Inhibitory Effect of Sambucus sieboldiana var. pendula (Nakai) Extract on the mRNA and Protein Expression of iNOS and COX-2 in Raw 264.7 Cells

RAW 264.7 세포에서 말오줌나무 추출물의 iNOS, COX-2 단백질 및 mRNA 발현 억제 효과

이진영¹, 유단희¹, 채정우²*

1호서대학교화장품과학과

2경기도산림환경연구소

Received _: April 28, 2017 / Revised _: June 5, 2017 / Accepted _: June 5, 2017

서 론

최근생활환경및식생활의변화로인해암

^,

^,

^,

^.

^,

요인으로인한면역조절이상으로유발된염증이지속됨에 따라아토피

^,

^[1, 2].

염증

(inflammation)

^,

다

^[3].

촉진되며

^,

Inhibitory Effect of Sambucus sieboldiana var. pendula (Nakai) Extract on the mRNA and Protein Expression of iNOS and COX-2 in Raw 264.7 Cells

Jin-Young Lee

, Dan-Hee Yoo

, and Jung-Woo Chae

*

1

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

2

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

This study examined a new functional cosmetic material possessing application possibility of Sambucus sieboldiana var. pendula (Nakai) (SS) extract. For this, we analyzed the toxic effect of the SS extract on macrophages (RAW 264.7 cells) by performing MTT assay. Results of the MTT assay showed ≥100% cell via- bility after treatment with 500 μg/ml SS extract. To determine the anti-inflammatory activity of the SS extract, we examined its inhibitory effect on lipopolysaccharide (LPS)-induced NO production in RAW 264.7 cells by performing Griess assay. Result of the Griess assay showed that the SS extract inhibited LPS- induced NO production in a concentration-dependent manner. Next, we examined the effect of the SS extract on the production of proinflammatory factors inducible NOS (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 cells. First, we determined the inhibitory effect of 50, 100, and 500 μg/ml SS extract on iNOS and COX-2 protein expression by performing western blot analysis, with β-actin as a posi- tive control. Results of western blotting showed that treatment with 500 μg/ml SS extract decreased iNOS and COX-2 protein expression by 31.2% and 54.7%, respectively. Next, we determined the inhibitory effect of 50, 100, and 500 μg/ml SS extract on iNOS and COX-2 mRNA expression by performing reverse transcrip- tion-polymerase chain reaction (PCR), with GAPDH as a positive control. Results of reverse transcription- PCR showed that treatment with 500 μg/ml SS extract decreased the mRNA expression of iNOS and COX-2 by 72.2% and 89%, respectively. These results suggest that the SS extract is a highly valuable natural com- pound because of its functional components and anti-inflammatory activity.

Keywords: Sambucus sieboldiana var. pendula (Nakai), anti-inflammatory, iNOS, COX-2

*Corresponding author

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

© 2017, The Korean Society for Microbiology and Biotechnology

증반응을개시하거나약화시킨다

^.

lipopolysaccharide (LPS)

toll like receptor 4 (TLR4)

heterodimerization

nuclear factor (NF-

^B)

^.

^NF-

^B

cytokine, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)

^.

nitric oxide (NO)

L-arginine

NO synthase (NOS)

^,

arachidonic acid (AA)

cyclooxygenase (COX)

prostaglandin (PG)

^[4

^6].

NO

^NOS

^iNOS

^NO

,

^{[7, 8].}

^AA

^PG

^PGE

^,

^[9].

서양에서딱총나무속식물은관목이면서꽃이많이피고 열매가아름다워조경수로개발되어인기가높으며꽃과열 매는다양한용도로활용되고있다

^.

^(Sambucus sieboldiana var. pendula (Nakai))

^.

^,

^[10

^12].

^.

^LPS

RAW 264.7

^.

재료 및 방법

시료준비

실험에사용된말오줌나무잎과줄기는울릉도에서채취 하였으며

^,

¹⁰

양의

^70%

²⁴

등액과침전물을분리하여동일한방법으로

³

^.

(Whatman No.2)

EYELA evaporator

²⁰

^.

시약 및 기기

세포배양및세포독성측정에사용된세포주는대식세 포인

^{RAW 264.7}

(Korea Cell Line Bank)

^,

haemacytometer (Marienfeld, Germany), 3-[4,5-dimethyl-thiazol-2-yl]-2,5- diphenyl-tetrazoliumbromide (MTT)

Sigma Chemical Co. (USA)

, dimethyl sulfoxide (DMSO)

Bioshop (Canada)

^.

lipopolysaccaride (LPS), griess regent

Sigma Chemical Co. (USA)

^.

