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1 J. Exp. Biomed. Sci. 2010, 16(2): 91~95 Anti-inflammatory Properties of Meso-dihydroguaiaretic Acid in Lipopolysaccharide-induced Macrophage Yong-Jae Kim *, Yeo-Jin Kang * and Tack-Joong Kim Division of Biological Science and Technology, College of Science and Technology, Yonsei University, Wonju , Korea Meso-dihydroguaiaretic acid (MDGA) is a medicinal herbal product isolated from the bark of Machilus thunbergii Sieb. et Zucc. (Lauraceae). It exhibits a neuroprotective effect and also exerts cytotoxicity to certain cancer cells. In the present study, we investigated whether or not MDGA inhibits inflammatory reaction through the inhibition of nitric oxide (NO) generation. The results showed that MDGA (5~25 μm) inhibited 100 ng/ml lipopolysaccharide (LPS)- induced NO generation in macrophage Raw cells in a concentration-dependent manner. We also measured the cytotoxic effects of MDGA on Raw cells and found no evidence of cytotoxicity. The inhibition of NO generation by MDGA was consistent with the inhibitory effect on the expression of inducible nitric oxide synthase (inos). In addition, MDGA inhibited the LPS-induced gene expression of interleukin-1β (IL-1β) as well as tumor necrosis factor-α (TNF-α). The present results may provide that MDGA has anti-inflammatory properties through inhibition of the toll-like receptors (TLRs) pathway, and suggest that MDGA can be used as an anti-inflammatory agent. Key Words: Meso-dihydroguaiaretic acid, Toll-like receptor, Nitric oxide 서 염증반응은외부병원균에대한숙주의방어체제로알려져있다. 선천면역은대부분의생물체에서보이는면역체계로, 숙주에침입한병원균을인식하고이를제거하기위한반응이일어나며, 생체내에질병을일으킬수있는병원균중의하나인그람음성균의세포외벽은 lipopolysaccharide (LPS) 로이루어져있다 (Ulevitch et al., 2004). 대식세포의세포벽에는이와같은분자를인식하여활성화하는 toll-like receptor (TLR) 들을갖추고있다 (Yun et al., 2009). TLR family는현재 13개의종류가밝혀져있으며, 이중 LPS를인지할수있는수용체는 TLR4 이다 (Yun et al., 2009). TLR4에의해서 LPS가인식되면그아래의분자들의활성화가생겨신호전달과정이일어나게된다 (Moon and Pyo, 2007). * 접수일 : 2010년 4월 10일 / 수정일 : 2010년 4월 30일채택일 : 2010년 4월 30일 교신저자 : 김택중, ( 우 ) 강원도원주시흥업면매지리 234, 연세대학교과학기술대학생명과학기술학부 Tel: , Fax: ktj@yonsei.ac.kr * Yong-Jae Kim and Yeo-Jin Kang contributed equally to this work 론 TLR4 신호전달경로에는 MyD88 의존성신호전달과비의존성신호전달두가지과정이있다. MyD88 의존성신호전달은 TLR4의 toll/interluekin-1 receptor (TIR) domain 과 MyD88이상호조절하여 IL-1 receptor-associated kinase (IRAK) 을활성화시킨다. IRAK는 tumor necrosis factor receptor-associated factor 6 (TRAF6) 와복합체를이루어 TRAF6를활성화하여신호전달을일으킨다 (Takeda and Akira, 2004). Nitric oxide (NO) 는체내에서혈관확장자로서조절을하고조직을손상시킨다. 이와같은 NO를합성하는효소가 nitric oxide synthase (NOS) 이다. NOS는 neural NOS (nnos), endothelial NOS (enos), inducible NOS (inos) 의종류가있다 (Min et al., 2009). 이중 nnos와 enos는체내에항상발현되어생체내 NO의양을소량으로존재하게하여항상성을유지하는조절자역할을한다. 그러나 LPS에의해유도되는 inos는 L-arginine을 citrulline 으로전환하며 NO를대량으로생성하게된다 (Min et al., 2009). NO가과량으로생성되게되면류마티스관절염이나천식, 파킨슨병등의염증성질병에노출되게되며, 이러한외부병원균의침입에대한 inos의발현은염증반응을조절한다 (Min et al., 2009). 후박나무 (Machilus thunbergii) 에서추출한 Meso

