Red Ginseng Ethanol Extract Suppressed Ag I/II-induced Up-expression of Inflammatory Mediators in RAW 264.7 Macrophages

홍삼에탄올추출물의 염증유발인자에 대한 억제효과

최경민¹, 황승미¹, 임지예¹, 고은실¹, 박종혁², 문정혜², 이민정³, 장지은³, 차정단¹*

1

(

)

2

(

)

3

(

)

Received: February 4, 2015 / Revised: June 1, 2015 / Accepted: June 1, 2015

고려인삼

(Panax ginseng C. A. Meyer)

⁶⁰

^70%

^,

^, polyacetylene,

(acidic peptide)

(ginsenosides)

[3, 8, 9].

^,

(ginsenoside)

^,

(acidic polysaccharide), panaxydol

polyacetylene

^,

^(maltol)

유하고있다

^{[3, 38].}

조과정중화학구조가변환되어새로운사포닌성분이생성 되기도하고일부생리활성성분의함량이증가하기도한 다

^{[22, 35].}

MG-Rb1, MG-

Rb2, MGRc, MG-Rd

(triterpeneoligoglygoside)

^-

^(malonyl- ginsenoside; MG)

^,

^MG

malonic acid

^G- Rg3, GRg2, G-Rh2, G-Rs1, G-Rs2, Rh4

[19, 29, 35].

^,

^,

^,

[10, 32, 33].

^,

^,

^,

G-Rg3, G-Rf

^G-Rh2

^{[4, 11].}

염증은여러가지형태의감염이나생체내대사산물중의 자극성 물질에 대한 생체 방어반응이며

^,

^, prostaglandin E

(PGE

), lysosomal enzyme, free radicals

^{[6, 28].}

tumor necrosis factor-

^(TNF-

⁾ Red Ginseng Ethanol Extract Suppressed Ag I/II-induced Up-expression of Inflammatory Mediators in RAW 264.7 Macrophages

Kyung-Min Choi

, Seung-Mi Hwang

, Ji-Ye Lim

, Eun-Sil Ko

, Jong-Hyuk Park

, Jung-Hye Moon

, Min-Jung Lee

, Ji-Eun Jang

, and Jeong-Dan Cha

*

1

Department of Research Development, Institute of Jinan Red Ginseng, Jinan 567-801, Republic of Korea

2

Imsil Research Institute of Cheese Science, Imsil 566-881, Republic of Korea

3

Berry&Bio Food Research Institute, Gochan 585-943, Republic of Korea

In this study, we investigated the effects of 40% ethanol extract of Red Ginseng (RGE) on the productions of inflammatory pro- teins in Antigen I/II (Ag I/II)-N, a recombinant protein isolated from Streptococcus mutans -stimulated in RAW 264.7 cells. RGE inhibited the expression of Ag I/II-N-induced pro-inflammatory mediators, both mRNA and protein synthesis levels, without any cytotoxic effects. Moreover, RGE significantly inhibited Ag I/II-N induced NF- κB translocation into the nucleus by preventing the degradation of inhibitor κB-α. In conclusion, RGE down regulates the expression of pro-inflammatory genes involved in the synthesis of NO and iNOS in Ag I/II-N-stimulated RAW 264.7 cells by suppressing NF- κB activity.

Keywords: Panax ginseng, red ginseng, ginsenosides, anti-inflammation, inflammatory mediators, cytokines

*Corresponding author

Tel: +82-63-432-0913, Fax: +82-63-432-0910 E-mail: [email protected]

© 2015, The Korean Society for Microbiology and Biotechnology

와같은친염증성사이토카인의발현이유도되고

, inducible nitric oxide synthase (iNOS)

cyclooxygenase-2 (COX-2)

를코딩하는유전자의발현을 자극하게되어

nitric oxide

(NO)

^PGE

^[27].

, iNOS, COX-2, TNF-

, Interleukin-6 (IL- 6)

nuclear factor-

^{B (NF-}

^B)

^,

^NO

^shock

^,

^, mutagenesis,

[2, 26, 28, 37].

홍삼으로부터분리된산성다당체성분은대식세포로부터

iNOS

^NO

되었으며

^,

^NO

TNF-

[12, 24, 40].

^NF-

^B

AP-1

^MAPKs

[24].

Streptococcus mutans

착관련단백질로알려진

^{Ag I/II}

사용하였다

^.

S. mutans

S. mutans

단백질인

^{Ag I/II}

과정이시작되는가장중요한단계라할수있다

^.

^S.

mutans

중이며

^,

^{Ag I/II}

어지고있다

^{[17, 21].}

인발현변화를통한항염증효과를규명하고자한다

^.

홍삼을

^40%

에농도별로처리한후

²⁴

^MTT

하여세포독성을확인하였다

^{. NO}

^PGE

찰하기대식세포에염증유발물질인구강미생물인

Streptococcus mutans

Ag I/II-N

^(RGE)

²⁴

양하였다

^{. NO}

Griess reaction

^{[14, 18].}

^,

PGE

^ELISA

다

^.

^{, RGE}

¹²⁰

^g/ml

^{. NO}

^{, 10}

^g/ml

^58.44%

^{(Fig. 1).}

^{, PGE}

^{, 10}

^g/ml

^43.67%

(Fig. 1). NO

^iNOS

게분비된

^NO

^{, DNA}

손상

,

,

^,

[2, 34, 36].

염증관련사이토카인

^{, TNF-}

^{, IL-1}

^{, IL-6}

^IL-10

^RGE

Ag I/II-N

^.

ELISA

^.

^,

백질발현변화를확인하기위해

^RGE

세포를모아

RIPA buffer

^.