¹

iNOS, COX-2

²

anti-rabbit IgG-HRP

Santa Cruz (USA)

^{, mRNA}

^cDNA polymerase kit, GoScript

Reverse Transcription kits

Promega (USA)

^.

ELISA reader (Tecan, Austria), PCR (C-100, Bio-Rad, USA), image quant LAS 4,000 (GE Healthcare Bio-Sciences AB, Sweden), CO

incubator (Vision scientific, Korea), pH meter (Mettler-Toledo AG, Swltzerland), centrifuge (Hanil Science Industrial Co., Korea), autoclave (JS Research Inc., Korea), Davinch-Chemi

imager CAS- 400SM System (Davinch-K Co., Korea)

^.

세포 배양

세포 배양은

10% fetal bovine serum (FBS)

^1%

penicillin/streptomycin (100

^/ml)

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

^, 37

^{, 5% CO}

incubator

^.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay

세포생존율측정은

Carmichael

^[13]

. 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}

^.

^.

세포생존율

^{(%) =}

Nitric oxide (NO) 생성 억제 활성 측정

RAW 264.7 cell

^NO

^Green

^[14]

^griess

^NO

. 6 well plate

^RAW 264.7 cell

^{1 × 10}

^cell/well

^{. 37}

^CO

incubator

²⁴

^{1× PBS}

²

^. Lipopolysaccharide (LPS) 10

^g/ml

^normal

²

²⁴

^,

^griess

96 well plate

¹⁰

^{540 nm}

^{. NO}

^.

NO

^(%) =

Western blot

iNOS, COX-2

cell line RAW 264.7

100 mm tissue culture dish

^{1 × 10}

cells/well

cell seeding

²⁴

^.

^LPS

¹

^g/ml

²

²⁴

⁴⁸

^PBS

²

^. Radio-immunoprecipitation assay (RIPA) buffer 10 ml

complete mini 1 tab

¹⁰⁰

^l

⁴

16,110 ×g

²⁰

^.

BCA protein assay kit

²⁰

^l

의단백질을

10% SDS-PAGE

였다

^.

^transfer

^(BIORAD)

polyvinylidene fluoride (PVDF) membrane

¹

blocking buffer (5% skim milk in TBST)

. iNOS, COX-2,

^-actin

¹

⁴

^overnight

^,

¹⁰

^tris- buffered saline and tween 20 (TBST)

³

^. iNOS

^COX-2

²

anti-rabbit,

^-actin

²

anti-mouse

^1:1,000

²

^{. 3}

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 water

^{1 ml}

16,110 ×g

⁵

. Diethylpyrocarbonate (DEPC)-treated water

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 iNOS, COX-2

^mRNA

^PCR

^.

primer sequences

^{Table 1}

^{. PCR tube}

5× 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

^.

결과 및 고찰

MTT assay에 의한 세포 생존율 측정

세포생존율을확인하기위하여진행한

3-(4,5-dimethylthiazol- 2-yl)2,5-diphenyltetrazolium bromide (MTT)

⎝ ⎠

⎛ ⎞ 100×

Table 1. Sequence of the primers used for RT-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

기질로생세포의미토콘드리아내의호흡연쇄효소에의해 환원되어암적색의

^formazan

^.

반응이일어나지않고생세포에서생성된

^formazan

성량을측정하는시험법이다

^[15].

말오줌나무 추출물의 세포생존율을 대식세포인

^RAW 264.7

^{MTT assay}

^{Fig. 1}

^.

⁵⁰⁰

^g/ml

^108%

^,

western blot

reverse transcription-polymerase chain reaction (RT-PCR)

⁵⁰⁰

^g/ml

^.

Nitric oxide (NO) 저해활성 측정

활성산소중하나이며염증유발에중요한역할을하는것 으로알려진

^NO

^.

^{Fig. 2}

^LPS

^LPS

^NO

^NO

^{. 1,000}

^g/ml

^29.0%

, RAW 264.7

^.

iNOS 및 COX-2 단백질 및 mRNA 발현 억제 효과 측정

nitric oxide (NO), prostaglandin E

(PGE

),

^cytokine

^.

^NO

L-arginine

^NO

synthase (NOS)

^{, NOS}

endothelial NOS, neuronal NOS, inducible NOS (iNOS)

^.

^iNOS

^NO

^[16].

