137

서 론

(Chronic Obstructive Pulmonary

Disease: COPD) 7.7%

, 14.5 101,2).

(Disability Adjusted

Life's Years) 1990 12 2020 5

3

3,4).

COPD

, , ,

.

5). COPD

.

Protective Effects of Gamipalmi-hwan on Elastase-inducedApoptosis of A549 Cells

Ji-Seok Oh, Yang-Chun Park

Division of Respiratory System, Dept. of Internal Medicine, College of Oriental Medicine, Daejeon University

Objective: This study aimed to evaluate the protective effects of Gamipalmi-hwan (GPH) on elastase-induced lung

cell injury.Materials and Methods: As an in vitro model of emphysema, the current study was performed to investigate potential

activity of GPH in regulating injury responses of A549 human type II cell line mediated by elastase treatment.

Results: GPH treatment increased the number of A549 cells which was reduced by elastase digestion. Elastin protein

level, which was reduced by elastase treatment, was increased by GPH treatment. Labeling intensity with caspase 3

protein in elastase-treated cells was reduced by GPH treatment. Both Erk1/2 and Cdc2 protein levels, which were

decreased by elastase treatment, were increased to a level similar to that of the normal cells. mRNA levels encodingIL-1 and TNF- were increased by elastase and then down-regulated by GPH.β αConclusion: The present data suggest that A549 cells are subjected to inflammatory damage by elastase and can be

recovered by GPH treatment. Further studies examining the protective activity of GPH in elastase-treated lung tissue

would be useful for therapeutic strategies of emphysema treatment.

Key Words : Gamipalmi-hwan (Jiaweibawei-wan), emphysema, herbal medicine

加味八味丸

오지석 박양춘,

대전대학교 한의과대학 폐계내과학교실

Original article

Received 15 January 2010：⋅ Revised 2 March 2010：⋅ Accepted 2 March 2010：⋅박양춘Correspondence to (Yang-Chun Park)：⋅

충북 청주시 상당구 용담동 대전대학교청주한방병원 내과173-9 1

Tel +82-43-229-3704, Fax +82-43-253-8757, E-mail omdpyc@dju.kr： ： ：

31 2 (2010 3 )J Korean Oriental Med 2010;31(2):137-148

31 2 (2010 3 )

138

COPD

,

6).

7)

, , ,

, , , ,

, , ,8)

COPD

.9)

.

, ,

, ,

, , ,6)

.

in vitro

A549

elastase

.

재료와 방법

재료1.

1)

A549

aneuploid cell line ,

.

2)

(Gamipalmi-hwan, GPH)

( )

1 (Table 1).

構成藥物 生藥名 (g)用量熟地黃 Rehmanniae Radix Preparat 16.0山 藥 Dioscoreae Rhizoma 8.0山茱萸 Corni Fructus 8.0牧丹皮 Moutan Cortex 6.0澤 瀉 Alismatis Rhizoma 6.0白茯苓 Poria 6.0白 朮 Atractylodis macrocephalae Rhizoma 6.0附 子 Aconiti iateralis preparata Radix 4.0肉 桂 Cinnamomi Cortex 4.0淫羊藿 Epimedii Herba 4.0菟絲子 Cuscutae Semen 4.0補骨脂 Psoraleae Fructus 4.0胡桃肉 Juglandis Semen 4.0杏 仁 Armeniacae Arrarum 4.0蘇 子 Perillae Fructus 4.0蘿葍子 Raphani Semen 4.0神 麯 Massa medicata Fermentata 4.0麥 芽 Hordei Fructus Germiniatus 4.0白 果 Ginkgo Semen 10.0Total 110.0

Table 1.

(312)

1 : elastase A549

139

1 1,000

2

(Rotary evaporator, Buchi B-480, Switzerland)

, (Freeze dryer, Eyela

FDU-540, Japan)

(-84 )

. 21.8 g

19.8 % .

3)

elastase Sigma

, 80 unit

-20 . Anti-Elastin rabbit pAb (Ca-

lbiochem, USA), cleaved caspase-3 (Cell signaling,

USA), fluorescein goat anti-mouse IgG (Invitrogen,

USA), rhodamine red-X goat anti-rabbit IgG (Invitr-

ogen, USA), Hoechst 33258 (Invitrogen, USA),

anti-cyclin B1 developed in rabbit IgG fraction of

antiserum (Sigma, USA), p-Erk 1/2 (Cell Signaling,

USA), total Erk1/2 (Cell Signaling, USA), actin

(MP Biomedicals, USA), goat anti-rabbit IgG-HRP

(Santa Cruz Biotechnology, USA) .

방법2.

1)

A549 10% heat-inactivated fetal bovine

serum (FBS) penicillin (100 units/ml)/streptomycin

(100 g/ml) RPMI 1640 media (Lonza,μ

USA) 37 , 5% CO2 . Cell

75 cm2flask (SPL, Korea)

, 3 .

2) MTT assay

MTT[3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltet

razolium bromide] .