분리된단백질정량은

^BCA

였고

^{, 40}

^g

⁸

12% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE)

, PVDF membrane

^blotting

^, iNOS, COX-2, ERK, JNK, P38,

^NF-

^B

Fig. 1. Effects of the ethanol extract of red ginseng (RGE) on the production of NO and PGE

in Ag I/II-N-stimulated RAW 264.7

cells. The cells were pretreated with the indicated doses of RGE for 1 h before stimulating the cells with Ag I/II-N 0.25 μg/ml for a further

24 h. (A) The nitrite concentration in the culture supernatants was then determined for NO production. (B) The PGE

concentration in the

culture supernatants was determined, as described in the Materials and methods. The results are reported as a mean ± SE of triplicate

experiments. ***p < 0.001 vs. Ag I/II-N treatment alone (ANOVA, Scheffe’s test).

다

. Horse radish peroxidase

chemiluminescence detection system (Fisher)

^.

iNOS

^NO

^iNOS

^{, RGE 5}

^g/ml

^iNOS

음을확인할수있었다

^{(Fig. 2).}

^,

^PGE

소인

^{, COX-2}

^RGE

증이유발된

RAW 264.7

^,

iNOS

(Fig. 2). iNOS

COX-2 mRNA

Fig. 2. Effects of the ethanol extract of red ginseng (RGE) on the protein and mRNA expression of iNOS and COX-2 in the Ag I/II-N-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with the indicated doses of RGE for 1 h followed by stimulation with 0.25 μg/ml Ag I/II-N for another 12 h. The induction of iNOS and COX-2 proteins and mRNA was determined by Western blot analysis (A) and RT-PCR (B). A representive result from three independent experiments is shown.

Fig. 3. Effects of the ethanol extract of red ginseng (RGE) on Ag I/II-N-induced TNF- α, IL-1β, IL-6, and IL-10 production and mRNA

expressions. Cells were pretreated with different concentrations of RGE for 1 h, then with 0.25 μg/ml Ag I/II-N, and incubated

for 6-24 h. The levels of TNF-α, IL-1β, IL-6, and IL-10 in macrophage culture medium were measured by EIA kits according to the man-

ufacturer’s instructions (R&D Systems).

의존적으로감소됨을확인하였다

^.

TNF-

TNF receptor (TNFR)

^{, IL-} 6

^IL-6

^(receptor)

^{[7, 23].}

분비는

^TNF-

^IL-6

다양한세포와조직에기능저해를일으켜염증성장질환

^,

[15, 16]. RGE

Ag I/II-N

발된

RAW 264.7

사이토카인생성을확인하였다

^.

^RGE

⁵

^g/ml

에서

^TNF-

^50%

었으며

^{, IL-1}

^IL-6

¹⁰

^g/ml

^50%

^46%

^{. IL-10}

¹⁵

^g/ml

35%

(Fig. 3).

MAPKs

을하는단백질인산화효소가연속되는경로로서

^,

^,

^,

^,

멸등을매개한다

[21, 39]. ERK

해활성화되고

^,

^, NF-

^B

^{, p38}

삼투압과관련된세포스트레스에의해활성화되며

^{, TNF-}

^stability

translation

[13, 39].

^MAPKs

과

^{, ERK}

^p38

확인되었으나

^{, JNK}

^{(Fig. 4).}

LPS

^TNF-

^I

^B-

분해되고

^NF-

^{B p65}

전자의프로모터에존재하는

^NF-

^{B element}

[20, 30, 31].

에서 관련전사인자

^NF-

^{B p65}

^,

RGE

^p65

^RGE

^,

^p65

^I

^B-

^{(Fig. 5).}

위결과는

iNOS

Ag I/II-N

^ERK

^p38

^NF-

^{B p65}

의해유도되기때문에이농도에서

^NF-

^{B p65}

단되어

^iNOS

^.

최근연구에의하면

^NSAID

기능저하를 일으키며 특히

^,

^PGE

prostacyclin

용이보고되고있다

^{[5, 14].}

COX-2

^NSAID

Fig. 4. The anti-inflammatory effect of the ethanol extract of red ginseng (RGE) is closely related to the suppression of MAPK-mediated signaling pathways in the Ag I/II-N-stimu- lated RAW 264.7 cells. A, RAW 264.7 cells were pretreated with the indicated concentrations of RGE for 1 h followed by stimulation with 0.25 μg/ml Ag I/II-N for another 4 h. The effects of the RGE on Ag I/II-N induced phosphorylation of MAPK were determined by Western blot analysis. The results from three independent experiments were quantified by densitometry.

Fig. 5. Effects of the ethanol extract of red ginseng (RGE) on the activation of NF- κB in Ag I/II-N-stimulated RAW 264.7 cells. Nuclear extracts were prepared from RAW 264.7 cells that had been pretreated with the indicated doses of the RGE for 1 h before being stimulated with 0.25 μg/ml Ag I/II-N for another 2 h.

The nuclear and cytosolic proteins were subjected to immunoblot

analysis in order to measure the level of nuclear translocated p65

protein. The results from three independent experiments were

quantified by densitometry.

되므로부작용이좀더적을것으로판단하여

^,

유래의

^silymarin

⁶

(shinbaro)

^COX-2

노력이진행되고있다

[1, 18, 25].

에탄올추출물

^(RGE)

^TNF-

^{, IL-1}

^IL-6

^iNOS

^COX-2

^{, NF-}

^B

^ERK

^p38

^.

요 약

본연구에서

^,

S. mutans Ag I/II

^40%

^.

Ag I/II

mRNA

^.

^NF-

^p65

^.

^40%

^NF-

^B

NO

^iNOS

며

^,

^.

Acknowledgments

This research was supported by the Ministry of Knowledge Economy (MKE-R0002038).

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