Effects of Haengso-tang and Chwiyeon-tang on Expression of Respiratory Mucin Gene and Secretion of Airway Mucus

서 론

(mucus)

1) .

杏蘇湯 取淵湯

강원제, 서운교

Original Article

Effects of Haengso-tang and Chwiyeon-tang on Expression of Respiratory Mucin Gene and Secretion of Airway Mucus

Won-Je Kang, Un-Kyo Seo

Dept. of Oriental Internal Medicine, College of Oriental Medicine, Dongguk University

Objectives: In this study, the author investigated whether Haengso-tang (HST) and Chwiyeon-tang (CHT) affect both in vitro mucin secretion and MUC5AC gene expression in airway epithelial cells and in vivo mucin secretion from animal model for airway mucus hypersecretion.

Materials and Methods: Confluent HTSE cells (non-labeled) were chased for 30 min in the presence of HST and CHT to assess the effects of the agents on mucin secretion by enzyme-linked immunosorbent assay (ELISA), with removal of oriental herbal medicine extract from each agent-treated sample by centrifuge microfilter. Also, the effects of the agents on TNF- or EGF-induced MUC5AC gene expression in human airway epithelial cells (NCI-H292) were investigated. The author also induced hypersecretion of airway mucus by exposure of rats to SO2 for 3 weeks. Effects of orally-administered HST and CHT during 1 week on in vivo mucin secretion from tracheal goblet cells of rats were assessed using ELISA.

Results:

(1) HST significantly decreased in vitro mucin secretion from cultured HTSE cells. However, CHT did not affect in vitro mucin secretion from HTSE cells;

(2) CHT significantly inhibited the expression levels of EGF- or TNF-alpha-induced MUC5AC gene in NCI-H292 cells. However, HST did not affect the expression levels of EGF- or TNF-alpha-induced MUC5AC gene in NCI-H292 cells;

(3) CHT significantly inhibited hypersecretion of in vivo mucin. However, HST did not affect hypersecretion of in vivo mucin.

Conclusion: These results suggest that CHT can not only affect the secretion of mucin but also the expression of the mucin gene and could be helpful for treating pulmonary disease caused by secretion of mucin.

Key Words : HTSE, NCL-H292, MUC5AC, Haengso-tang , Chwiyeon-tang

2008 4 18 2008 6 30

: , 3 87-2

2

(Tel : +82-31-710-3751, Fax : +82-31-710-3780, E-mail : [email protected])

2007

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(viscoelasticity)

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12-13)

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실 험

1.

재료 1)

NCI-H292 American Type Culture Coll-

ection (Manassas, VA, USA) .

2)

8-10 Golden Syrian 6

SD rat ( ) (Gyeoggi

-do, Korea)

.

3)

, ¹¹⁾ , ¹³⁾ Table 1, 2

. 800 1,000

2 100

3 , 80

.

, 0.22μm filter

, 4 .

4)

Epidermal growth factor (EGF), tumor necrosis

factor-alpha (TNF-alpha), trypsin-EDTA, ethidium

bromide, HEPES, dimethyl sulfoxide (DMSO),

diethyl-pyrocarbonate (DEPC), Pronase (Type XIV),

insulin, transferrin, hydrocortisone, sodium selenite,

Tween 20, bovine serum albumin (BSA), testi-

cular hyaluronidase (Type VI-S), retinoic acid,

gentamicin, sodium dodecyl sulfate, Sepharose CL -4B, 3,3',5,5'-tetramethyl-benzidine peroxide solution (TMB) Sigma (St. Louis, MO, USA) , penicillin-G, streptomycin, Joklik-modified Minimal Essential Medium (S-MEM), Dulbecco's Modified Eagle's Medium (DME), fetal bovine serum (FBS), Medium 199 (M199), RPMI 1640 GIBCO-BRL

(Grand Island, NY, USA) , [6-3H] glucosamine (39.2 Ci/mmol) Amersham (Piscataway, NJ, USA) , mouse anti-human total mucin clone 17Q2 Covance (Berkley, CA, USA) , mouse

anti-MUC5AC clone 45M1 HRP-Goat Anti- Mouse IgG Conjugate NeoMarkers (Freemont, CA, USA) , type I collagen Regenmed (Seoul, Korea) , Easy-Blue RNA extraction kit INTRON biotechnology (Gyeonggi-do, Korea)

, Accuprep RT premix kit Accuprep PCR premix kit Bioneer (Daejeon, Korea) ,

sodium acetate reagent

grade ,

Lee ^17-18)

2 .

Constituent herbs Scientific name Dose(g)

杏仁 Rehmanniae Radix Preparat 4.00

紫蘇葉 Raphani Semen 4.00

桑白皮 Mori Cortex 4.00

陳皮 Citri Pericarpium 4.00

半夏 Pinelliae Rhizoma 4.00

貝母 Fritillaria cirrhosa D. Don 4.00

白朮 Atractylodis Macrocephalae Rhizoma 4.00

五味子 Schizandrae Fructus 4.00

甘草 Glycyrrhizae Radix 2.00

生薑 Zingiber officinale Rosc 5.00

Total Amount 39.00

Table 1.

