocortisone (10-7 M) LPS + hydrocortisone (10-6 M) ** LPS + hydrocortisone (10-5 M) ** LPS + hydrocortisone (10-4 M) ** LPS + Jacho (50 / ) ** LPS + Jacho (100 / ) * LPS + Jacho (250 / ) ** LPS + Jacho (500 / ) ** LPS + Jacho (1000 / ) ** Fig. 2. Effects of Jacho on IL-6 by LPS-stimulated in macrophages Cells were pretreated for 1 hour with drug. At the end of pretreatment, macrophage were incubated with LPS 100 for 12 h and media collected and analyzed as described under material and methods. Drugs were used 50, 100, 250, 500, Hydrocortisones(+) were used 10-8, 10-7, 10-6, 10-5, 10-4 M. Data are presented as means standard error. *p<0.05, ** p<0.01 and ++ p<0.01 indicate statistically significant differences from the LPS-treated group. (p<0.01). 50 / 1000 /. 3. IL-1 Macrophage cell LPS (100 ng/ml) proinflammatory cytokine IL-1 ELISA (Table )(Fig 3). Hydrocortisone IL-1. Hydrocortisone hydrocortisone 5 (10-8, 10-7, 10-6, 10-5, 10-4 M) IL-1 (p<0.01), 5 (50, 100, 250, 500, 1000 / ) IL-1 (p<0.01). IL / 114

6 (573) Table 3. Effects of Jacho on LPS-induced IL-1 in Macrophages Treatment of cells IL-1 (pg/ml) Normal LPS LPS + hydrocortisone (10-8 M) ** LPS + hydrocortisone (10-7 M) ** LPS + hydrocortisone (10-6 M) ** LPS + hydrocortisone (10-5 M) ** LPS + hydrocortisone (10-4 M) ** LPS + Jacho (50 / ) LPS + Jacho (100 / ) LPS + Jacho (250 / ) LPS + Jacho (500 / ) LPS + Jacho (1000 / ) Fig. 3. Effects of Jacho on IL-1 by LPS-stimulated in macrophages Cells were pretreated for 1 hour with drug. At the end of pretreatment, macrophage were incubated with LPS 100 for 24 h and media collected and analyzed as described under material and methods. Drugs were used 50, 100, 250, 500, Hydrocortisones(+) were used 10-8, 10-7, 10-6, 10-5, 10-4 M. Data are presented as means standard error. ** p<0.01 and ++ p<0.01 indicate statistically significant differences from the LPS-treated group /. 4. IL-10 Macrophage cell LPS (100 ng/ml) antiinflammatory cytokine IL-10 ELISA (Table )(Fig 4). Hydrocortisone hydrocortisone 10-8 M 4 (10-7, 10-6, 10-5, 10-4 M) IL-10 (p<0.01).. 5 (50, 100, 250, 500, 1000 / ) IL-10 (p<0.01). 115

7 (574) 26 3 ( ) Table 4. Effects of Jacho on LPS-induced IL-10 in Macrophages Treatment of cells IL-10 (pg/ml) Normal LPS LPS + hydrocortisone (10-8 M) LPS + hydrocortisone (10-7 M) ** LPS + hydrocortisone (10-6 M) ** LPS + hydrocortisone (10-5 M) ** LPS + hydrocortisone (10-4 M) ** LPS + Jacho(50 / ) * LPS + Jacho(100 / ) ** LPS + Jacho(250 / ) ** LPS + Jacho(500 / ) ** LPS + Jacho(1000 / ) ** Fig. 4. Effects of Jacho on IL-10 by LPS-stimulated in macrophages Cells were pretreated for 1 hour with drug. At the end of pretreatment, macrophage were incubated with LPS 100 for 24 h and media collected and analyzed as described under material and methods. Drugs were used 50, 100, 250, 500, Hydrocortisones were used 10-8, 10-7, 10-6, 10-5, 10-4 M. Data are presented as means standard error. *p<0.05 and ** p<0.01 indicate statistically significant differences from the LPS-treated group., 34), %, 13% 35,10). 36,11,37,38). 10, 38)., 4 11). 1 anaphylaxis type IgE,,, 1 116