96 well plate 1 × 105

A549

plating , (0.01 - 1 mg/ml)

37 , 5% CO2 incubator 24

. well MTT solution

4 , 570nm

.

.

(%) = × 100

3)

(double immunofluorescence

staining) , coverslip poly-L-

ornithine (0.1 mg/ml, Sigma, USA) laminin (0.02

mg/ml, Collaborate Research, USA)

pre-coating . 1 × 105

A549

pre-coating coverslip plating , (i)

( ), (ii) elastase (2unit), (iii) elastase

0.3 mg/ml 5 mg/ml

37 , 5% CO2 incubator 24

. Coverslip

blocking buffer 4 16

. 1 2.5% BSA, 2.5%

horse serum blocking buffer

1:500 , 4

. 1

PBST (PBS plus 0.1% triton X-100)

, 2.5% BSA, 2.5% horse serum

blocking buffer Fluorescein-goat anti-mouse

(green) Rhodamine-goat anti-rabbit antibody (red)

1:400 1 30 2

. 2 3

PBST . Hoechst

2 0.25% Hoechst

33258 PBST

PBST .

(Zeiss fluorescent microscope)

, images

Adobe Photoshop (version 5.5) green

red

. Photoshop program Layer blending

(313)

31 2 (2010 3 )

140

mode options images

.

4) Western blot

A549 60 mm culture dish 2 × 105cells

/ml (i) ( ), (ii) elastase

(2unit), (iii) elastase 0.3 mg/ml

5 mg/ml 24 37 , 5%

CO2 incubator . A549

137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, 2

mM KH2PO4 (pH 7.4) PBS 50

- 200 l triton lysis buffer (20 mM Tris, pH 7.4,μ

137 mM NaCl, 25 mM -glycerophosphate, pHβ

7.14, 2 mM sodium pyrophosphate, 2 mM EDTA, 1

mM Na3VO4, 1% Triron X-100, 10% glycerol, 5 μ

g/ml leupeptin, 5 g/ml aprotinin, 2 M benzamidine,μ μ

0.5 mM DTT, 1 mM PMSF)

. sample

, 10 g western analysisμ

. 12% SDS-polyacrylaminde

gel (1.5M Trizma base, 10% sodium dodecyl sulfate,

30% acrylamide, 10% ammonium sulfate, TEMED)

PVDF membrane (Pall

Corporation, USA) . Antibody

3% bovine serum

albumin (BSA, Sigma, USA), 0.1% Tween 20

TBS buffer membrane 1

4 16 blocking

buffer .

membrane washing 1 blocking

buffer (1 × TBS buffer, 3% BSA, 0.1% Tween 20)

1:1000 30

. membrane goat anti-

rabbit IgG (Santa Cruz biotechnology, USA)

anti-mouse IgG (Santa Cruz biotechnology, USA)

horseradish peroxidase 1:1000

30

. membrane

western blotting detection system

Kodak Scientific Imaging Film

(Eastman Kodak Co, USA) .

5) RNA RT-PCR

A549 60 mm culture dish 2 × 105cells

/ml (i) ( ), (ii) elastase

(2unit), (iii) elastase 0.5 mg/ml

6 , 24

. RNA 6 , 24 easy-

BLUE (iNtRon Biotechnology, Korea)

. 1 ml easy-BLUE

, 10 ,

200 l chloroformμ

13,000 rpm 10 .

400 l 2-propanolμ

10 , 13,000 rpm 5

. RNA 1 ml 75%

ethanol , 10,000 rpm 5

. 75% Ethanol , diethylprocarbonate

(DEPC) 260 nm

,

. cDNA 1 g oligo-dTμ

(MMLV-RT, Promega, USA)

. PCR Taq polymerase (Promega, USA)

primer , PCR

Target Gene Primer Sequence Pruduct Size (bp)

IL-1β Forward GCT GAT GGC CCT AAACAG 672Reverse GAAGAC GGGCAT GTT TTC

TNF-α Forward AGCCCA TGT TGT AGCAAACC 516Reverse GGT TGAGGG TGT CTGAAGGA

Table 2.

(314)

1 : elastase A549

141

94 -5min, 94 -30sec, 55 -30sec, 72

-30sec, 72 -7min 30 cycle .

primer Homo IL-1 (Bioneer, Korea),β

Homo TNF- (Bioneer, Korea) .α

primer Table 2 .

결 과

세포 생존력에 미치는 영향1.

MTT

assay . A549

0.5 mg/ml

90% 1

mg/ml 80%

(data not shown).

A549 elastin elastase

0.3 mg/ml 0.5 mg/ml

Hoechst

.

. elastase

9.5

90%

(Fig. 1).

단백질 생성수준의 변화2. Elastin

A549 elastase elastin

elastin

western blot

, elastase

elastin

, elastase

0.3 0.5 mg/ml

elastin (Fig. 2A). Elastin

Hoechst

elastin

elastase elastin

. elastase

Intact GPH(0.3mg/ml) GPH(0.5mg/ml)

Elastase+GPH(0.3mg/ml)

Elastase+GPH(0.5mg/ml)Elastase

A

100

Num

ber o

f cells

80

60

40

20

01 2 3 4 5 6

****

B

Fig. 1.