2 H H 3 3 CO HO 상온에서반응시켰다. NaNO 2 standard curve를사용하여 NO의생성량을 microplate reader (BioTek Instruments Inc., Winooski, VT, USA) 를이용해 550 nm에서측정하였다. 세포독성 OCH 3 3 OH Fig. 1. The structure of meso-dihydroguaiaretic acid (MDGA). dihydroguaiaretic acid (MDGA) 는 glutamate나 staurosporine 에의해유도되는신경독성에대해신경보호제로서효과가있다고보고되었다 (Ma et al., 2005, Ma et al., 2006). 그리고, MDGA는피부노화예방을위한 matrix metalloproteinase 억제효과가보고되었다 (Moon and Jung, 2005; Moon and Jung, 2006). 최근, MDGA는골수에서파생된비만세포에서다른염증과알러지매개물질인 prostaglandins D 2 (PGD 2 ), leukotriene C 4 (LTC 4 ) 를억제함을확인하였다 (Moon et al., 2008). 본연구에서후박나무에서분리한 MDGA를이용하여 Raw 세포에서항염증효과를연구하였으며, MDGA 의세포독성을포함해 LPS에의해유도된 NO의생성량변화와염증관련유전자의발현변화를분석비교하였다. 재료및방법세포배양 MDGA는 Dr. Sang Hyun Sung (Seoul National University, Seoul, Korea) 으로부터공급받았다 (Ma et al., 2004, Fig. 1). Raw 세포는 10% FBS, 100 U/ml penicillin, 100 μg/ ml streptomycin이들어있는 Dulbecco's modified Eagle's medium 에서배양하였다. 세포는 5% CO 2 배양기에서배양하였다. Nitrite Assay Raw 세포에 24 well에서 MDGA를처리하고 LPS (100 ng/ml) 를 18시간동안처리하였다. 상층액을동량의 Griess 시약 (1% sulfanilamide, 0.1% naphthylethylenedaiamine dihydrochloride, 2.5% phosphoric acid) 와혼합하여, 10분간 Raw 세포에 96 well에서 MDGA를 17시간동안처리하였다. EZ-Cytox kit (Daeil Lab., Seoul, Korea) 를 10 μl 씩넣어주고 1시간동안 37 에서배양시킨후, microplate reader를이용하여 450 nm에서흡광도를측정하였다. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Raw 세포에 24 well에서 MDGA를처리하고 LPS (100 ng/ml) 를 18시간동안처리하였다. Trizol 시약을이용하여 total RNA를추출하였다. Total RNA는 1 μg으로정량하여 PreMix RT/PCR kit (Bioneer, Daejeon, Korea) 를이용하여 PCR을수행하였다. 이때사용한 primer: inos (sense) 5'-ATG GCT TGC CCC TGG AAG TTT CTC-3'; inos (antisense) 5'-CCT CTG ATG GTG CCA TCG GGC ATC TG-3'; IL-1β (sense) 5'-CAG GATGAG GAC ATG AGC ACC-3'; IL-1β (antisense) 5'-CTC TGC AGA CTC AAA CTC CAC-3'; TNF-α (sense) 5'-ATG AGC ACA GAA AGC ATG ATC-3'; TNF-α (antisense) 5'-TAC AGG CTT GTC ACT CGA ATT-3'; β-actin (sense) 5'-GTG GGC CGC CCT AGG CAC CAG-3'; β-actin (antisense) 5'-GGA GGA AGA GGA TGC GGC AGT-3' 이고, PCR은 94, 5 min 후에 94, 45 sec; 56, 45 sec ; 72, 1 min을 40 cycle 수행하고 72, 10 min 수행하였다. PCR 산물은 2% agarose gel에서 EtBr에의해자외선으로관찰하였다. 통계분석실험결과는평균 ± 표준편차로표시하였으며, 통계처리는 One-way ANOVA, Dunnett's test를따랐다. P value가 <0.05와 <0.01인것을통계학적으로의미가있다고간주하였다. 결과및고찰대식세포에서 LPS에의해유도된 NO 생산에있어 MDGA 의억제효과대식세포가 LPS를인식하게되면, 이를세포내 TLR4 에서인지하여, 세포내신호전달과정이생긴다. 이과

A Fig. 2. Effect of MDGA on LPS-induced NO production in Raw 264.7 cells.

The isolated supernatant analyzed for NO levels by Griess reaction. Each 100 μl of culture supernatant was mixed with an equal volume of Griess reagent.

) from three independent experiments, each performed in triplicate ( * P<0.05, ** P<0.001). B 정에서 MyD88 의존적인신호전달이생기게되고, 이들의결과로염증매개물질들을방출한다 (Yun et al., 2009). 우리는먼저대식세포로알려진 Raw 264.

