10

26 3 (2005 9 ) J Korean Oriental Med 2005;26(3):110-123 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 264.7 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 264.7. 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 264.7. 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 : 2005 5 31 : 2005 7 1 : 2004 8 10 :. (130-702) 1 (Tel: 02-958-9172, Fax: 02-958-9171, Email: aokop@hanmail.net ) 1963 Gell Coombs 20~30 1,2,3,4) 20% 5,6,7), 110

(569) 8,9,10)., 4 11). 1 anaphylaxis type IgE,,., Th1 Th2, IgE cytokine,, cytokine chemokine 12). 13-20).. 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 264.7 cell 50, 100, 250, 500, 1000 /. 111

(570) 26 3 (2005 9 ) 2) Lipopolysaccharide(LPS) Hydrocortisone Sigma (St. Louis, MO, U.S.A.). 3) Macrophage 264.7 cell dulbecco s minimum eagle s medium (DMEM, 10% fetal bovine serum (FBS), penicillin; 100 U/ml, streptomycin; 100 U/ml), macrophage 264.7 cell 24 well plate 2 10 5 /well 5% CO2, 37. 1) macrophage 264.7 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 264.7 cell 2 10 5 24 well plate overnight incubation, medium. 1 LPS (100 ng/ml) stimulation. TNF-, IL-6 12, IL-1 IL-10 24 medium 2,000 rpm, 4 10. 3) 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<0.01. 5 (50, 100, 250, 500, 1000 / ) TNF-. TNF- 112

(571) Table 1. Effects of Jacho on LPS-induced TNF- in Macrophages 264.7 Treatment of cells TNF- (pg/ml)(mean S.E) Normal 272.05 5.72 LPS 2278.33 52.47 LPS + hydrocortisone (10-8 M) 2208.00 114.24 LPS + hydrocortisone (10-7 M) 1763.00 106.67** LPS + hydrocortisone (10-6 M) 1559.33 84.99** LPS + hydrocortisone (10-5 M) 1616.33 55.03** LPS + hydrocortisone (10-4 M) 1269.50 101.43** LPS + Jacho (50 / ) 1928.83 48.94** LPS + Jacho (100 / ) 1952.33 67.61** LPS + Jacho (250 / ) 1743.20 225.73* LPS + Jacho (500 / ) 1982.17 82.48* LPS + Jacho (1000 / ) 1861.17 51.83** Fig. 1. Effects of Jacho on TNF- by LPS-stimulated in macrophages 264.7 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, 1000. 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-6 10-7 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

(572) 26 3 (2005 9 ) Table 2. Effects of Jacho on LPS-induced IL-6 in Macrophages 264.7 Treatment of cells IL-6 (pg/ml) Normal 8.14 1.45 LPS 97.16 2.09 LPS + hydrocortisone (10-8 M) 113.73 2.48++ LPS + hydrocortisone (10-7 M) 90.77 3.25 LPS + hydrocortisone (10-6 M) 74.88 0.47** LPS + hydrocortisone (10-5 M) 81.34 2.23** LPS + hydrocortisone (10-4 M) 40.34 1.53** LPS + Jacho (50 / ) 82.13 3.29** LPS + Jacho (100 / ) 89.41 2.80* LPS + Jacho (250 / ) 69.85 8.30** LPS + Jacho (500 / ) 77.96 1.69** LPS + Jacho (1000 / ) 58.60 1.62** Fig. 2. Effects of Jacho on IL-6 by LPS-stimulated in macrophages 264.7 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, 1000. 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-1. 50 / 114

(573) Table 3. Effects of Jacho on LPS-induced IL-1 in Macrophages 264.7 Treatment of cells IL-1 (pg/ml) Normal 1.91 0.11 LPS 4.07 0.21 LPS + hydrocortisone (10-8 M) 3.31 0.09** LPS + hydrocortisone (10-7 M) 2.27 0.19** LPS + hydrocortisone (10-6 M) 1.98 0.15** LPS + hydrocortisone (10-5 M) 2.09 0.04** LPS + hydrocortisone (10-4 M) 1.63 0.10** LPS + Jacho (50 / ) 7.74 0.24++ LPS + Jacho (100 / ) 6.25 0.30++ LPS + Jacho (250 / ) 6.17 0.13++ LPS + Jacho (500 / ) 6.07 0.23++ LPS + Jacho (1000 / ) 5.99 0.20++ Fig. 3. Effects of Jacho on IL-1 by LPS-stimulated in macrophages 264.7 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, 1000. 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. 1000 /. 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

(574) 26 3 (2005 9 ) Table 4. Effects of Jacho on LPS-induced IL-10 in Macrophages 264.7 Treatment of cells IL-10 (pg/ml) Normal 339.87 13.43 LPS 902.27 64.36 LPS + hydrocortisone (10-8 M) 797.43 46.94 LPS + hydrocortisone (10-7 M) 591.37 18.31** LPS + hydrocortisone (10-6 M) 471.45 8.16** LPS + hydrocortisone (10-5 M) 546.62 12.66** LPS + hydrocortisone (10-4 M) 370.83 3.85** LPS + Jacho(50 / ) 719.38 36.26* LPS + Jacho(100 / ) 579.25 28.77** LPS + Jacho(250 / ) 601.85 39.26** LPS + Jacho(500 / ) 557.72 17.31** LPS + Jacho(1000 / ) 535.20 30.67** Fig. 4. Effects of Jacho on IL-10 by LPS-stimulated in macrophages 264.7. 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, 1000. 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), 2000 24%, 13% 35,10). 36,11,37,38). 10, 38)., 4 11). 1 anaphylaxis type IgE,,, 1 116

(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). 13-20).,.,,,,, 23).,,, 23).,, 23).,,,,, 23). 23).,,, 23).,,, Staphylococcus aureus,, 25). Shikonin, Acetylshikonin, Alkannan, Isobutyrylshikonin, - Dimethyl acryloylshikonin, -Hydrxy isvaloryl shikonin, Teracryl shikonin 24). shikonin 117

(576) 26 3 (2005 9 ) 28,29).,,. Celsus (rubor), (tumor), (calor), (dolor) 4.. 40).,,,. 1965 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

(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-. 250 /. 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-1. 50 / 1000 /.. Hydrocortisone hydrocortisone. IL-1.. IL-1. macrophage LPS 24 IL-1, 24 119

(578) 26 3 (2005 9 ) 12 36. 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-10. 55) 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 264.7 cell. 1. TNF-. 2. IL-6. 3. IL-1. 4. IL-10.. 1. Rothe MJ, Grant-kels JM. Atopic dermatitis: an update. J Am Acad Dermatol. 1996;35:1-13. 2. Ruzika T. Ring J, Przybilla B. Handbook of atopic eczema. Heidelberg Germany: Springer verlag. 1991. 3. Wuthrich B. Clinical aspects, epidemiology, and prognosis of atopic dermatitis. Ann Allergy Asthma Immunol. 1999;83:464-470. 4. Barnetson RSC, Rogers M. Childhood atopic eczema. BMJ. 2002;324:1376-1379. 5. Kyu Han Kim, Kyong Chan Park. Clinical 120

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