179

서 론

, ,

1-3) ceramide

2008 2 21

2008 3 4

, 814

(Tel:+82-31-961-9085,

E-mail:heenthsu@duih.org)

淸肌散

구영희, 홍승욱

Original Article

Therapeutic Effects of Cheonggi-san Extract on NC/Nga Mice with Atopic Dermatitis-like Skin Lesions

Young-Hui Ku, Seung-Ug Hong

Dept. of Oriental Medical Ophthalmology, Otolaryngology & Dermatology, College of Oriental Medicine, Dongguk University

Background and Objectives :Atopic dermatitis is a recurrent or chronic eczematous skin disease with severe

pruritus,and has increased in Korea. Although the pathogenic mechanisms of atopic dermatitis are yet unknown,

recently skin barrier dysfunction and hyperresponsive Th2 cells in the acute phase have been reported as important

mechanisms. Cheonggi-san(CGS) is used in oriental clinics for treatingacute skin lesions of eczema or urticaria. There

have been no studies on the therapeutic mechanism of CGS for curing atopic dermatitis. We aimed to find out the

therapeutic effects of its internaluse on atopic dermatitis-like skin lesions, induced in NC/Nga mice by the mite antigen D. pteronyssinus and disrupting skin barrier.

Materials and Methods :The NC/Nga mice were classified into three groups: control group, atopic dermatitis

elicitated group(AD), and CGS treated group (CT). Atopic dermatitis-like skin lesions were induced on the back of

female NC/Nga mice, 12 weeks of age, by tape stripping, 5% SDS applied to disrupt skin barrier and painting 3

times a week with D. pteronyssinus crude extract solution for 3 weeks. CT was treated with CGS orally after atopic

dermatitis was elicitated. We observed changes of skin damage, mast cells, substance P, angiogenesis, skin barrier, Th2 cell differentiation, nuclear factor-κB(NF-κB) p65 activation and COX-2 in NC/Nga mice with atopic

dermatitis-like skin lesions.

Results :The skin damages as eczema were seenin AD, but mitigated in CT. The degranulated mast cells in dermal

papillae increased in AD, but decreased in CT. The substance P positive reacted cells in CT remarkably decreased.

The angiogenesis increased in AD, but decreased in CT. The decrease of lipid deposition and ceramide in AD was

seen, but anincrease of lipid deposition and ceramide in CT was seen.The distribution of IL-4 positive reacted cells in dermal papillae increased in AD, but decreased in CT. The

distribution of NF-κB p65 positive reacted cells & COX-2 positive reacted cells in CT decreased.

Conclusion :The results may suggest that the CGS per os decreases the dysfunction of the skin barrier, inhibits

Th2 cell differentiation and inhibits NF-κB p65 activation in NC/Nga mice with atopic dermatitis-like skin lesions.

Key Words : NC/Nga mice, atopic dermatitis, skin barrier, Th2 cell, Cheonggi-san.

29 1 (2008 3 )J Korean Oriental Med 2008;29(1):179-191

(180) 29 1 (2008 3 )

180

2,4)Th2

2,3,5).

6),

7),

8),

9)

, 10)

.

, , 10).

, , 11)

. 12)

, 13,14)

, 2,3,5)

Th2

.

15)In vitro Th2

EL 4 RAW 264.7

.

Th2

In vivo NC/Nga

mice

.

재료 및 방법

1. 실험 재료

1)

6

NC/Nga 2

Herb Scientific Name Amount (g)

荊芥 Nepetae Herba 4g

防風 Ledebouriellae Radix 4g

人蔘 Ginseng Radix alba 4g

柴胡 Bupleuri Radix 4g

前胡 Anthrisci Radix 4g

羌活 Angelicae Koreanae Radix 4g

獨活 Araliae Cordatae Radix 4g

川芎 Cnidii Rhizoma 4g

赤茯 Hoelen rubra 4g

桔梗 Platycody Radix 4g

枳殼 Auranti Fructus 4g

甘草 Glycyrrhizae Radix 4g

天麻 Gastrodiae Rhizoma 4g

薄荷 Mentae Folium 4g

蟬退 Cicadae Periostracum 4g

生薑 Zingiberis Rhizoma 6g

Total amount 66g

Table 1.

(181)1 :

181

20 g

. (CON ),

(AD ),

(CT ) , 10

.

2) ,

16)

. (Cheonggisan; CGS) 2

500 2

. rotary evaporator

50

CT 3 2.5 / /day

.

2. 실험 방법

1)

4

conventional condition

.

12

cellophane tape(3M, USA) 5

(stratum corneum) (desqumatation)

. (surfactant)

5% sodium dodeecyl sulfate(SDS : Sigma,

USA) 1 20 lipid

lamella Dermatophagoides pteronyss-

inus crude extract solution(10 / , Woongbee

meditech, Korea) 25 3 3

.