3 A Fig. 2. Effect of MDGA on LPS-induced NO production in Raw cells. Cells were plated at cells/ml in 24 well plates and stimulated with LPS (100 ng/ml) in the absence or presence of MDGA (5 μm, 10 μm, and 25 μm) for 18 h. The isolated supernatant analyzed for NO levels by Griess reaction. Each 100 μl of culture supernatant was mixed with an equal volume of Griess reagent. The culture supernatant incubated at room temperature for 10 min. The absorbance at 550 nm was measured by ELISA. The results are presented as the mean values (± S.D.) from three independent experiments, each performed in triplicate ( * P<0.05, ** P<0.001). B 정에서 MyD88 의존적인신호전달이생기게되고, 이들의결과로염증매개물질들을방출한다 (Yun et al., 2009). 우리는먼저대식세포로알려진 Raw 세포에서 MDGA가 LPS에의해유도된 NO의생성을억제할수있는지연구하였다. NO의생성량을측정하기위하여 Raw 세포에, MDGA를각각 5, 10, 25 μm이되도록전처리하고 30분후, LPS를 100 ng/ml의농도로처리하여 18시간배양하였다. 실험결과, Raw 세포에서 LPS 처리에의해 0.36 μm에서 μm로 NO의양이증가됨을확인할수가있었다. 또한 MDGA에의해농도 5, 10, 25 μm에의해각각 9.90, 6.86, 1.32 μm까지농도의존적으로 NO의생성량이줄어드는것을확인할수있었다 (Fig. 2). 이러한연구결과는 MDGA가대식세포에서 LPS에의해유도되는신호전달과정에관여하여농도의존적으로염증매개물질인 NO를억제한다는것을보여준다. MDGA 의세포독성효과 MDGA에의한 NO 생성량의감소가세포독성에의한세포수의감소가원인이었는지확인하기위해세포생존률실험을진행하였다. Raw 세포에 MDGA를각 5, 10, 25 μm를처리하여, NO 생성량측정과동일한조건으로 18시간배양하였다. 우선광학현미경을이용하여 Fig. 3. Effect of MDGA on viability of Raw cells. Cells were treated with MDGA (5 μm, 10 μm, and 25 μm) for 18 h. (A) Cell morphology was observed under an optical microscope. a: Control, b: 5 μm MDGA, c: 10 μm MDGA, d: 25 μm MDGA. (B) Cell viability was examined by EZ-Cytox kit. The results were expressed as percentage of surviving cells relative to control cells. 세포의형태와생존률을확인한결과 MDGA의처리한세포군과처리하지않은세포군에서특별한변화를관찰할수없었다 (Fig. 3A). 생화학적인방법을이용한 Ez- Cytox kit에의해세포생존률을확인한결과, 세포생존률은 MDGA 5, 10, 25 μm 처리한실험군에서감소하지않음을확인하였다 (Fig. 3B). 이는현미경을이용한형태학적으로관찰한결과와일치한다. 이러한연구결과들은 MDGA가세포에직접적인독성을나타내지않고, 염증매개물질인 NO를억제한다는것을나타낸다. LPS 에의해유도된염증매개자들에있어서 MDGA 의효과 NO 생성량의억제와관련하여 MDGA의세포내작용기작을확인하기위해, 우리는 LPS에의해유도되는염증매개자들의유전자발현량에대한 MDGA의효과에대한실험을진행하였다. 면역반응중에생성되는

A B C Fig. 4. Effect of MDGA on the LPS-induced expression of inos, TNF-α and IL-1β in Raw 264.7 cells.

Total RNA (1 μg) was prepared and analyzed by RT-PCR as described in Materials and methods. RT-PCR performed for inos (A), TNF-α (B), IL-1β (C) and β-actin expression.

inos에의해세포내 NO의양이과도하게증가되고, NO는세포내전달자역할을함으로써중추신경계, 심혈관계, 면역시스템등에중요한역할을하게된다 (Oh et al., 2008). 그러므로, 먼저 LPS에의해유도된 inos 유전자발현량의변화를분석하였다.

4 A B C Fig. 4. Effect of MDGA on the LPS-induced expression of inos, TNF-α and IL-1β in Raw cells. Cells were plated at cells/ml, stimulated with LPS (100 ng/ml) in the absence or presence of MDGA (5 μm, 10 μm, and 25 μm) for 18 h. Total RNA (1 μg) was prepared and analyzed by RT-PCR as described in Materials and methods. RT-PCR performed for inos (A), TNF-α (B), IL-1β (C) and β-actin expression. The β-actin gene was used for normalization and relative activities were quantified by scanning densitometry, with the shown values representing the mean ± SD of three different assays. ( ** P<0.001). inos에의해세포내 NO의양이과도하게증가되고, NO는세포내전달자역할을함으로써중추신경계, 심혈관계, 면역시스템등에중요한역할을하게된다 (Oh et al., 2008). 그러므로, 먼저 LPS에의해유도된 inos 유전자발현량의변화를분석하였다. 그결과, MDGA를 5, 10, 25 μm 처리한실험군에서 inos 유전자발현이각각 11, 32, 65% 가감소함을확인하였다 (Fig. 4A). LPS 에의해유도되는 TLR4 신호전달을통해 NF-κB가활성화되어 TNF-α, IL-1β 등염증을유발시키는 proinflammatory cytokine이나염증매개물질들이생성된다 (Yun et al., 2009). 그렇기때문에, TNF-α 와 IL-1β의유전자발현에있어 MDGA의효과를확인한결과, MDGA는 TNF-α 유전자의발현량을농도의존적으로감소시켰으며 (Fig. 4B), IL-1β 유전자발현에서도 MDGA에의해농도의존적인감소의결과를확인할수있었다 (Fig. 4C). 이들의결과는 MDGA가 LPS에의해유도되는 proinflammatory cytokine으로알려진 inos, IL-1β와 TNF-α의유전자발현억제를통해항염증반응이나타남을알수있었다. 이번연구를통해 MDGA가 LPS에의해유도되는

