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1
서 론
,
35001,2)
.
0.5-2.0% ,
5-10% 13).
4)1, 2
65% ,
12-15% ,
.
,
托裏消毒飮
이상문, 홍승욱
동국대학교 한의과대학 안이비인후피부과학교실
Original Article
The Effects of Taklisodok-eum Extract on Acute Sinusitis-induced mouse
Sang-Moon Lee, Seung-Ug Hong
Dept. of Oriental Medical Ophthalmology, Otolaryngology & Dermatology, College of Oriental Medicine, Dongguk University
Background and Objectives: Sinusitis is a common disease in the otorhinolaryngology field. It is inflammatory change
of the mucous membrane which surrounds the sinus. The aim of this study was to investigate the anti-inflammatory
effects of the Taklisodok-eum (TSE) extract on the mouse model with acute sinusitis induced by S. pneumoniae.
Methods: Thirty six-week-old male BALB/c mice were divided into three groups: the normal group, the group
inoculated with S. pneumoniae which caused allergic rhinitis (control group), and the group treated with the TSE extract after it was treated the same as the control group (sample group).
Results: NF-κB activation was suppressed, and iNOS COX-2 production were inhibited by TSE in acute sinusitis.
Apoptosis was increased by TSE in acute sinusitis. The number of eosinophils in the sample group noticeably
decreased when compared to the control group. In the general morphologic change, the increase of damaged
respiratory ciliated epithelia & eosinophils’ infiltration was lessin the sample group. Goblet cells were maintained in
the sample group. MMP-9, HSP-70 and BrdU decreased in the sample group. Apocrine secretion decreased in the sample group.
Conclusion: The findings in this study show that TSE reduces acute sinusitis through its anti-inflammatory effects,
such as the inhibition of NF-κB activity.
Key Words : Taklisodok-eum, acute sinusitis, anti-inflammatory effects, NF-κB
접수:2008년 6월 4일 수정:2008년 8월 7일 채택:2008년 8월 8일
교신저자 : 홍승욱(Seung-Ug Hong)
경기도 고양시 일산동구 식사동 814번지 동국대학교 일산불교한방병원 안이비인후피부과
Tel : +82-31-961-9085, Fax : +82-31-961-9339, E-mail : [email protected]
29 4 (2008 9 )J Korean Oriental Med 2008;29(4):01-12
29 4 (2008 9 )
2
,
,
, 5,6)
.
,
, , , 7),
.
8,9) 10-12),
9,10,12),
13),
14),
15)
,
.
S. pneumoniae
NF-κB
.
재료 및 방법
1. 실험 재료
1)
6
BALB/C 2
20g
. ,
S. pneumoniae ( ),
S. pneumoniae
( ) , 10
.
2) , MTT assay
(Taklisodok-eum,
TSE)
(Table 1). TSE 2 500
3 .
rotary evaporator 50
2.5 / 5
.
2. 실험 방법
1)
S.
pneumoniae .
ovalbumin(OVA : chicken egg
Herbal Name Scientific Name Dose(g)
金銀花 Lonicerae Flos 12
陳皮 Citri Pericarpium 12
黃 蜜水炒 Astragali Radix 8
天花粉 Trichosanthis Radix 8
防風 Ledebouriellae Radix 4
當歸酒洗 Angelicae gigantis Radix 4
川芎 Cnidii Rhizoma 4
白芷 Angelicae Dahuricae Radix 4
厚朴薑汁炒 Magnoliae Cortex 4
桔梗 Platycodi Radix 4
穿山甲炒成珠 Manitis Squama 4
角刺炒 Gleditsiae Spina 4
Total amount 72
Table 1.
S. Pneumoniae Infection
OVA/Alum ExposureOVA/Alum IP Injection Tissue obtain
Day : -14 -7 -1 1 2 3 4 5 7
Fig. 1.
(584)
1 :
3
albumin, grade V, Sigma, USA)
, PBS Al(OH)3 gel( , Sigma)
1:1 OVA 1 PBS Al(OH)3 gel
1 0.1% OVA
. , , 1,
7, 14 0.1% OVA 0.1 /20g
. 5 0.1% OVA
50
,
.
2 S.
pneumoniae 6×107 colony-forming unit
(naris) . 5
sodium pentobarbital vascular
rinse 10% (neutral buffered
formalin : NBF) .