Constituent herbs Scientific name Dose(g)

當歸 Angelica sinensis (Oliv.) Diels 120.00

玄蔘 Scrophularia ningpoensis Hemsl 40.00

辛夷 Magnolia Liliflora Desr 8.00

柴胡 Bupleurum chinense DC 4.00

梔子炒 Gardenia Jasminoides Ellis 12.00

貝母 Fritillaria cirrhosa D. Don 4.00

Total Amount 188.00

Table 2.

2.

방법 1)

(HTSE)

16-23)

. 8 10

,

. Ca ²⁺ , Mg ²⁺ -free

Minimum Essential Medium (S-MEM)

0.1% pronase , 4

16 . 16

, 10% fetal bovine serum

S-MEM 200×g

2 . cell

pellets insulin (5μg/ ), transferrin (5μg/ ), epid- ermal growth factor (12.5ng/ ), hydrocortisone (0.1μM), sodium selenite (0.01μM), fetal bovine serum (5%, V/V) (FBS), retinoic acid (0.1μM), penicillin G (100U/ ), streptomycin (100μg/ ), gentamicin (50μg/ ) Dulbecco's Modified Eagle's Medium (DME) M199 1:1

. Type I collagen collagen gel (0.15 / ² ) 24-well plate 10 ⁴ cells/ ²

.

, 95% , 5% CO 2 , 37 3

2 .

1, 3, 5, 7 .

2) HTSE

HTSE well 200

24 (pretre-

atment sample, PT) .

well 0.5 PBS 2

,

10 120 PBS 200 well

32 30 . 30

, treat- ment

sample (T) . sample

, T sample

(Centrifuge microfilter,

Centricon) mucin

-70 .

3)

Shao ²⁴⁾ , Song ²⁵⁾

ELISA . ,

PT T sample sample PBS

1/10 , sample ELISA

96-well plate 100 ,

2 . 2 PBS-

Tween 20 (PBS-T, 0.05%) 200 /well

, well 3 . 2%

BSA in PBS-T 200 well ,

1 . 1 PBS-T

200 3 , total mucin

monoclonal antibody mouse antihuman total mucin clone 17Q2 2% BSA 1:1,000

well 100 , 1

. 1 PBS -T 3

, 2 HRP-Goat Anti- Mouse IgG Conjugate 2% BSA 1:5,000

well 100 1

. PBS-T 3 3,3',5,5'-tetra- methyl-benzidine peroxide solution (TMB) 100

well , 5 1N H 2 SO 4

50 , . 450nm

well

, .

4) NCI-H292

MUC5AC

NCI-H292 .

95% , 5% CO 2 37

HEPES (25mM), penicillin G (100 U/

), streptomycin (100μg/ ), FBS (10%, V/V)

RPMI 1640 ( )

, 1 2 subculture .

24-well culture plate , well 2.5×10 ⁵ cells/well

. , FBS 0.3

% 24

, serum (serum-

free medium) .

TNF-alpha 0.1nM (500U/ )

EGF 25ng/ 30

20 200

well (24-well plate ) , 37

24 .

5) NCI-H292 total RNA

NCI total RNA Ka-

rlinsey ²⁶⁾ 24

PBS 2 .

trypsin-EDTA

, 1.5

microtube

. total RNA

INTRON biotechnology Easy-Blue RNA extraction kit (total RNA isolation reagent)

(0.5 /4×10 ⁵ cells) lysis ,

5 . 5 , microtube

chlorofrom , 15 vortexing

2-3 4 , 13,000 rpm (Hanil centrifuge, MICRO 17 R Korea) 10

400 microtube

. isopropanol

10 4 , 13,000 rpm 10

RNA . diethylpyro-

carbonate (DEPC) 75% ethanol 4 , 10,000 rpm 10

. RNA 5

, 20 RNase-free water , spectrophotometer (Beckman, DU -650, Coulter Inc, CA, USA) 260nm

RNA

(1.0A260=single strand RNA 40 / ).

6) Polymerase Chain Reaction (PCR) primer

PCR primer

( ) (Daejeon, Korea) , . NCI

-H292 human MUC5AC

sense primer 5‘-TGA

TCA TCC AGC AGC AGG GCT-3', antisense

primer 5’-CCG AGC TCA GAG

GAC ATA TGG G-3' , primer

PCR 500 bp . β-actin

sense primer

5'-TAC AAC GAG CTG CGT GTG GCC-3' , antisense primer 5'-CAA CGG AAC CGC CTC GTT GC-3' primer

DNA 500 bp .