2)

D. pteronyssinus crude extract solution

sodium pentobarbital

vascular rinse 10% (neutral

buffered formalin : NBF)

. 10% NBF

24

paraffin 5

. Hematoxyline Eosin

.

ceramide NBF

10% formol-calcium 1 oxi-

dation . 30% sucrose cryo-pro-

tection (Microm, Germany)

10 .

1/2 Karnovsky 1%

OsO4 . Uranyl acetate 90

epon

. ultramicrotome(LKB, Nova, Germany)

60-80

(Hitach H-600, Japan) .

3)

BrdU

.

5 , 3

, 1 5-bromo-2‘

-deoxyuridine(BrdU, Sigma) 50 /

. 4 37 2N HCl

20 DNA-denaturation

. 0.1M borate

0.01%

proteinase K(DAKO) normal goat

serum 1 . 1

mouse anti-mouse BrdU(1:50, Amer- sham, UK)

4 incubation chamber 72

biotinylated goat anti-mouse IgG(DAKO)

10 link . Streptavidin peroxidase

(DAKO) 10 0.05%

3,3’- diaminobenzidine(DAB) 0.01% HCl

0.05M tris-HCl (pH 7.4)

(182) 29 1 (2008 3 )

182

, hematoxylin

.

Luna’s

method .

aldehyde fuchsin 30 .

Weigert’s iron hematoxylin methyl

orange 1 , 5

.

substance P

goat anti mouse substance P(1:250, Santa

Cruz Biotec, USA)

.

4)

x4 .

Optimas 5.2 image sharpen low-

filter binary

morphology invert

intensity 180-200 .

Histogram pixel .

10% NBF Wright’s

stain solution 30 .

IL-8 chemokine

macrophage inflammatory protein(MIP)-2

rabbit anti-mouse MIP

-2(1:100, Santa Cruz Biotec)

.

5) PKC

Sudan black B

,

absolute propylene glycol 10

sudan black B 2 . 85%

propylene glycol 10

nuclear fast red 1

. 3

glycerin jelly

.

26% protein kinase

C (PKC) ceramide

mouse anti-mouse

ceramide(1:100, Sigma)

.

Nuclear factor(NF)-κB

protein kinase

C(PKC) rabbit anti-

mouse PKC(1:250, Santa Cruz Biotec)

.

6) IL-4

IL-4 goat anti-

mouse IL-4(1:250, Santa Cruz Biotec)

.

7) NF-kB COX-2

NF-kB

NF-κB p65(1:250,

Santa Cruz Biotec)

. COX-2

rabbit anti-mouse COX

-2(1:250, Santa Cruz Biotec)

.

8)

Relative intensity

Optimas 5.2 (Optima Co., USA)

(image analysis) .

Sigma plot 2000(Sigma) t

test p<0.05

.

(183)1 :

183

결 과

1. 피부손상에 대한 치료 효과

1)

AD

(eczema) ,

(blood clot)

(Fig. 1- ).

,

(hyperplasia) ,

.

lipid lamella ,

. CT

.

, ,

(Fig. 1- , ).

BrdU , AD

CT BrdU

(Fig. 1- ).

AD 401.7%

CT AD 52.9%

(Table 2).

AD (subcutaneous layer)

,

(degranulated type) .

AD

①

② ④ ⑤ ⑥

③

①

② ④ ⑤ ⑥

③

CT

DEREPI

EPI

DER DER

Fig. 1.

AntibodyGroup

CON AD CT

BrdU 1,145±23 5,745±48 2,659±49*

Substance P 1,495±16 14,091±196 9,124±97*

BrdU : 5-bromo-2‘-deoxyuridine

CON : No-treated group, AD : Atopic dermatitis elicitated group, CT : CGS treated group after atopic dermatitis elicitation

* : P < 0.05 compared with AD.

Table 2.

(184) 29 1 (2008 3 )

184

CT AD

, (Fig. 1- ).

substance P

, AD

CT substance P

(Fig. 1- ). AD

842.5% CT

AD 35.2%

(Table 2).

2. 혈관신생성 억제 효과

AD

, CT AD

(Fig. 2- ).

AD 146.2%

, CT AD 41.9%

(Table 3).

2)

AD

.

CT

AD (Fig. 2- ).

3) Angiogenic chemokine

MIP-2 AD CT

(Fig. 2- ), AD

711.1% CT

AD 44.8%

(Table 3).

① ② ③

① ② ③

EPI

DER

DER

Fig. 2.

AntibodyGroup

CON AD CT

Angiogram 11,321±415 27,873±514 16,178±139*

MIP-2 947±28 7,681±104 4,237±49*

MIP-2 : macrophage inflammatory protein-2

* : P < 0.05 compared with AD.

Table 3.

(185)1 :

185

3. 피부장벽 기능 회복과 PKC 억제 효과

lipid lamella lipid body

AD

, (stratum granulosum)

(stratum corneum) (des-

qumation)

(Fig. 3- ). Sudan black B

lipid blot AD

79.9% CT AD

305.41%

(Table 4).

ceramide AD

76.6% CT

AD 91.1%

(Table 4),

(Fig. 3- ).