(decalcification solution, BBC, UK) 24
5 .
Hematoxylin Eosin
(Fig. 1).
2) NF-κB
NF-κB
NF-κB
NF-κB p65(1:250,
Santa Cruz Biotec)
.
iNOS COX-2
mouse anti-mouse iNOS(1:250, Santa
Cruz Biotec) rabbit anti-mouse COX-2(1:250,
Santa Cruz Biotec)
.
NF-κB Apoptosis
in situ apoptosis detection kit(Apoptag,
Intergen, USA) TUNEL(terminal deoxynucleotid
transferase-mediated dUTP-biotin nick-end labelling)
. proteinase K
5 proteolysis equilibration buffer
20 . strength TdT
enzyme(36 TdT enzyme : 72 reaction buffer)
37 humidified chamber 1
strength stop/wash buffer 10
. Anti-digoxigenin-peroxidase 1
DAB . Hematoxylin
.
3) Eosinophil
Eosinophil
, Hinkelmann Neubauer
(Superior, Germany) .
4)
(neutral mucin)
periodic acid-schiff
reaction (PAS) stain periodic
acid 5 Schiff reagent 15
. sulfurous rinse
2 3 Hematoxylin 1
.
MMP-9
Type IV collagen matrix metallopr-
oteinase (MMP)-9 goat
anti mouse MMP-9(1:100, Santa Cruz Biotec, USA)
.
blocking serum 10% normal goat
serum (1:20, DAKO, Denmark) 12
. 1
(585)
29 4 (2008 9 )
4
mouse anti-mouse MMP-9(1:100, Santa
Cruz Biotec) 4 humidified chamber 72
, biotinylated goat anti-mouse
IgG(DAKO) 4 humidified chamber 24
link . Avidin biotin complex(DAKO) 1
0.05% 3,3’-diamin-
obenzidine(DAB) 0.01% HCl 0.05 M
tris-HCl (pH 7.4) , hema-
toxylin
.
(heat shock protein,
HSP) HSP70
rabbit anti-mouse HSP70(1:200, Santa Cruz Biotec)
.
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,
Amersham, UK) 4 incubation chamber
72
.
Apocrine
Apocrine Phloxine-tartr-
azine . Mayer’s hematoxylin
5 phloxine 30
. tartrazine
.
5)
relative intensity
Optimas 5.2(Optima Co.,
USA) (image analysis)
. Sigmaplot 2000(Sigma, USA)
Student t-test , p <
0.05 .
실험 결과
1. NF-B 활성 억제와 염증 효소 생성 억제
1) NF-κB
(1) NF-κB
NF-κB
p65
,
ObjectiveGroup
Normal Control Sample
NF-κB p65 743±25 7204±158 2895±87*
iNOS 649±38 4104±114 1786±61*
COX-2 720±39 4393±129 1522±69*
Apoptosis 1312±59 3129±272 4137±115*
(image analysis for 200,000 particles / range of intensity : 80-120)Normal, No-treated group; Control, Sinusitis elicited group; Sample, TSE-treated group after acute sinusitis elicitation; *, P < 0.05 compared with Control. Abbreviations : NF-κB p65, nuclear factor (NF)-κB p65; iNOS, induce Nitric oxide synthase; COX-2, cyclooxygenase-2.
Table 2. κ
(586)
1 :
5
. NF-κB p65
869.6% ,
59.8%
(Table 2, Fig. 2- ).
(2)
iNOS
,
. iNOS
532.4% ,
56.8%
(Table 2, Fig. 2- ).
Prostaglandin COX-2
,
. COX-2
510.1%
, 65.4%
(Table 2, Fig. 2- ).
(3)
Apoptosis ,
. Apoptosis
138.5% ,
32.2%
(Table 2, Fig. 2- ).
Sample
Control
RM RM RM RM
RM RM RM RM
① ② ③ ④
① ② ③ ④
LP
LP
Fig. 2. κ
κ κ
EO
SIN
OPH
IL (I ul)
2500
2000
1500
1000
500
Normal ControlGROUP
Sample
Fig. 3.
(587)
29 4 (2008 9 )
6
2) Eosinophil
Eosinophil , 2310
±138 / 875±25 / 163.6%
, 1345±77 /
41.8% (Fig. 3).
