Inhibitory Efficacy of Dystaenia takeshimana Extract on iNOS, COX-2 Protein and mRNA Expression in Raw 264.7 Cell

Raw 264.7 세포에서 섬바디나물 추출물의 iNOS, COX-2 단백질 및 mRNA 발현 억제 효과

이진영¹, 유단희¹, 주다혜¹, 채정우²*

1호서대학교한방화장품과학과

2

(

)

Received: October 27, 2016 / Revised: November 23, 2016 / Accepted: November 23, 2016

서 론

염증은체내의세포가물리적충격이나세균감염등의자 극에의한피부손상으로부터인체를보호하기위해반응하 는생체방어기전이다

^[12].

통한초기단계로서단기간치료가가능한급성염증

^(acute

inflammation)

과도한방어반응으로염증반응이지속되는만성염증

^(chronic

inflammation)

^[20].

^,

^,

^[4,

21].

다양한면역세포가관여하지만

^[17]

^,

inducible nitric oxide synthase (iNOS)

^(NO)

cyclooxygenase-2 (COX-2)

prostaglandin E

(PGE

)

촉진인자들을생성한다

^[5].

Inhibitory Efficacy of Dystaenia takeshimana Extract on iNOS, COX-2 Protein and mRNA Expression in Raw 264.7 Cell Jin-Young Lee

, Dan-Hee Yoo

, Da-Hye Joo

, and Jung-Woo Chae

*

1

Department of Herbal Cosmetic Science, Hoseo University, Chungnam 31499, Republic of Korea

2

Gyeonggi-do Forest Environment Research Institute, Osan 12408, Republic of Korea

In this study, the anti-inflammatory activities of the 80% ethanol extract of Dystaenia takeshimana (DT) were investigated using Raw 264.7 cells treated with lipopolysaccharide (LPS). The effect of DT extract on the production of pro-inflammatory factors (iNOS, COX-2) in LPS-stimulated Raw 264.7 macrophages was examined. The cytotoxic effect of DT extract on macrophage cells (Raw 264.7) was examined by the 3-[4, 5- dimethyl-thiazol-2-yl]-2, 5-diphenyl-tetrazoliumbromide (MTT) assay. Treatment with DT extract showed 100% or more cell viability at the concentration 1,000 μg/ml. The inhibitory effect of DT extract on protein expression of inducible NOS (iNOS) and cyclooxygenase-2 (COX-2) was measured by western blotting using the concentrations 50, 100, and 500 μg/ml, with β-actin used as the positive control. Consequently, the pro- tein expression of iNOS, and COX-2 as observed by western blotting, was decreased by 56%, 61.6%, respec- tively with 500 μg/ml DT extract. Inhibition of iNOS and COX-2 mRNA expression was measured by reverse transcription- polymerase chain reaction (PCR) using DT extract concentrations 50, 100, and 500 μg/ml, with GAPDH used as a positive control. Consequently, the mRNA expression of iNOS and COX-2 as observed by reverse-transcription-PCR was decreased by 77.9% and 83.3%, respectively at 500 μg/ml concen- tration of DT extract. In conclusion, DT extract may affect inflammatory factors as a potential anti-inflam- matory agent.

Keywords: Dystaenia takeshimana extract, anti-inflammatory, iNOS, COX-2

*Corresponding author

Tel: +82-31-8008-6658, Fax: +82-31-374-2492 E-mail: [email protected]

© 2016, The Korean Society for Microbiology and Biotechnology

부터분리한내독소이며

^,

lipopolysaccharide (LPS)

LPS binding protein (LBP)

toll-like receptor 4 (TLR4)

^MAPKs

NF-

^B

^[18],

^,

^[15].

^,

^,

^,

작용을초래하고제한성이있다

^[19].

은계속해서보다안전하고효과적으로항염효과를지니는 천연소재의항염증물질을찾고자노력하고있다

^.

섬바디

(Dystaenia takeshimana)

⁽

^),

⁽

^),

⁽

⁾

,

(Nakai)

^{[6, 14].}

^{2 m}

⁴

⁵

^.

