Effect of Whitmania pigra whitman on the Allergic Inflammatory Response

서 론

( , Whitmania pigra WHITMAN,

WP) ( ; Hirudividae)

Whitmania pigra WHITMAN ( ) Hirudo niponica WHITMAN

Whitmania acranulata WHITMAN

Effect of Whitmania pigra whitman on the Allergic Inflammatory Response

Ju Yong Lee ¹ , Eun Kyoung Kim ¹ , Hyun A Oh ¹ , Hyun-Sam Lee ¹ , Youngjoo Sohn ³ , Hyuk-Sang Jung ¹ , Yoon-Bum Kim ¹ , Seong Kyu Park ¹ , Nak-Won Sohn ²

1

College of Oriental Medicine, Institute of Oriental Medicine, Kyung Hee University,

2

Graduate School of East-West Medicine, Kyung Hee University,

3

College of Oriental Medicine, Sangji University

Objectives: Whitmania pigra whitman (WP) has been used in herbal medicine to treat various conditions, such as eczema, skin burns and frostbites in herbal medicine. The purpose of this study is was to investigate the effect of WP on anti-allergy mechanism.

Methods: To clarify the mechanism, the effect of WP on vascular permeability of rat cutaneous tissue and histamine and cytokines (IL-6, IL-8, TNF-α) released from mast cells were observed.

Results: The results were

1. the pretreatment with WP significantly decreased the compound 48/80-induced degranulation and histamine release from RPMC

2. WP did not inhibit the anti-DNP IgE-induced increment of vascular permeability of rat cutaneous tissue 3. WP significantly reduced the PMA plus A23187-induced increment of expression of IL-6, IL-8, and TNF-α in

HMC-1 cells.

Conclusions: The present study providesevidence that WP acid inhibits mast cell-derived inflammatory allergic reactions by blocking histamine release and pro-inflammatory cytokine expression, and suggest the mechanisms of action. Furthermore, in vivo and in vitro anti-allergic effect of WP suggests a possible therapeutic application of this agent in inflammatory allergic diseases.

Key Words : Whitmania pigra whitman , HMC-1 cells, histamine, passive cutaneous anaphylaxis (PCA), cytokine

2008 3 4 2008 5 13

: , 283

(Tel:+82-33-741-9303, Fax:+82-33-732-2124, E-mail:[email protected])

- (20060401)

.

This work (research) was supported by a grant from KyungHee-Pacific Beauty & Health Research Center, Republic of Korea.

水蛭

이주용 ¹ , 김은경 ¹ , 오현아 ¹ , 이현삼 ¹ , 손영주 ³ , 정혁상 ¹ , 김윤범 ¹ , 박성규 ¹ , 손낙원 ²

Original Article

,

, ,

, , ¹⁾ .

hirudin ,

gelatin

. hirudin

histamin

heparin .

2) , ³⁾ ,

4) ,

, ,

5-10)

.

11-16)

. (allergy)

17) . (type

hypersensitivity) allergen

, IgE

(mast cell)

18) . ,

, TNF-α, IL-1, IL-4,

IL-6, IL-8, IL-13

19) .

compound 48/

80 ²⁰⁾

2 cAMP

cGMP

, ²¹⁾ .

,

.

IgE

(passive cutaneous anaphylaxis,

PCA) ²²⁾ .

comp-

ound 48/80 (Rat

peritoneal mast cell)

. Human Mast Cell line (HMC-1 Cell) PMA plus A23187

IL-6, IL-8, TNF-α

.

재료 및 방법

1.

실험동물

( ) 200-

300 g Sprague-Dawley (SD) .

, 21-23 , 40-60%

, 12

.

2.

약물 1)

( : WP) 5-6 ,

, ,

( , )

.

300 g 3 L 1,500 ml

80% ethanol 2

. vacuum rotary

evaporator 10

g . in vitro

(WP) saline (5, 10, 50, 100,

500 1000 /ml) ,

in vivo 100 g

3.33 mg 1 ml .