(315)

31 2 (2010 3 )

142

0.5 mg/ml

elastin

(Fig. 2B). Western

blot ,

elastin

elastase

. Elastase

elastin

(Fig. 2C).

단백질 생성수준의 변화3. Caspase 3

A549 elastase

caspase 3 . Elastase

Intact Elastase

Elastase+GPH(0.3mg/ml)

Elastase+GPH(0.5mg/ml)

A

Elastase

Elastase+GPH(0.3mg/ml)

B

Fig. 3.

Intact Elastase

Elastase+GPH(0.3) Elastase+GPH(0.5)

A

Elastase Elastase+GPH(0.5)Intact

B

ElastaseIntact

Elastase+GPH(0.3

mg/ml)

Elastase+GPH(0.5

mg/ml)

elastin

actin

C

Fig. 2.

(316)

1 : elastase A549

143

caspase

. Elastase

caspase

elastase

(Fig. 3A). Caspase

Hoechst

, caspase

Hoechst

caspase

Hoechst

(Fig. 3B).

4. 세포의생존및증식관련단백질생성수준의

변화

Erk1/2

A549 , phospho-Erk1/2

elastase

, elastase

,

Erk1/2

(Fig. 4).

A549

cell cycle

Cdc2 cyclin B1

. Cdc2

,

elastase

,

(Fig. 5A). Cyclin B1 elastase

,

(Fig. 5B).

Intact Elastase

Elastase+GPH(0.3mg/ml)

Elastase+GPH(0.5mg/ml)

Cdc2

actin

Intact Elastase

Elastase+GPH(0.3mg/ml)

Elastase+GPH(0.5mg/ml)

Cyclin B1

actin

A B

Fig. 5.

Elastase+GPH(0.3mg/ml)ElastaseIntact

Elastase+GPH(0.5mg/ml)

p-Erk1/2

Erk1/2

Fig. 4.

(317)

31 2 (2010 3 )

144

및 발현수준의 변화6. IL-1 TNF- mRNAβ α

Elastase A549

IL-1β

TNF- mRNA RT-PCRα

, elastase 6

elastase IL-1 TNF- mRNAβ α

, elastase

mRNA

(Fig. 6). Elastase 24

IL-1 TNF- mRNAβ α

(data not shown).

고 찰

COPD

,

,10).

COPD

11).

COPD . elastase

elastin

3,12,13).

elastase

elastin

. elastin

3).

, ,

, ,

COPD

6).

,6).

7), , , ,

, , , , , ,

COPD

9)

.

in vitro

A549 elastase

. Elastin

elastin

Elastase+GPH(0.5mg/ml)ElastaseIntact

IL-1

TNF-α

actin

Fig. 6. β α

(318)

1 : elastase A549

145

14,15).

elastase elastase

16,17).

1 mg/ml

80%

elastin, caspase

3 .

A549

,

90%

0.3 - 0.5 mg/ml elastase

.

elastase

Hoechst staining

20%

30%

. Elastin

elastase

. elastase

elastin

.

Elastase

caspase 3

.

, ,

18,19).

caspase Bcl-2

Bcl-2

caspase20-22)

. caspase 3

DNA

, ,

. Caspase

3 caspase

. elastase

elastase caspase

3 . Elastase

caspase 3

.

elastase

. Caspase 3

.

,

Caspase 8 9 Bcl-2

.

Erk1

/2 (External signal-regulated protein kinase)

. Erk1/2 mitogen

activated protein kinase (MAP kinase)

42 44 kDa

23-26). Erk1/2

.

Erk1/2

CREB (cAMP responsiveness element binding

protein) MEF-2

27,28).

A549

(319)

31 2 (2010 3 )

146

phospho-Erk1/2

elastase

.

.

cell cycle

Cdc2 cyclin B1

. Cdc2 Cell division cycle 2

Cdk (cyclin-dependent kinase)

Cdk

Cdc2 cyclin

cell

cycle G2 (growth phase 2) M (mitosis;

)29). Cdc2 kinase

,

Cdc2

. Cdc2

Cdk2, 4, 6 G1 (growth phase 1)

S (DNA synthesis; DNA )

Cdc2

cell cycle30,31)

. elastase

Cdc2

. Cdc2

cyclin B1 Cdc2

. Cdc2 protein kinase

in vitro kinase assay

Cdc2

.

Elastase

IL-1 TNF-β α

RT-PCR .

IL-1 TNF-β α

IL-1 TNF- COPDβ α32).

elastase A549 IL-1β

TNF- mRNAα

. elastase

.

mRNA

ELISA

.

,

COPD

in vivo

.

결 론

.

1. A549 elastase

.

2. A549 elastase

elastin .

3. A549 elastase

caspase 3 .

4. A549 elastase

Erk1/2 Cdc2 .

(320)

1 : elastase A549

147

5. A549 elastase

IL-1 TNF- mRNAβ α

.

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