³

²

^.

7

⁸

³

^{4 mm}

⁵

^,

⁸

⁹

[2, 11, 13].

^,

^.

^,

^,

^,

^,

^,

^,

^,

^{[9, 16].}

^LPS

^,

^.

재료 및 방법

시료 준비

실험에사용된섬바디나물은울릉도에서채취하였으며

^,

^.

¹⁰

의

^80%

²⁴

액과침전물을분리하여동일한방법으로

³

^.

(Whatman No.2)

EYELA evaporator

²⁰

^.

세포 배양

세포 배양은

10% fetal bovine serum (FBS)

^1%

penicillin/streptomycin (100 U/ml)

^Dulbeco's modified eagle’s medium (DMEM)

^, 37

^{, 5% CO}

incubator

^.

MTT assay에 의한 세포 생존율 측정

세포생존율측정은

Carmichae [1]

. Raw 264.7

96 well plate

5 × 10

cells/well

^{0.18 ml}

^,

^{0.02 ml}

³⁷

^{, 5% CO}

incubator

²⁴

^.

^.

^{5 mg/ml}

^MTT

0.02 ml

⁴

well

DMSO 0.15 ml

³⁰

ELISA reader

^{540 nm}

^.

^.

Nitric oxide (NO) 생성 억제 활성 측정

RAW264.7 cell

^NO

^Green

^[3]

^griess

^NO

. 6 well plate

^{Raw 264.7} cell

^{1 × 10}

^cell/well

^{. 37}

^CO

incubator

²⁴

1X phosphate buffered saline (PBS)

²

^{. LPS 10}

^g/ml

^normal

²

²⁴

^,

^griess

96 well plate

¹⁰

^{540 nm}

^{. NO}

.

Western blot

iNOS, COX-2

cell line Raw 264.7

100 mm tissue culture dish

^{1 × 10}

cells/well

cell seeding

²⁴

^cell

^.

^LPS

¹

^g/ml

²

²⁴

⁴⁸

세포생존율 %( )=⎝⎛1 시료첨가군의 흡광도–---무첨가군의 흡광도-⎠⎞×100

No 억제능 %( )=⎝⎛1 시료첨가군의 흡광도–---무첨가군의 흡광도-⎠⎞×100

후다시배지를제거하고

^PBS

²

^{. Complete} mini 1 tab

¹⁰⁰

^l

Radio-immuno- precipitation assay (RIPA) buffer 10 ml

⁴

16,110 × g

20

^.

^BCA protein assay kit

^{, 20}

^l

^10%

acrylamide gel

^.

^transfer

^(BIORAD)

polyvinylidene fluoride (PVDF) membrane

¹

blocking buffer (5% skim milk in TBST)

^. iNOS, COX-2,

^-actin

¹

⁴

^over night

^,

¹⁰

tris-buffered saline and tween 20 (TBST)

³

^{. iNOS}

^COX-2

²

anti-rabbit,

^-actin

²

anti-mouse

^1:1,000

²

^. 3

^washing

LAS 4,000

.

Total RNA

세포를

100 mm culture dish

^{1 × 10}

cells/well

^cell seeding

²⁴

^LPS

¹

^g/ml

2

²⁴

안배양하였다

^.

trizol lysis buffer

^well

^{1 ml}

^lysis

chloroform 200

^l

²⁰

^.

16,110 × g

²⁰

isopropanol 500

^l

^.

16,110 × g

²⁰

^,

^75%

EtOH-diethylpyrocarbonate (DEPC) water

1 ml

16,110 × g

⁵

^.

DEPC

⁵⁰

^l

⁹⁶ well plate

^{RNA 5}

^l

¹⁹⁵

^l

260 nm, 280 nm

^{total RNA}

. Oligo (dT) 15 primer (500

^{g/ml) 1}

^l,

^{RNA (2}

^g)

nuclease free water

¹⁰

^l

75

⁵

5× reaction buffer, MgCl

, PCR nucleotide mix, rnasin inhibitor, reverse transcriptase, nuclease free water

²⁵

⁵

^{, 42}

60

^{, 70}

¹⁵

^cDNA

^.

