The Effects of Taklisodok-eum Extract on Acute Sinusitis-induced mouse

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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

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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

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 Exposure

OVA/Alum IP Injection Tissue obtain

Day : -14 -7 -1 1 2 3 4 5 7

Fig. 1.

albumin, grade V, Sigma, USA)

, PBS Al(OH)

gel( , Sigma)

1:1 OVA 1 PBS Al(OH)

gel

1 0.1% OVA

. , , 1,

7, 14 0.1% OVA 0.1 /20g

. 5 0.1% OVA

50

, .

2 S.

pneumoniae 6×10

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

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

,

Objective Group

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

. 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

EOSINOPHIL (I ul)

2500

2000

1500

1000

500

Normal Control GROUP

Sample

Fig. 3.

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

Objective Group

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 RM RM

LP RM

RM

RM

Fig. 4.

(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- ).

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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

(TNF-α, IL-1), / (LPS, dsRNA), (ROI, UV, adriamycin, )

IκB NF-κB

NF-κB ( : 5’-GGGpuNNPyPyCC-3’)

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IKK(IKKα, β)

IκB (serine residue)

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NF-κB Eosinophil

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NF-κB p65, iNOS COX-2

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Apoptosis ,

Apoptosis ,

Apoptotic Eosinophil .

Eosinophil ,

, .

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Eosinophil

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PAS

.

Type IV collagen MMP-9

HSP70

, BrdU

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Apocrine

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MMP-9 (undul-

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HSP70 ,

Eosinophil .

NF-κB

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결 론

S. pneu- moniae

NF-κB

. 1.

NF-κB p65

, iNOS

COX-2

. Apoptosis

. 2. Eosinophil

. 3.

,

, MMP-9, HSP70, BrdU

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Apocrine .

NF-κB

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