5 inos mrna 의억제를통해 NO의생산을억제하는효과를확인하였다. 또 LPS에의해유도되는 pro-inflammatory cytokine인 IL-1β mrna와 TNF-α mrna의발현량도억제하는것을확인하였다. 최근 Moon et al. 은 MDGA가골수에서파생된비만세포에서다른염증매개물질인각각 COX-2와 5-LOX에의해생성되는 PGD 2, LTC 4 를억제함을보고하였다 (Moon et al., 2008). 이는이번결과와함께 MDGA가염증및알러지성질병의치료제로서개발가능성이있음을의미한다. 이번실험결과를통해 MDGA가대식세포에서 TLR 신호전달에의해조절되는염증반응이나류마티스관절염이나천식, 파킨슨병등의여러가지염증성질병들의치료제로서사용될수있는가능성을제시하지만, MDGA에의한 Raw 세포에서의이러한세포내반응을조절하는인자는매우다양하며복잡하기때문에본연구를바탕으로더명확하고구체적인신호전달경로를파악하는것이필요하다. 그러므로앞으로항염증작용과관련된 MDGA의신호전달경로에대한지속적인연구가수행되어야할것이다. 감사의글본연구를위해 MDGA를제공해주신 Dr. Sang Hyun Sung (Seoul National University, Seoul, Korea) 과 Dr. Seung Hyun Kim (Elcomscience Co. Ltd., Seoul, Korea) 께감사의말씀을전합니다. REFERENCES Ma CJ, Kim SR, Kim J, Kim YC. Meso-dihydroguaiaretic acid and licarin A of Machilus thunbergii protect against glutamateinduced toxicity in primary cultures of a rat cortical cells. Br J Pharmacol : Ma CJ, Lee MK, Kim YC. Meso-Dihydroguaiaretic acid attenuates the neurotoxic effect of staurosporine in primary rat cortical cultures. Neuropharmacology : Ma CJ, Sung SH, Kim YC. Neuroprotective lignans from the bark of Machilus thunbergii. Planta Med : Min HY, Kim MS, Jang DS, Park EJ, Seo EK, Lee SK. Suppression of lipopolysaccharide-stimulated inducible nitric oxide synthase (inos) expression by a novel humulene derivative in macrophage cells. Int Immunopharmacol : Moon EY, Pyo S. Lipopolysaccharide stimulates Epac1-mediated Rap1/NF-kappaB pathway in Raw murine macrophages. Immunol Lett : Moon HI, Chung JH. Meso-dihydroguaiaretic acid from Machilus thunbergii SIEB et ZUCC., and its effects on the expression of matrix metalloproteinase-2, 9 cause by ultraviolet irradiated cultured human keratinocyte cells (HaCaT). Biol Pharm Bull : Moon HI, Jung JC. Effect of meso-dihydroguaiaretic acid from Machilus thunbergii Sieb et Zucc on MMP-1 expression in heat shock-induced cultured primary human fibroblasts. Phytother Res : Moon TC, Seo CS, Haa K, Kim JC, Hwang NK, Hong TG, Kim JH, Kim do H, Son JK, Chang HW. Meso-dihydroguaiaretic acid isolated from Saururus chinensis inhibits cyclooxygenase- 2 and 5-lipoxygenase in mouse bone marrow-derived mast cells. Arch Pharm Res : Oh JH, Lee TJ, Park JW, Kwon TK. Withaferin A inhibits inos expression and nitric oxide production by Akt inactivation and down-regulating LPS-induced activity of NF-kappaB in RAW cells. Eur J Pharmacol : Takeda K, Akira S. TLR signaling pathways. Semin Immunol : 3-9. Ulevitch RJ, Mathison JC, da Silva Correia J. Innate immune responses during infection. Vaccine : Yun SM, Park SJ, Lee AN, Ahn SI, Youn HS. Oak Wood Vinegar Suppresses the Expression of Cyclooxygenase-2 Induced by TLR4 Agonist. J Exp Biomed Sci :

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