2)

HEPES-Tyrode (10 mM HEPES, 136 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 2.75 mM MgCl 2 , 5.6 mM glucose, 11 mM NaHCO 3 , 0.6 mM NaH 2 PO 4 , 1.0 /ml bovine serum albumine

3 pH 7.4 ), comp-

ound 48/80 (5 /ml : Sigma Chemical Co., St.

Louis Mo), anti-DNP IgE (0.5 ug[50 ul] ; Sigma Chemical Co., St. Louis Mo), DNP-human serum albumine (HSA) (100 ug[100 ul] ; Sigma Chemical Co., St. Louis Mo), saline, Iscove's Modified Dulbecco's Medium (IMDM) (Grand Island, NY, USA), PMA, Calcium ionophore A23187 (Sigma Chemical Co., St. Louis Mo), 3-[4,5-dimetylthizol -2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma Chemical Co., St. Louis Mo), avidin-HRP, Tetramethylbenzidine (TMB) substrate (PharMi- ngen, Sandiego, CA, USA), Anti-humam TNF-α antibody (Ab), biotinylated anti-human TNF-α Ab, recombinant human TNF-α (R&D systems, Minneapolis, MN, USA), Anti-human IL-8 Ab, biotinylated anti-human IL-8, recombinant human IL-8 (PharMingen, Sandiego, CA, USA), Anti- human IL-6 Ab, biotinylated anti-human IL-6, recombinant human IL-6 (PharMingen, Sandiego, CA, USA)

3. in vivo study

1) PCA

PCA Kim ²³⁾ ,

. anti-DNP IgE PCA

pentothal sodium (

, )

anti-DNP IgE 0.5

(50 ) . 48 h DNP-

Human serum albumin 100 (100 ) 4% Evans blue (1:4)

. DNP-HSA (challenge) 1 h WP 3.33 mg/100 g 1 ml D.W

.

4% Evans blue 30

.

3-4 mm 5 ml formamide

40 oven 24 h (overnight) Evans

blue . Evans blue

density spectrophotometer (backman instruments Inc, DU530, U.S.A.) 620 nm

Evans blue .

4. in vitro study

1) SD histamine

(1) SD-Rat

24)

.

10 ml HEPES-Tyrode buffer (

) 90

.

800 rpm 10 min

, HEPES-Tyrode 1×10 ⁶ cells/ml

.

(2) SD-Rat

200 HEPES-Tyrode

25 WP 25 37

10 . WP comp-

ound 48/80

WP 10

compound 48/80 25

20 .

(3) SD-Rat

200 50

(slide glass, 22×60 mm)

5 . 1,000

(Olympus,

Japan) .

.

,

5 , .

(%) = (

/ ) × 100

(4) SD-Rat histamine

1×10 ⁶ cells/ml

200 .

histamine

200 HEPES-Tyrode 25

, compound 48/80

histamine HEPES-

Tyrode 25 10

compound 48/80 ( : 0.5 /ml) 25 . WP

histamine

WP ( : 100, 500 1000 /ml) 25 10

HEPES-Tyrode 25

. WP compound 48/80 histamine

WP 25 10 compound 48/80 (0.5

/ml) 25 .

4 3000 rpm

histamine enzyme immunoassay for histamine kit (Oxford, USA)

.

2) Human mast cell MTS assay cytokine

(1)

Human mast cell line (HMC-1 Cell) 10%

fetal bovine serum (FBS), 100 U/ml penicillin, 100 /ml streptomycin Iscove's Modified

Dulbecco's Medium (IMDM) CO2

incubator (5% CO2, 37 )

2 3 .

(2) MTS assay

cell proliferation assay kit (Promega, Madison, WI, U.S.A.)

MTS assay , ²⁵⁾

. HMC-1 Cells (1×10 ⁵ cells/100 ) 96 well plate CO 2 incubator (5%

CO2, 37 ) 1 WP

(5, 10, 50, 100, 500 1000 /ml) 24 . well MTS solution 20

37 2 .

formazan

ELISA reader (Molecular Device, Sunnyvale, CA, U.S.A.) wave length 490 nm

. (3) Cytokine assay

HMC-1 Cells (3×10 ⁵ cells/ml) 24 well plate CO2 incubator (5% CO2, 37 ) 30 min WP (5, 10, 50, 100,

500 1000 /ml) 1 .