Reverse transcription-polymerase chain reaction (RT- PCR)

iNOS, COX-2

^mRNA

polymerase chain reaction (PCR)

^.

^primer sequences

^{Table 1}

^{. PCR tube}

5X green GoTaq

flexi buffer, MgCl

, PCR nucleotide mix (10 mM), primer, GoTaq DNA polymerase, nuclease free water,

cDNA

^PCR

^{. GAPDH,} iNOS

⁹⁶

²

^{, 96}

¹⁰

^{, 64}

³⁰

^, 72

¹

^{, 72}

¹⁰

(40 cycles), COX-2

⁹⁶

²

^{, 94}

¹⁰

^{, 51}

³⁰

^{, 72}

¹

^{, 72}

¹⁰

(40 cycles)

^{. PCR}

0.002% ethidium bromide

1.5% agarose gel

100 V

⁴⁰

^{LAS 4,000}

^.

통계처리

모든실험은

³

^,

SPSS10.0 (Evanston, USA) software

^,

^(analysis of variance ANOVA)

^{= 0.05}

^Turkey’s HSD test

^.

결과 및 고찰

대식세포(Raw264.7)의 생존율 확인

섬바디나물추출물에의한대식세포

^(Raw264.7)

^{MTT assay}

^{Fig. 1}

^.

^LPS

macrophage cell

^1,000

^g/ml

100%

Vit. C

^1,000

^g/ml

^60%

^.

따라서이하의

western blot

^RT-PCR

Fig. 1. Cell viability of extract from Dystaenia takeshimana on

macrophage cell (Raw264.7). After Raw 264.7 cells (5 × 10

cells) were started in medium for 24 h the cells were treated with

5, 10, 50, 100, 500 and 1,000 µg/ml of extracted of Dystaenia

takeshimana for 24 h. Results are means ± S.D. of triplicate data.

및

^mRNA

^{Vit. C}

^{Vit. C}

^80%

^50, 100, 500

^g/ml

^.

Nitric oxide (NO) 저해활성 측정 결과

본연구에서는활성산소중하나이며염증유발에중요한 역할을하는것으로알려진

^NO

^.

^{Fig. 2}

^LPS

군은

^LPS

^NO

며

^,

^NO

^.

^1,000

^g/ml

서

^31.4%

^,

주에서의염증발현을억제시키는것에섬바디나물추출물이 효과가있음을확인할수있었다

^.

Western blot을 통한 iNOS 및 COX-2 단백질 발현 억제 효

염증유발인자인

iNOS, COX-2

측정하기위하여

western blot

^.

house keeping gene

^-actin

positive control

^.

Fig. 3, 4

^LPS

^iNOS

COX-2

^,

대조군인

^{Vit. C}

^{Vit. C}

나물추출물에서

^iNOS

^COX-2

^.

Fig. 2. Effect of Dystaenia takeshimana extract on production of nitric oxide in RAW 264.7 cell. Effect of 80% ethanol extracts of DT on NO production in LPS-induced RAW 264.7 cells. RAW 264.7 cells (1 × 10

cells) were treated with DT 80% ethanol extract and LPS (1 µg/ml) for 24 h. NO production was deter- mined in culture supernatant by griess reagent. Con: control, in Raw 264.7 cells treated with LPS, Nor: normal, in Raw 264.7 cells not treated with LPS. Results are means ± S.D. of triplicate data.

Fig. 3. iNOS protein expression rate of extract from Dystaenia takeshimana on macrophage cell (Raw264.7). After Raw 264.7 cells (1 × 10

cells) were started in serum free medium for 1 h the cells were treated 50, 100 and 500 µg/ml of extract from Dystae- nia takeshimana for 24 h. Con: control, in Raw 264.7 cells treated with LPS, Nor: normal, in Raw 264.7 cells not treated with LPS.