8 (575) IgE. IgE,.., interleukins(il)-1,3,4,5,6,10,13,16 TNF-, granulocyte macrophage colony stimulating factor, transforming growth factor-, platelet derived growth factor, nerve growth factor, macrophage chemotactic protein-1, macrophage inhibitor protein-1, lymphotactin.,,,,,,.,,,. 39), Th1 Th2, IgE cytokine,, cytokine chemokine 12). IgE Th2 cytokine target 39) ).,.,,,,, 23).,,, 23).,, 23).,,,,, 23). 23).,,, 23).,,, Staphylococcus aureus,, 25). Shikonin, Acetylshikonin, Alkannan, Isobutyrylshikonin, - Dimethyl acryloylshikonin, -Hydrxy isvaloryl shikonin, Teracryl shikonin 24). shikonin 117

9 (576) 26 3 ( ) 28,29).,,. Celsus (rubor), (tumor), (calor), (dolor) ).,,, Gordon blastogenic factor 150, 40). TNF- IL-6, IL-1. PGE2, CSF (acute-phase protein) 40). histamine 41)... hydrocortisone. Hydrocortisone.,,. 42,43). 5 (50, 100, 250, 500, 1000 / ) hydrocortisone 5 (10-8, 10-7, 10-6, 10-5, 10-4 M)., CD4 T 1 T (Th1) 2 T (Th2) 44), Th1 interferon-gamma (IFN- ), tumor necrosis factor- (TNF- ), interleukin-2 (IL-2) 45), Th2 IL-4, IL-5, IL-10, IL-13 46). IL-4 Th1 IFN- Th2. T IL-4, IL-5 IFN- Th2 39). Th2 Th1 Th2 47). TNF- IL-6, IL-1 TNF-, Th2 Th0 118

10 (577) 48). TNF- endocrine hormone, (endogenous pyrogen), (hepatocyte) (acute phase reactant protein) (lymphopenia) (immunodeficiency),. TNF-,, mononuclear phagocytes 49). T B cell co-stimulator, (apoptosis). LPS TNF-,. (septicemia) LPS TNF- (DIC, systemic Shwartzman reaction) 49). TNF- 5 TNF /. TNF- hydrocortisone 10-8 M 10-7 M. TNF-. IL-6 T,, B,,. B, 40). IL-6 hydrocortisone, IL-6,. IL-6 B. Hydrocortisone IL-1. IL-1 lymphocyte activating factor CD4 T IFN-. IL-1 I 49,50). Hydrocortisone hydrocortisone 5 (10-8, 10-7, 10-6, 10-5, 10-4 M) IL-1, 5 (50, 100, 250, 500, 1000 / ) IL-1. IL / 1000 /.. Hydrocortisone hydrocortisone. IL-1.. IL-1. macrophage LPS 24 IL-1,

11 (578) 26 3 ( ) IL-10 Th1 (cytokine synthesis inhibitory factor: CSIF) 51), Katsikis 52) IL-10 Th2 Th1. IL-10 T, B, B (human major histocompatibility complex, MHC) class II, MHC class II T. IL-10 IL-1, TNF-, IL-6, IL-8, IL-12 53), T INF- IL-2 54). IL ) 56,48,57) IL-10, IL-10 mrna 55) 58,55-59). IL-10 Th1 IL-10 55,60). 5 (50, 100, 250, 500, 1000 / ) IL-10.,, TNF- IL-6. IL-10 TNF- IL-6. hydrocortisone hydrocortisone., in vivo., macrophage cell. 1. TNF-. 2. IL IL IL Rothe MJ, Grant-kels JM. Atopic dermatitis: an update. J Am Acad Dermatol. 1996;35: Ruzika T. Ring J, Przybilla B. Handbook of atopic eczema. Heidelberg Germany: Springer verlag Wuthrich B. Clinical aspects, epidemiology, and prognosis of atopic dermatitis. Ann Allergy Asthma Immunol. 1999;83: Barnetson RSC, Rogers M. Childhood atopic eczema. BMJ. 2002;324: Kyu Han Kim, Kyong Chan Park. Clinical 120

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ÀÇÇÐ°�ÁÂc00Ì»óÀÏ˘ Common Allergic Diseases in Children Sang - Il Lee, M.D. Department of pediatrics Sungkyunkwan University School of Medicine, Samsung Medical Center E - mail : silee@smc.samsung.co.kr Abstract Allergy