다른 염증인자 중 하나인

cyclooxygenase (COX)

arachidonic acid

prostaglandins

COX-1

^COX-2

^,

향을나타낸다

^{. COX-1}

기능을 하지만

^COX-2

^.

COX-2

^PGE

^,

^,

진시키는것에깊이관여하고있는것으로알려져있다

^[17].

본연구에서는염증발현인자인

^iNOS

^COX-2

western blot

^.

^{RAW 264.7}

^50, 100, 500

^g/ml

²⁴

^{Fig. 3}

^.

^,

house keeping gene

^-actin

positive control

^.

^{Fig. 3}

^{50, 100,} 500

^g/ml

RAW 264.7

^LPS

^iNOS

^COX-2

^{, 500}

^g/ml

^31%, 54%

^.

^C

Fig. 1. Cell viability of extract from Sambucus sieboldiana var.

pendula (Nakai) on macrophage cell (RAW 264.7). 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 Sambucus sieboldiana var. pendula (Nakai) for 24 h.

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

Fig. 2. Effect of Sambucus sieboldiana var. pendula (Nakai) on production of nitric oxide in RAW 264.7 cell. Effect of 70%

ethanol extracts of Sambucus sieboldiana var. pendula (Nakai) on NO production in LPS-induced RAW 264.7 cells. RAW 264.7 cells (1 × 10

cells) were treated with Sambucus sieboldiana var.

pendula (Nakai) 70% ethanol extract and LPS (1 µg/ml) for 24 h.

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

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

하였을때대조군보다감소하는것으로보아단백질발현억 제효과가있음을확인할수있었다

^.

또한

iNOS, COX-2

^mRNA

reverse transcription-PCR

^.

house keeping gene

^GAPDH

positive control

사용하였다

^.

50, 100, 500

^g/ml

^LPS

^iNOS

^COX-2

mRNA

^{Fig. 4}

다

^{. Fig. 4}

^mRNA

^,

^C

^iNOS

Fig. 3. iNOS, COX-2 protein expression rate of extract from

Sambucus sieboldiana var. pendula (Nakai) on macrophage cell (RAW 264.7). (A) iNOS protein expression rate of Sambucus sieboldiana var. pendula (Nakai) extract. (B) COX-2 protein expres- sion rate of Sambucus sieboldiana var. pendula (Nakai) extract.

After RAW 264.7 cells (1 × 10

cells) were started in serum free medium for 1 h the cells were treated with 50, 150 and 500 µg/ml of extract of Sambucus sieboldiana var. pendula (Nakai) for 24 h.

Each values represents mean ± S.D. of three individual experi- ments (Significant as compared to control. *p < 0.05, **p < 0.01).

Fig. 4. iNOS, COX-2 mRNA expression rate of extract from Sambucus sieboldiana var. pendula (Nakai) on macrophage cell (RAW 264.7). (A) iNOS mRNA expression rate of Sambucus sieboldiana var. pendula (Nakai) extract. (B) COX-2 mRNA expres- sion rate of Sambucus sieboldiana var. pendula (Nakai) extract.

After RAW 264.7 cells (1 × 10

cells) were started in serum free medium for 1 h the cells were treated with 50, 150 and 500 µg/ml of extract of Sambucus sieboldiana var. pendula (Nakai) for 24 h.

Each values represents mean ± S.D. of three individual experi-

ments (Significant as compared to control. *p < 0.05, **p < 0.01).

만유사한결과를나타냈고

^COX-2

^iNOS

^COX-2

^.

Kim

^[18]

⁵⁰

^g/ml

^NO

^46.75%

^.

^iNOS

^,

^.

요 약

본연구는새로운기능성화장품소재를개발하기위해천 연물재료인말오줌나무추출물활용가능성연구하였다

^.

^,

^MTT assay

^{, 500}

^g/ml

^100%

^.

^{, LPS}

^NO

^griess

^.

^NO

^.

^LPS

^{RAW 264.7}

(iNOS, COX-2)

^.

^{, iNOS}

^COX-2

50, 100, 500

^g/ml

^western blot

^,

^-actin

^. iNOS

^COX-2

^mRNA

50, 100, 500

^g/ml

^RT-PCR

^,

^GAPDH

^.

^{, western} blot

^iNOS

^COX-2

⁵⁰⁰

^g/ml

31.2%, 54.7%

, iNOS, COX-2

^mRNA

^RT- PCR

⁵⁰⁰

^g/ml

72.2%, 89%

정도로감소하였다

^.

^.

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