3)
(1)
(Fig. 4- ).
(2)
(respiratory ciliated epithelium)
(nonciliated epithelial secretory cell)
, Eosinophil
pre-polypoid .
(Fig. 4- ).
(3)
(goblet cell) PAS
ObjectiveGroup
Normal Control Sample
MMP-9 125±60 6863±142 839±57*
HSP70 842±32 7278±104 2518±51*
BrdU 947±29 5849±125 2822±64*
(image analysis for 200,000 particles / range of intensity : 80-120)Normal, No-treated group; Control, Sinusitis elicited group; Sample, TSE-treated group after acute sinusitis elicitation; *, P < 0.05 compared with Control. Abbreviations : MMP-9, matrix metalloproteinase-9; HSP70, heat shock protein70; BrdU, 5-bromo-2'-deoxyuridine.
Table 3.
RM
①
②
③
Sample
Control
①
②
④
③
④
⑤
⑥
⑤
⑥
⑦
⑦
LP
RM
LP
RM
RM
RM
RM
RMRM
LP RM
RM
RM
Fig. 4.
(588)
1 :
7
(Fig. 4- ).
(4) MMP-9
Type IV collagen MMP-9
,
. MMP-9
5390.4% , 87.8
% (Table 3, Fig. 4- ).
(5)
HSP70
,
. HSP70
764.4%
, 65.4%
(Table 3, Fig. 4- ).
(6)
BrdU
,
. BrdU
517.6%
, 51.9%
(Table 3, Fig. 4- ).
(7) Apocrine
(hyperplasia) Apocrine
(Fig. 4- ).
고 찰
,
, , , 16)
.
30-60%17)
, ,
, X-
, CT MRI18)
.
,
.
, ,
,
, ,
, , C 19)
.
, ,
, ,
, , ,
,
, , , , 5).
, 20)
“
” .
,
, , 7).
, 8,9) 10-12)
, 9,10,12)
, 21)
, 8),
8),
9),
9),
10),
11),
11,12) .
,
(589)
29 4 (2008 9 )
8
“ ” “ ”22)
.
. 23)
“
” .
“ ” , “ ” , “ ”
, “ ”
24).
, , ,
, , , , ,
, ,
,
, , , , , , ,
, , ,
,
.
,
, , , ,
, 25,26)
, 27)
, 28,29)
, 22,30)
, 31) 32)
.
in vivo
S. pneumoniae
, NF-κB
.
.
Hilding33)
,
34,35).
36).
33-35,37).
,
Bomer 38)
38-40).
40).
S. pneumoniae
.
1986 B cell immunoglobulin κ light chain
NF-κB
p50 subunit family(p50, p52) p65 subunit family
(p65, c-Rel, RelB) (homodimer)
(heterodimer) ,
(inhibitor) IκB (Iκ
Ba, IκBβ, IκBγ, IκBε, Bcl3)
. cytokine
(590)
1 :
9
(TNF-α, IL-1), / (LPS, dsRNA),
(ROI, UV, adriamycin, )
IκB
NF-κB
NF-κB ( : 5’-GGGpuNNPyPyCC-3’)
, anti-apoptosis 41)
. IκB IκB
IKK(IKKα, β)
IκB (serine residue)
, IκB 26S proteasome
. NF-κB TNF-α, IL
-1β proinflammatory cytokine LPS
iNOS COX-2
42).
NF-κB Eosinophil
cytokine 43)
.
NF-κB p65, iNOS COX-2
.
iNOS COX-2
.
NF-κB NF-κB Antiapoptotic 44)
Apoptosis ,
Apoptosis
,
Apoptotic Eosinophil
.
Eosinophil
,
,
.
,
, .
Eosinophil
.
,
PAS
.
Type IV collagen MMP-9
HSP70
, BrdU
.
Apocrine
.
MMP-9 (undul-
ation) ,
HSP70 ,
Eosinophil
.
NF-κB
.
,
. ,
.
.
, ,
(591)
29 4 (2008 9 )
10
,
.
.
결 론
S. pneu-
moniae
NF-κB
.
1.
NF-κB p65
, iNOS
COX-2
. Apoptosis
.
2. Eosinophil
.
3.
,
, MMP-9, HSP70, BrdU
.
Apocrine .
NF-κB
,
.
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