서 론
, chromosome DNA mRNA (transcription)
.
. (FDA)
50 10%
丹蔘
김희은, 민상연, 김장현
Original Article
In Vitro Study of Anti-inflammatory and Analgesic Effects of Salvia Miltiorrhiza (SM) Extracts Using Luciferase Reporter Gene Assay
Hee-Eun Kim, Sang-Yeon Min, Jang-Hyun Kim
Department of Pediatrics, College of Oriental Medicine, Dongguk University
Objectives: In order to identify the anti-inflammatory and analgesic properties of Salvia miltiorrhiza (Dan-Sam), widely used in Korean traditional medicine, an in vitro screening system was designed using pGL3, a luciferase reporter vector, and the tumor necrosis factor (TNF)-α and cyclooxygenase (COX)-2 as target genes.
Methods: The promoter regions of each gene were generated by PCR using the human chromosome as template DNA, and inserted into pGL3 vector with Kpn I and Hind . The final construct was transfected into human myelomonocytic leukemia cells (U-937) that could be differentiated and activated by phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). Using this system, the anti-inflammatory and analgesic effects of several herbal extracts regarded to have the medicinal effects of diminishing body heat and complementing Qi were tested. The chemicals PD98059 and berberine chloride were used as controls of the transcriptional inhibitors of TNF-α and COX-2, respectively.
Results: Salvia miltiorrhiza (Dan-Sam) demonstrated significant decrease of TNF-α and COX-2 mRNA in the in vitro assay system. In MTT assay, Salvia miltiorrhiza (Dan-Sam) did not significantly inhibit the survival and proliferation of human myelomonocytic leukemia cells (U-937).
Conclusions: Salvia miltiorrhiza (Dan-Sam) was found to exhibit the significant medicinal properties of anti-inflammatory and analgesic effects.
Key Words : Salvia miltiorrhiza, anti-inflammatory effect, analgesic effect.
2008 4 25 2008 7 15
: , 1090-1
,
(Tel : +82-54-770-1260, Fax : +82-54-770-2281, E-mail : [email protected])
TM
TM salicylate
1).
bio-compatibility
.
luciferase green fluorescence protein(GFP)
bio-luciferase
. luciferase bioluminescence ATP D-luciferase oxoluciferin
.
. luci-
ferase
,
, 2-4).
cytokine TNF-α , TNF-α LPS(lip- opolysaaccharide)
,
. LPS TNF-α
5). ,
mRNA .
LPS, TNF-α
.
(Labiatae) Salvia miltiorrhiza
, 6).
7).
8)
, 9) Glucose Oxidase
, 10) , 11)
apoptosis ,
. ,
in vitro
,
1 . ,
, , ,
TNF-α COX-2 mRNA
in vitro assay system
, ,
.
실 험
1. 시약 및 약재
PMA(phorbol 12-myristate 13-acetate), LPS(lipopolysaccharide from Escheichia coli), PD98059, berberine chloride
Sigma(St Louis, MO, USA) ,
pGL3-basic vector Promega .
(Salvia miltiorrhiza, SM), (Prunus persica Batsch, PP), (Astragalus membranaceus, AM),
(Achyranthes bidentata, AB)
protocol .
2. 세포배양
human leukemia
monocyte U-937
, ( )
RPMI1640 (Gibco, Ltd, UK) 10%(v/v) fetal bovine serum, penicillin 100 units/ml, streptomycin 100μg/ml
37 5% CO2
.
3. 관련 유전자 발현 변화 양상의 측정
2μg total RNA 65 15 denat-
uration , 200U moloney murine leukemia virus reverse transcriptase(GIBCO BRL, MD, USA)
20μl
cDNA mixture
. cDNA 10mM Tris-HCl(pH8.3), 50mM KCl, 1.5mM MgCl2, 0.2mM dNTP, 0.4mM primer 0.5U Taq polymerase(TaKaRa, Shiga,
Japan) 20μl PTC-100
Programmable Thermal Controller(MJ Research,
Waltham, MA, USA) ,
primer Genbank
(Table 1).
