[논문]홍삼추출액은 lipoteichoic acid로 자극된 소교세포에서 Akt 및 MAPK 의존적으로 heme oxygenase-1 발현을 유도함으로써 NO 생성을 억제함

신지은; 이경민; 김지희; 이스칸더 마디; 김영희

doi:10.5352/jls.2019.29.4.402

홍삼추출액은 lipoteichoic acid로 자극된 소교세포에서 Akt 및 MAPK 의존적으로 heme oxygenase-1 발현을 유도함으로써 NO 생성을 억제함
A Formulated Korean Red Ginseng Extract Inhibited Nitric Oxide Production through Akt- and Mitogen Activated Protein Kinase-dependent Heme Oxygenase-1 Upregulation in Lipoteichoic Acid-stimulated Microglial Cells 원문보기

생명과학회지 = Journal of life science, v.29 no.4 = no.228, 2019년, pp.402 - 409

신지은 (부산대학교 자연과학대학 분자생물학과) , 이경민 (부산대학교 자연과학대학 분자생물학과) , 김지희 (BK21플러스 장수해양바이오사업단) , 이스칸더 마디 (부산대학교 자연과학대학 분자생물학과) , 김영희 (부산대학교 자연과학대학 분자생물학과)

초록
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생삼을 쪄서 건조시킨 홍삼은 전통적으로 사용되고 있는 약재로서 면역, 내분비 및 중추신경계 작용을 증진시키며 염증을 억제하는 효과가 있는 것으로 알려져 있다. 본 연구에서는 그람 양성균의 세포벽 성분인 lipoteichoic acid (LTA)에 의한 염증반응에 홍삼추출액(RGE)이 항염증 효과를 가지는지 관찰하고 그 작용 기전을 연구하였다. BV-2 소교세포에서 RGE는 세포에 독성을 유도하지 않으면서 LTA로 인한 nitric oxide (NO)의 생성과 inducible nitric oxide synthase (iNOS) 발현을 억제하였으며, NF-kB p65의 핵으로의 이동과 IkB-a의 분해 또한 억제하였다. 한편, RGE는 농도의존적으로 heme oxygenase-1 (HO-1)의 발현을 유도하였으며, HO-1 siRNA를 처리했을 때는 RGE가 iNOS의 발현을 억제하지 못하였다. RGE는 HO-1의 발현에 관여하는 전사인자인 nuclear factor E2-related factor 2 (Nrf2)를 핵으로 이동을 촉진시켰다. 또한 RGE에 의한 HO-1의 발현은 phosphatidylinositol-3-kinase(PI-3K) 및 MAPK 억제제에 의해 감소되었으며, RGE가 Akt와 ERK, p38, JNK의 인산화를 유도하였다. 이상의 결과를 종합해보면, RGE는 PI-3K/Akt 및 ERK, p38, JNK 신호전달과정을 통해 HO-1의 발현을 유도함으로써 NO와 같은 염증매개물질의 생성을 억제한다는 것을 알 수 있다. 그러므로 홍삼추출액은 그람 양성균에 의한 신경염증과 염증관련 신경계 질환의 치료제로서 사용될 수 있을 것이라 사료된다.

Abstract ▼ AI-Helper

Korean red ginseng made from steaming and drying fresh ginseng has long been used as a traditional herbal medicine due to its effects on the immune, endocrine, and central nerve systems and its anti-inflammatory activity. In this study, we investigated the molecular mechanism responsible for the anti-inflammatory effects of a formulated Korean red ginseng extract (RGE) in response to lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria. RGE inhibited LTA-induced nitric oxide (NO) secretion and inducible nitric oxide synthase (iNOS) expression in BV-2 microglial cells, without affecting cell viability. RGE also inhibited nuclear translocation of nuclear factor kappa B ($NF-{\kappa}B$) p65 and degradation of $I{\kappa}B-{\alpha}$. In addition, RGE increased the expression of heme oxygenase-1 (HO-1) in a dose-dependent manner, and the inhibitory effect of RGE on iNOS expression was abrogated by small interfering RNA-mediated knockdown of HO-1. Moreover, RGE induced nuclear translocation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor that regulates HO-1 expression. Furthermore, the phosphoinositide-3-kinase (PI-3K) inhibitor and mitogen-activated protein kinase (MAPK) inhibitors suppressed RGE-mediated expression of HO-1, and RGE enhanced the phosphorylation of Akt, extracellular signal-regulated kinases (ERKs), p38, and c-JUN N-terminal kinases (JNKs). These results suggested that RGE suppressed the production of NO, a proinflammatory mediator, by inducing HO-1 expression via PI-3K/Akt- and MAPK-dependent signaling in LTA-stimulated microglia. The findings indicate that RGE could be used for the treatment of neuroinflammation induced by grampositive bacteria and that it may have therapeutic potential for various neuroinflammation-associated disorders.

주제어

표/그림 (5)

그림 Fig. 1. RGE inhibits NO production and iNOS expression in LTA-stimulated microglial cells.
그림 Fig. 2. RGE inhibits LTA-induced NF-κB activation.
그림 Fig. 3. RGE-induced HO-1 suppresses iNOS expression.
그림 Fig. 4. Effects of RGE on nuclear translocation of Nrf2.
그림 Fig. 5. Involvement of Akt and MAPKs in RGE-mediated expression of HO-1.

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제안 방법

To investigate whether RGE could abrogate LTA-mediated neuroinflammation, we examined the effects of RGE on NO production expression of in BV-2 microglial cells. Stimulation of BV-2 microglial cells with LTA increased NO synthesis and iNOS expression, whereas RGE pretreatment significantly attenuated the LTA-induced NO synthesis and iNOS expression in a dose-dependent manner (Fig.
To investigate whether RGE induces HO-1 expression in microglia, BV-2 cells were incubated with various concentrations of RGE. The HO-1 protein level was significantly increased after 6 hr by RGE in a dose-dependent manner (Fig.

대상 데이터

Louis, MO, USA). A formulated Korean red ginseng extract (Hansamin Gold) was purchased from Nonghyup Red Ginseng (Seoul, Korea). HO-1 siRNA, and antibodies for iNOS, HO-1, NF-κB p65, inhibitor of kappa B-alpha (IkB-a), histone deacetylase 3 (HDAC3), ERK, JNK, p38, Akt, α-tubulin, and b-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

이론/모형

NO synthesis in cell cultures was measured by a microplate assay method. To measure nitrite, 100 ml aliquots were removed from conditioned medium and incubated with an equal volume of the Griess reagent (1% sulfanilamide/0.
The cytotoxicity of RG was assessed using the microculture tetrazolium (MTT)-based colorimetric assay. The remaining cells after Griess reaction were used for MTT assay.

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