25 nM PMA plus 1 μM A23187 7 stimulation 3000 rpm, 4 , 5 min

. TNF-α, IL-6, IL-8 sandwich enzyme-linked immunosorbent

assay . 96-well ELISA

plate (Nunc, Denmark) anti-human TNF- α, IL-6, IL-8 monoclonal antibody 100

coating (TNF-α : RT, overnight, IL-6, IL-8 : 4 , overnight). Well plate 0.05%

Tween-20 PBS washing 1%

BSA, 5% sucrose, 0.05% NaN3 PBS

200 1 blocking .

WP sample TNF-α, IL-6, IL-8 standards 100

37 2

. Well plate PBS washing TNF- α, IL-6, IL-8 biotinylated detection Ab

100 37 2

. Well plate washing avidin-perox-

idase 37 30 min

washing tetramethylbenzidine (TMB)

substrate 30 min

. Stop solution (2N H2SO4)

ELISA reader wave

length 450 nm O.D .

5.

통계처리

SPSS® for windows (version 10.0, SPSS, Inc., Chicago, U.S.A.)

oneway ANOVA ,

P value<0.05

post hoc test Scheffe's test .

실험성적

1.

비만세포의 형태와 탈과립 관찰

1) HEPES-Tyrode

, . 10 20

( ) 2

.

(Fig. 1A).

, 94.0%

6.0%

(Table 1, Fig. 1).

2) WP

WP (100, 500 1000 ug/ml)

, ,

, 30

(Fig. 1B, 1C, 1D).

6.0%, 10.0%, 9.8% (Table 1).

3) Compound 48/80

compound 48/80 (0.5 ug/ml)

, .

A

B

C

D

E

F

G

H Fig. 1.

Treatment

Type of mast cell (%)

Inhibition of degranulation (%)

WP ( ㎍/ml) Com 48/80 Normal degranulation

- - 94.0 6.0

100 - 94.0 6.0

500 - 90.0 10.0

1000 - 90.2 9.8

- + 2.0 98.0

100 + 10.0 90.0 10.0%

500 + 88.0 12.0 88.0%

1000 + 86.0 14.0 86.0%

a) Two hundred microliter of rat peritoneal mast cell (RPMC) suspension (10

cells/ml) was treated with 25 ㎕ of WP (100, 500 or 1000

㎍/ml) or HEPES-Tyrode solution at 37℃ for 10 min. 25 ㎕ of compound 48/80 (Com 48/80) solution (0.5 ㎍/ml) or HEPES-Tyrode solution was added into RPMC suspension pretreated either with WP or HEPES-Tyrode solution at 37℃ for 20 min.

b) Counted number (%) of RPMC in 10 Fields (×400). Mast cell degranulation (%) = (Degranulation mast cell / Total mast cell)×100 c) Inhibition of degranulation (%) = [1-(Com 48/80-induced degranulation of mast cells pretreated with WP / Com 48/80-induced degranulation of RPMC)]×100

Table 1.

(Fig. 1E).

98.0%

(Table 1).

4) WP compound 48/80

WP (100, 500

1000 ug/ml) 10 compound

48/80 (0.5 ug/ml) ,

,

. 20

, compound 48/80

(Fig. 1F, 1G). WP (100

ug/ml) Fig. 1E

(Fig. 1H).

WP compound 48/

80 WP

100 ug/ml 90.0%, 500 ug/ml 12.0%, 1000 ug/ml 14.0%

(Table 1). WP compound

48/80

.

2.

비만세포로터 유리된

양의 측정결과 compound 48/80 ( : 0.5 ug/ml)

histamine 10.08±0.36

, WP

compound 48/80

histamine 8.29±0.85 , 0.88±0.05 , 1.13±0.08 . compound 48/

80 histamine

WP

H is ta m in e re le as e (u g/ m l)

12.0

10.0

8.0

6.0

4.0

2.0

0.0

Normal Control 100 500 1000

Compound 48/80 (0.5㎍/ml) (WP ㎍g/ml)

*** ***

Fig. 2.