7) RNA

total RNA , (RT)

cDNA , (PCR)

. , total RNA 1 7

5 5 denaturation ,

RT premix kit

.

MUC5AC PCR ,

cDNA 2 PCR premix

kit .

, PCR 40 (PCR thermal cycler;

Takara MP-300, Japan) , denaturation 94 30 , annealing 60 30 , extension

72 30 .

8)

RNA

cDNA MUC5AC

. , PCR 10 10×gel loading buffer (0.25% bromphenol blue, 0.25% xylene cyanol FF, 50% glycerol) , Tris -acetate-EDTA buffer (40mM Tris-acetate, 1mM

EDTA) 1 / ethidium bromide

1.0% agarose gel . Gel

DNA band

, .

9)

200 , 60 , 30

2 .

,

nipple polyethylene duct

. duct

, duct

.

Pon ²⁷⁾ .

15% (V/V) Sodium metabisulfite (MBS)

,

. 3 MBS

150 ppm .

20 3,600ppm

Gastec detector kit (Tokyo, Japan) .

, 3 ( ),

3 1

( ) ,

5 .

,

, 1 4 1 3

, 1 5 , 3 .

,

1 3

28) .

10)

, 70kg

350g 2

. , 3

1 ( 5 )

10 11

, 1 4 ,

.

11)

Shao ²⁴⁾ , Harkema ²⁹⁾ .

, ,

(bronchoalveolar lavage, BAL)

5 . sample

PBS 1/10 , sample

ELISA 96-well plate 100

, 2 . 2

PBS-Tween 20 (PBS-T, 0.05%) 200

/well , well 3 .

2% BSA in PBS-T 200 well

, 1 . 1

PBS-T 200 3 ,

MUC5AC monoclonal antibody mouse anti-MUC5AC clone 45M1 2% BSA

1:1,000 well 100

, 1 . 1 PBS-

T 3 , 2 HRP-Goat Anti-

Mouse IgG Conjugate 2% BSA 1:5,000

well 100 1

. PBS-T 3

3,3',5,5'-tetramethyl-benzidine peroxide solution

(TMB) 100 well , 5 1N

50 , . 450

nm well ,

. 12)

Mean±SE ,

. unpaired Student's t-test , p<0.05

.

실험결과

1.

杏蘇湯이 일차배양

세포로부터의 뮤신

분비에 미치는 영향

40μl/200μl PBS

(Fig.1).

*

M uc in s ec re tio n (% )

150

Normal

HST(㎕/200㎕ PBS) 100

50

0

10 20 40

MUC5AC β-actin

N orm al E G F TN F E + H S T T+ H S T

Fig. 1. Fig. 2.

2.

杏蘇湯이

세포에서

또는

로 유도된

유전자 발현 증가에 미치는 영향

24 EGF

TNF-alpha MUC5AC

(Fig.2).

3.

杏蘇湯이 이산화황으로 유발된 생체내의 기

도점액 과다분비 상태에 미치는 영향

5 2

,

(Fig.3).

4.

取淵湯이 일차배양

세포로부터의 뮤신

분비에 미치는 영향

40 120μl/200μl PBS

(Fig.4).

5.

取淵湯이

세포에서

또는

로 유도된

유전자 발현 증가에 미치는 영향

24 EGF

TNF-alpha MUC5AC

(Fig.5).

6.

取淵湯이 이산화황으로 유발된 기도점액 과

다분비에 미치는 영향

5 2

,

(Fig.6).

고 찰

11)

In v ivo m uc in ( % )

500

Normal 200

0 400 300

100

SO

HST+SO

M uc in s ec re tio n (% )

150

Normal 50

0 100

20 40 80

CHT (㎕/200㎕ PBS)

Fig. 3. Fig. 4.

10)

“ ”

.

, .

12-13,30)

.

“

”

12) ,

13,30)

.

14)

, ,

. ¹⁵⁾

, ,

.

31)

, ³²⁾

,

.

.

. ,

(HTSE) MUC5AC

β-actin

N orm al E G F TN F E + H S T T+ H S T

+

*

In v ivo m uc in ( % )

400

Normal 0

300

200

100

SO

HST+SO

*

Fig. 5. Fig. 6.

(ELISA) . NCI-H292

(EGF) (TNF)

24

MUC5AC

RT-PCR .

.

HTSE 30

(Fig.1).

HTSE

(Fig.4).

NCI H292

. NCI-H292 MUC5AC

EGF TNF-alpha 30

24 MUC5AC

,

EGF TNF-alpha

(Fig.2) MUC5AC

(Fig.5).

,

(Fig.6). ,

(Fig.3).

,

,

.

, 30

,

. 5

,

.

결 론

.

(HTSE) 30

NCI-H292 24

EGF TNF-alpha MUC5AC

,

.

HTSE

, 24

NCI-H292 EGF TNF-alpha

MUC5AC ,

.

, ,

.

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