DNA 1.0% agarose gel
. gel band intensity Imag- eMaster TotalLab(Amersham Biosceinces, Pisca- taway, NJ, USA)
glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
. 4. Plasmid 제작
TNF-α COX-2 promoter site
Kpn I Hind
primer human chromosomal DNA template
PCR . PCR
product pGL3-basic vector(Promega Co., Madison, WI) subcloning TNF-α COX-2 promoter luciferase
(Fig. 1).
Name Oligonucleotide sequence (5'-3') Product size(bp)
human TNF-α promoter
F-CACGGAGGTACCTGCACCCTCGATGAAGC
R-GTGTGCCAAAAGCTTCCTTTATATGTCCCTGGG 1,287
human COX-2 promoter
F-GGCAGATGGTACCTTCTAGAAATTCATC
R-CCGAATAGAGAAGCTTCGTGACTTGG 7,270
human GAPDH F-ATCCCATCACCATCTTCCAG
R-CCTGCTTCACCACCTTCTTG 579
human TNF-α F-CCTCAACCTCTTCTGGCTCA
R-CTGGGCTCCGTGTCTCAA 344
human COX-2 F-CATTCTTTGCCCAGCACTTC
R-TACTCTGTTGTGTTCCCGCA 341
Table 1.
5. Transient transfection과 luciferase assay luciferase reporter vector U-937
transient transfection EffecteneTM (Qiagen Co., Valencia, CA) kit
reporter vector 2μg transfection control vector
pSV β-gal vector(Promega Co., Madison, WI) 1μg , 5 × 106 U-937 co-transfection . PMA-induced
, transfection 24
PBS , , , ,
PD98059, berberine chloride
12well plate 1 × 105 split
TNF-α, COX-2 promoter
Kpn I Hind III
Kpn I Hind III ori
Ampr
pGL3-Basic Iuc + f1 ori
Kpn I
TNF-α promoter
Hind III
Iuc + pGL3/
TNF-α promoter
pGL3/
COX-2 promoter
Hind III
Iuc + ori
ori
Ampr Ampr
f1 ori f1 ori
Kpn I COX-2 promoter
+
Fig. 1. α
α
Luciferase
assay 1 Luciferase
assay 2
Luciferase LPS
LPS /drugs PMA
/drugs PMA
macrophge monocyte
TNF-α, COX 2 promoter
Transient transfection
Fig. 2.
. 2 , well 25nM PMA , 24
lysate .
LPS-induced , transfection
24 PBS 25nM
PMA 12well plate 1 × 105 split
. 24 well ,
, , , PD98059, berberine chloride
PBS , 2 ,
well 5μg/ml LPS
. 24 lysate
Luciferase activity Luciferase Reporter Assay SystemTM(Promega Co., Madison, WI) kit
. data normalization lysate β-galactosidase assay
(Fig. 2).
6. 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny l tetrazoliumbromide (MTT) assay
U-937
MTT assay
. U-937 96 well plate 1 × 104 cells/
well split , 24 0, 0.01, 0.1,
0.5 mg/ml . 24
PBS 5mg/ml MTT well 10ul
, 37 , 5% CO2 incubator 1
MTT .
plate 450g 5
100ul 0.04N Hcl
10 . ELISA
leader 540nm . PMA
U-937
25nM PMA 24
.
결 과
1. RT-PCR을 이용한 U-937 세포에서의 TNF- α와 COX-2 mRNA 발현 분석
TNF-α COX-2 LPS
12). U-937 (macrophage) PMA
.
U-937 TNF-α COX-2
mRNA PMA
LPS
12-13)
. PMA
PMA , LPS
. ,
U-937 TNF-α COX-2
PMA LPS
RT-PCR .