WP 500 1000 /ml histamine

(Fig. 2).

3. WP

전처리 후

피부반응 억제 효과

PCA WP

( )

Evans blue .

anti-DNP IgE

0.5 (50 ul) 48 h

DNP-HSA 100 (100 ul) control Evans blue WP patch 183.22

±14.95 mm, 1.60±0.07 g

Evans blue 9024.58±467.02 /ml (Table 2). , DNP-HSA 100 (100 ul)

1h WP Evans

blue WP patch 160.21±11.07 mm,

3.20±0.10 g Evans blue

7952.26±585.55 /ml control ,

, (Table 2, Fig. 3).

4. HMC-1 Cells

에 대한

의 세포독성 측정 결과

WP HMC-1 Cell

WP 5, 10, 50, 100, 500, 1000

/ml 97.49±1.00, 93.78

±2.73, 93.49±2.61, 91.83±1.49, 89.08±3.51, 42.44

±5.79 (%) 5, 10, 50, 100 /ml , 500, 1000 /ml

Anti-DNP IgE ( ㎍)

Amount of dye ( ㎍/ml)

Area/min (mm)

Weight (g)

Inhibition (%)

None 330.24 ± 42.91 0.40 ± 0.00

Control 9024.58 ± 467.02 183.22 ± 14.95 1.60 ± 0.07 WP (3.33mg/100g) 7952.26 ± 585.55 160.21 ± 11.07 3.20 ± 0.10

a) Anti-DNP IgE (0.5 ㎍) was injected into dorsal skin sites. After 48 h, WP (3.33 mg/100g) was administered orally 1 h prior to the challenge with antigen (DNP-HSA).

b) Each amount of dye is presented as the mean±S.E. three independent experiments.

c) The WPtch of evans blue was measured 30 min after antigen (DNP-HSA) injection.

d) Weight of evans blue WPtch was measured 30 min after antigen (DNP-HSA 100ug) injection. *** p<0.001 compared with the control group (Anti-DNP IgE plus DNP-HSA).

Table 2.

A B C

Fig. 3.

. (Table 3).

5. IL-6

분비에 미치는

의 효과 PMA plus A23187 stimulation IL-6 0.82±0.04 ng/ml 0.03±0.00 ng/ml

. WP (5, 10, 50, 100 /ml) PMA plus A23187 stimulation

WP 5, 10, 50,

100 /ml 0.39±0.07 ng/ml, 0.37±0.04 ng/ml, 0.30±0.04 ng/ml, 0.25±0.03 PMA plus A23187 HMC-1 Cells IL-6

WP (P<0.001)

(Fig. 4).

6. TNF-

α 분비에 미치는

의 효과 PMA plus A23187 stimulation TNF-α 7.05±0.26 ng/ml

IL - 6 se cre tio n (n g/ m l)

0.9

Normal Control 5

PMA (25nM) plus A23187 (uM) (WP ㎍g/ml)

***

***

0.8 0.7 0.6 0.5 0.4 0.2 0.1 0.0

***

***

10 50 100

Fig. 4.

μ

Group Dose

( ㎍/ml)

Percent of Control Data (%)a) WP

Control 100 ± 1.5

WP

5 97.49 ± 1.00

10 93.78 ± 2.73

50 93.49 ± 2.61

100 91.83 ± 1.49

500 89.08 ± 3.51

1000 42.44 ± 5.79

a) The HMC-1 Cells (1×10

cells/ml) viability was evaluated using MTS colorimetric assay for 8 h incubation after pretreated with various concentration (1, 10, 50, 100, 500, 1000 ㎍/ml) of WP. Data compared with the untreated control. (% of control). Data represent the mean±S.E. (n=4).

Table 3.

0.44±0.13 ng/ml

. WP (5, 10, 50, 100 /ml) PMA plus A23187 stimulation

WP 5, 10,

50, 100 /ml 3.53±0.42 ng/ml, 3.08

±0.25 ng/ml, 2.93±0.13 ng/ml, 2.33±0.20 ng/ml PMA plus A23187 HMC-1 Cells

TNF-α WP

(P<0.001) (Fig. 5).

