서 론
, ,
1-3)
ceramide
2008 2 21
2008 3 4
, 814
(Tel:+82-31-961-9085, E-mail:[email protected])
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 .
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.
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)
, 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
.
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
DER EPI
EPI
DER DER
Fig. 1.
Antibody Group
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.
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.
Antibody Group
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.
3.
피부장벽 기능 회복과
PKC억제 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.
Antibody Group
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.
(Fig. 4).
5. NF-kB
활성 억제를 통한
COX-2생성 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.
Antibody Group
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.
,
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 cytokine
4)
,
T Th2 cytokine Th1 Th2 cytokine
5)
.
.
4)
.
2)
ceramide
.
18)
2)
.
6)
,
7)
, ,
8),
9),
19),
20).
21),
20)
,
.
22), ,
, , ,
.
7)
.
8),
19).
9), .
,
6).
11)
16)
.
, .
,
23)
.
21)
“ ”
.
10)
.
NC/Nga ,
.
,
.
lamella body lipid
lamella
24),
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-2
31)
. 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
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
.
3. lipid lamella
ceramide protein kinase C
.
4. IL-4 .
5. NF-κB
COX-2 .
참고문헌