Group mRNA Expression(%)
TNF-α COX-2
Control 0.5710207 0
LPS 0 0
PMA 38.2 33.5
PMA/LPS 74.7 71.5
Non-treated group (Control); LPS-treated group (LPS); PMA-treated group (PMA); PMA and LPS-treated group (PMA/LPS). Each PCR product was normalized to GAPDH.
Table 2. α
U-937 TNF-α COX-2
mRNA . LPS
mRNA
, PMA 40%,
PMA LPS
75% TNF-α COX-2 mRNA
. U-937
PMA , PMA
LPS
(Table 2, Fig. 3).
2. Luciferase reporter assay를 이용한 한약재 들의 염증 억제 효과 분석
vector transient transfection
U-937 TNF-α COX-2 promoter
luciferase assay .
(Salvia miltiorrhiza, SM), (Prunus persica, PP), (Astragalus membranaceus, AM), (Achyranthes bidentata,
AB) , PMA
LPS TNF-α COX-2
promoter
.
,
, positive control PD98059
(TNF-α ) berberine chloride
(COX-2 ,
) .
PMA U-937
, U-937 luciferase
PMA , 10
luciferase .
positive control PD98059 TNF-α promoter (luciferase
) 50% inhibition
,
berberine chloride
COX-2 promoter 10%
inhibition .
- - + + PMA - + - + LPS
mRNA Expression (%)
80 70 60 50 40 30 20 10 0
`1 2 3 4
GAPDH
TNF-α
COX-2
TNF-α COX-2
cont LPS PMA PMA/LPS
Fig. 3. α
α
PMA promoter
50% ,
. ,
, promoter
(Table 3, Fig. 4).
PMA LPS
,
LPS luciferase 20%
LPS ,
Group Concentration(mg/ml) Relative Luciferase Activity(%)
COX-2 TNF-α
Control 10.5±1.3 7.28±0.6
PMA 100 100
Berberine Chloride 86.7±4.9
PD98059 45.6±3.2
SM 0.5 30.3±1.8 41.1±8.7
PP
1 94.9±2.6 87.7±1.9
0.1 98.2±7.2 87.9±9.7
0.01 102.3±9.8 102.2±16.4
AM 1 125.5±8.1 113.6±6.6
0.01 104.8±0.9 95.2±8.5
AB 1 124.4±12.2 114.3±5.2
0.01 91.2±5.5 94.7±1.7
Transcriptional activities of TNF-α and COX-2 promoter in U-937 cells were assessed by measuring luciferase activities. Data were normalized by β-galactosidase assay. PMA, phorbol 12-myristate 13-acetate; SM, Salvia miltiorrhiza; PP, Prunus persica; AM, Astragalus membranaceus; AB, Achyranthes bidentata. Data were presented as mean ±SEM.
Table 3.
COX-2 Berberine
TNF-α
PD98059
Relative Luciferase Activity (%)
140 120 100 80 60 40 20 140 120 100 80 60 40 20 0
0
- + + + + + + + + + +
0.5 1 0.1 0.01 1 0.01 1 0.01
SM PP AM AB
mg/ml
Inhibitor
PMA
Fig. 4. α
β
antigen
U-937
( , purity,
)
. PD98059 berberine chloride
transcription inhibition
COX-2
Berberine
Relative Luciferase Activity (%)
140 120 100 80 60 40 20 0
- + +
0.5 SM
mg/ml
Inhibitor
100
20
TNF-α
PD98059 160
140 120 100 80 60 40 20
0 + + + + +
- + + + + + + +
PMA LPS
0.5 PP
0.1 0.5
AM 0.5 AB
Fig. 5. α
β
Group Concentration(mg/ml) Relative Luciferase Activity(%)
COX-2 TNF-α
Control 84.0±4.1 76.8±5.1
LPS 100 100
Berberine Chloride 75.8±3.8
PD98059 57.3±1.8
SM 0.5 81.8±4.4 82.3±3.0
PP 0.5 120.5±8.1 102.7±3.8
0.1 146.1±11.7 102.9±8.3
AM 0.5 146.3±17.1 92.3±19.5
AB 0.5 138.7±10 107.1±7.7
Transcriptional activities of TNF-α and COX-2 promoter in U-937 cells were assessed by measuring luciferase activities. Data were normalized by β-galactosidase assay. PMA, phorbol 12-myristate 13-acetate; LPS, lipopolysaccharide; SM, Salvia miltiorrhiza; PP, Prunus persica; AM, Astragalus membranaceus; AB, Achyranthes bidentata. Data were presented as mean ±SEM.