7. IL-8

분비에 미치는

의 효과 PMA plus A23187 stimulation IL-8 54.10±1.55 ng/ml

1.00±0.13 ng/ml

IL - 8 se cre tio n (n g/ m l)

60

Normal Control 5

PMA (25nM) plus A23187 (uM) (WP ㎍g/ml)

*** *** ***

***

10 50 100

50

40 30

20

10

0

Fig. 6.

TN F- a se cre tio n (n g/ m l)

8

Normal Control 5

PMA (25nM) plus A23187 (uM) (WP ㎍g/ml)

***

***

7 6 5 4 3 2 1 0

***

***

10 50 100

Fig. 5. α

μ

. WP (5, 10, 50, 100 /ml) PMA plus A23187 stimulation

WP 5, 10, 50,

100 /ml 26.06±1.06 ng/ml, 26.43±

1.87 ng/ml, 26.12±0.95 ng/ml, 24.188±0.34 ng/ml PMA plus A23187 HMC-1 Cells

IL-8 WP

( P<0.001) (Fig. 6).

고 찰

(Hirudo) ²⁶⁾

, , , , , ,

. , , ,

, .

, , , ,

, , ,

. ( ; Hirudividae)

Whitmania pigra WHITMAN ( ) Hirudo niponica WHITMAN Whitmania acranulata WHITMAN

. Hirudo niponica

WHITMAN ,

Whitmania pigra WHITMAN ,

.

27) .

, tessulin 1998 Theromyzon tessulatum

28) . Tessulin, therin antistasin-type lipopolysacaride

, tessulin, therin

therostasin ,

.

(allergy) (type I hyper sensitivity)

, IgE (mast cell)

. (Allergen)

, IgE ,

IgE Fc (FcεRI)

IgE . allergen

allergen IgE (cross link) ,

, Ca2+ channel ,

Ca2+ ,

prostaglandin leukotrien

, cAMP

. cAMP

. ,

(histamine) ,

,

29) .

Ehrlich ³⁰⁾ ,

,

.

(

, , , )

cytokine .

, , ,

.

Ca2+

Ca2+ ,

Ca2+

, ,

31) .

compound 48/80 ,

compound 48/80

, , .

, cAMP-phosphodiesterase

cAMP

32-33)

. PMA (phorbol 12-myristate-13-acetate) calcium ionophore A23187 phosphatidylin ositol cycle protein Kinase C (PKC) ,

34) . , compound 48/80

. , , compound 48/80

. compound 48/80

, compound

48/80

(competitive inhibition) ,

. Cytokine

,

. 1965 Gordon

blastogenic factor 150

, .

cytokine

cytokine TNF-α, IL-1, IL-4, IL-6, IL-8, IL-13 ^34-35) .

(5, 10, 50,

100 /ml) ,

(500, 1000 /ml) 5, 10, 50, 100 /m PMA plus A23187

IL-6, TNF-α, IL-8 .

IL-6

cytokine ,

T , , B , ,

, acute phase reaction, immune response, nerve cell function

, ,

36) . IL-6 IL-1, PDGF TNF-α

, ³⁷⁾ .

HMC-1 Cell PMA plus A23187

stimulation control IL-6

, PMA plus

A23187 IL-6

. TNF-α

cytokine , inflamm antory function ,

38) . TNF-α

ELAM -1

,

39) . TNF-α HMC-1 Cell

PMA plus A23187 stimulation control

TNF-α ,

PMA plus A23187 TNF-α

. IL-8

(chemotactic cytokine) , 2

(secondary mediator) (neut-

rophils, macrophage, T lymphocytes) ,

(chemokines) , IL-8

(neutrophils, macrophage, T lympho cytes)

39-40)

. HMC-1 Cell

PMA plus A23187 stimulation control

IL-8 ,

PMA plus A23187 IL-8 .

compound 48/80 PMA plus A23187

.

결 론

cytokine (IL-6, IL-8, TNF-α)

. , Compound 48/80 ,

. anti-IgE

. HMC-1 Cell PMA plus A23187

IL-6, IL-8, TNF-α

. compound

48/80 PMA plus A23187

.

.

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