PKC

.

AD CT PKC

(Fig. 3- ). AD

230.8% CT AD

37.5%

(Table 4).

4. IL-4 생성 억제 효과

IL-4

. AD

CT IL-4

. AD 522.8%

CT AD 40.2%

① ② ③

① ② ③

EPI EPI

EPI

DER

Fig. 3.

AntibodyGroup

CON AD CT

Sudan black B 5,398±52 1,083±48 4,390±105*

Ceramide 1,578±36 370±9 707±16*

Protein kinase C 341±15 1,128±75 705±12*

* : P < 0.05 compared with AD.

Table 4.

(186) 29 1 (2008 3 )

186

(Fig. 4).

5. NF-kB 활성 억제를 통한 COX-2 생성 억제

효과

NF-κB

p65

, AD CT

NF-κB p65

(Fig. 5- ). AD 489.4%

CT AD 46.5%

(Table 5).

COX-2

, AD CT COX-2

(Fig. 5- ). AD

554.4% CT

AD 63.1%

(Table 5).

고 찰

1995 15.3% 2000 17%17).

18),

, , 1).

AD

CT

DER

① ②

① ②

DER

EPI

DER

EPI

Fig. 4. Fig. 5.

AntibodyGroup

CON AD CT

NF-κB p65 11,321±415 27,873±514 16,178±139*

COX-2 947±28 7,681±104 4,237±49*

NF-κB p65 : nuclear factor-κB p65, COX-2 : cyclooxygenase-2* : P < 0.05 compared with AD.

Table 5.

(187)1 :

187

, 18). , ,

,

.

,

, 1).

5).

·

IL-4, IL-5,

IL-13 mRNA IFN-γ

IL-12 mRNA .

, Ig-E

Langerhans cell Inflammatory dendritic

epidermal cell .

IL-4, IL-13

mRNA IL-5, GM-

CSF, IL-12, IFN-γ mRNA

.

Th2 cytokine4),

T Th2 cytokine

Th1 Th2 cytokine5).

. 4).

2) ceramide

. 18)

2).6),

7), ,

8),

9),

19),

20)

. 21),

20)

,

. 22)

, ,

, , ,

.

7)

. 8)

, 19)

. 9)

,

.

, 6)

.

11)

16)

.

,

.

,

(188) 29 1 (2008 3 )

188

23).

21)“ ”

.

10)

.

NC/Nga

,

.

,

.

lamella body lipid

lamella24),

25). Ceramide,

, , triglyceride, steryl ester

cholesterol sulfate , ceramide

26%26). Ceramaide

27).

NC/Nga .

Chri-

stophers Mrowietz 28)

desqumatation Minehiro 29)

SDS(sodium lauryl

sulfate) .

D.

pteronyssinus

. Sudan black B

ceramide

ceramide

, 30) pruritic erythematous papules,

(subacute skin lesion), ,

, ,

.

angiogenic CXC chemokine

, IL-8

Mip-231). AD Mip-2 .

substance

P ,

, ,

32).

. AD

substance P

.

ceramide sphingosine

, sphingosine protein kinase

C (PKC)33),

ceramide PKC

34)

.

nuclear factor kappa B (NF-κB)

inhibitor IκB

protein (IκBa, IκBβ, IκBγ, IκBε, Bcl3)

.

cytokine (TNF-α, IL-1), bacterial/viral infection

(LPS, dsRNA), stress (ROI, UV, adriamycin,

) IκB protein

(189)1 :

189

NF-κB

target NF-κB binding site (consensus

sequence: 5’-GGGpuNNPyPyCC-3’)

, anti-apoptosis 35).

NF-κB TNF-α, IL-1β proinf-

lammatory cytokine LPS

COX-2 33,36,37)

.

(inducible isoform)

COX-2 mitogen prosta-

gladin

, 38). AD NF-κB p65

, COX-2

.

T cytokine

, IL-4 Th2 cell

IL-4, IL-5, IL-6

. IL-4 Th2 Janus

family tyrosine kinase (Jak) - signal transducers

and activators of transcription (STAT)

ligand

39). Th2 IL-4 IgE

IgE 40). AD

IL-4

, IL-4

.

Th2 cytokine

NC/Nga

.

lipid lamella

.

,

, Substance P ,

. Ceramide

PKC

NF-κB

, COX-2

. IL-4

Th2 .

, IL-4 Th2

, NF-κB

.

결 론

Th2

NF-kB

NC/Nga mite antigen

D. pteronyssinus

, , , IL-4 , NF-κ

B COX-2

.

1. (

, )

,

substance P

.

2.

angiogenic chemokine

(190) 29 1 (2008 3 )

190

.

3. lipid lamella

ceramide

protein kinase C

.

4. IL-4 .

5. NF-κB

COX-2

.

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