Table 4.
PMA
. PMA
LPS
, PD98059 berberine chloride
(Table 4, Fig. 5).
3. MTT assay를 이용한 丹蔘의 세포 독성 측정
U-937 TNF-α COX-2 promoter
MTT assay .
U-937 0.01, 0.1, 0.5mg/ml
24 ,
. PMA
.
(Table 5, Fig. 6).
고 찰
SM Concentration(mg/ml)
Live Cell(%)
PMA(-) PMA(+)
0 100±7.0 100±10.6
0.5 97.3±5.3 96.2±12.8
0.1 100.1±1.8 112.5±2.9
0.01 102.8±5.4 96.4±4.4
After cells were differentiated with 100nM PMA for 24 hours, cells were exposed to 0, 0.5, 0.1, 0.01 mg/ml SM for 24 hours. Data were presented as mean ±SEM.
Table 5.
120 100 80 60 40 20 0
Live cell (%)
-
PMA - - -
0 0.5 0.1 0.01
SM (mg/ml)
120 100 80 60 40 20 0
Live cell (%)
+
PMA + + +
0 0.5 0.1 0.01
SM (mg/ml)
Fig. 6.
, chromosome DNA
mRNA (transcription)
.
promoter
cAMP steroid hormone signal molecule
(activation) (repression)
1).
inflammation TNF-α prostagladin(PG) COX-2
transcription activity
luciferase
promoter , U-937
.
TNF-α COX-2 LPS
12). U-937 (macrophage) PMA
.
U-937 TNF-α COX-2
mRNA PMA
LPS
12-13)
.
U-937
PMA , TNF-α COX-2 promoter inducer LPS
. , PMA TNF-α
promoter ,
LPS TNF-α promoter
14-15)
. PMA
PMA , LPS
.
, U-937
TNF-α COX-2 PMA LPS
RT-PCR
, U-937 TNF-α COX-2
mRNA . LPS
mRNA
, PMA 40%,
PMA LPS
75% TNF-α COX-2 mRNA
. U-937
PMA , PMA
LPS
. Luciferase reporter assay (Salvia miltiorrhiza, SM), (Prunus persica, PP), (Astragalus membranaceus, AM), (Achyranthes
bidentata, AB) ,
PMA
promoter 50%
, . ,
, , promoter
. , PMA LPS
, PD98059 berberine chloride
. ,
3
. MTT assay
U-937 0.01,
0.1, 0.5 mg/ml 24 ,
.
.
, PMA LPS TNF
-α COX-2 promoter ,
PMA LPS TNF-α COX-2
promoter
,
.
, TNF-α
COX-2 mRNA assay
system
,
system in vitro
,
,
.
결 론 ,
, , , TNF-
α COX-2 mRNA
in vitro assay system , ,
.
1. U-937 TNF-α COX-2 mRNA
PMA , LPS
RT-PCR .
2. in vitro assay
system TNF-α COX-2 mRNA
.
3. , ,
TNF-α COX-2 mRNA .
4. MTT assay U-937
. in vitro
,
. screening
system 1
.
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, p.419-420, 1995.
7. , , , , ,
, p.855-862, 1997.
8. , , , ,
. , 17(4):1008
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9. , , , , Glucose Oxidase
. , 17(1):136-139,
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10. , , , ,
. , 15(6):
876-880, 2001.
11. , , ,
apoptosis . , 14
(2):22-47, 2000.
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Biochem Biophys Res Commun, 244:143- 148., 1998
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