Results are means ± S.D. of triplicate data (Significant as com- pared to control. *p < 0.05, **p < 0.01).

Fig. 4. COX-2 protein expression rate of extract from Dystaenia takeshimana on macrophage cell (Raw264.7). After Raw264.7 cells (1 × 10

cells) were started in serum free medium for 1 h the cells were treated 50, 100 and 500 µg/ml of extract from Dystae- nia takeshimana for 24 h. Con: control, in Raw 264.7 cells treated with LPS, Nor: normal, in Raw 264.7 cells not treated with LPS.

Results are means ± S.D. of triplicate data (Significant as com-

pared to control. *p < 0.05, **p < 0.01).

RT- PCR

염증유발인자인

iNOS, COX-2 mRNA

측정하기위하여

^RT-PCR

^.

포의 여러 조건에서도 그 발현 정도의 차이가 거의 없는

house keeping gene

^GAPDH

positive control

^.

Fig. 5, 6

^LPS

^iNOS

와

COX-2 mRNA

며

^,

^{Vit. C}

^{Vit. C}

^iNOS

COX-2 mRNA

^.

iNOS

에서섬바디나물추출물은

^LPS

^NO

[7, 10], Kim

^COX-2

5-lipoxygenase (5-

LOX)

^,

eicosanoid

고보고한바있다

^[8].

나물은항염증소재로서의가능성이있다고판단된다

^.

요 약

본연구에서는섬바디나물의항염증효과를알아보기위 하여

^LPS

Raw 264.7

나물

^80%

^.

추출물을

^LPS

Raw 264.7

(iNOS, COX-2)

^.

^MTT

^.

^, 1,000

^g/ml

^100%

^.

50, 100, 500

^g/ml

iNOS

^COX-2

western blot

^,

^-actin

^.

^,

western blot

을통해측정한

iNOS, COX-2

Fig. 5. iNOS mRNA expression rate of extract from Dystaenia takeshimana on macrophage cell (Raw264.7). After Raw264.7 cells (1 × 10

cells) were started in serum free medium for 1 h the cells were treated 50, 100 and 500 µg/ml of extract from Dystaenia takeshimana for 24 h. Con: control, in Raw 264.7 cells treated with LPS, Nor: normal, in Raw 264.7 cells not treated with LPS. Results are means ± S.D. of triplicate data (Significant as compared to control. *p < 0.05, **p < 0.01).

Fig. 6. COX-2 mRNA expression rate of extract from Dystaenia takeshimana on macrophage cell (Raw264.7). After Raw264.7 cells (1 × 10

cells) were started in serum free medium for 1 h the cells were treated 50, 100 and 500 µg/ml of extract from Dystaenia takeshimana for 24 h. Con: control, in Raw 264.7 cells treated with LPS, Nor: normal, in Raw 264.7 cells not treated with LPS. Results are means ± S.D. of triplicate data (Significant as compared to control. *p < 0.05, **p < 0.01).

Table 1. Sequence of the primers used for PCR.

Gene Primer Sequence (5’ → 3’)

GAPDH Sense TGA AGG TCG GTG TGA ACG GAT TTG GC Anti-sense CAT GTA GGC CAT GAG GTC CAC CAC COX-2 Sense GGA GAG ACT ATC AAG ATA GT

Anti-sense ATG GTC AGT AGA CTT TTA CA

iNOS Sense AAT GGC AAC ATC AGG TCG GCC ATC ACT

Anti-sense GCT GTG TGT CAC AGA AGT CTC GAA CTC

500

^g/ml

56%, 61.6%

^.

50, 100, 500

^g/ml

iNOS, COX-2

의

^mRNA

^RT-PCR

해측정하였으며

^,

^GAPDH

^.

^,

^RT-PCR

^iNOS, COX-2

^mRNA

⁵⁰⁰

^g/

ml

77.9%, 83.3%

^.

^,

^.

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