배초향이 RAW 264.7의 염증인자 생성에 미치는 영향

박완수 ( Wansu Park ) 대한본초학회 2022 大韓本草學會誌 Vol.37 No.1

Objectives : The aim of this study was to investigate the effect of water extract of Agastache rugosa (AR) on productions of inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 mouse macrophages. Methods : Cell viabilities were measured with MTT assay. The production of nitric oxide (NO) from RAW 264.7 cells was measured with Griess reagent assay. The production of cytokines in RAW 264.7 cells was measured with multiplex cytokine assay. Results : AR showed no cytotoxicity on RAW 264.7 cells. AR at concentrations of 25, 50, 100, and 200 μg/mL significantly inhibited NO production in LPS-stimulated RAW 264.7 cells. AR at concentrations of 50, 100, and 200 μg/mL significantly inhibited productions of TNF-α and IL-1β in LPS-stimulated RAW 264.7 cells; AR at concentrations of 50 and 200 μg/mL significantly inhibited productions of RANTES (CCL5) in LPS-stimulated RAW 264.7 cells; AR at concentrations of 100 μg/mL significantly inhibited productions of macrophage inflammatory protein-1β in LPS-stimulated RAW 264.7 cells; AR at concentrations of 50, 100, and 200 μg/mL significantly increased productions of IP-10 (CXCL10) in LPS-stimulated RAW 264.7 cells; AR at concentrations of 100 and 200 μg/mL significantly increased MCP-1 (CCL-2) in LPS-stimulated RAW 264.7 cells; AR at concentrations of 50 and 100 μg/mL significantly increased productions of IL-10 in LPS-stimulated RAW 264.7 cells. Conclusions : AR might have immunomodulatory effects on productions of NO, cytokines, and chemokines in LPSstimulated RAW 264.7 mouse macrophages.

LPS로 자극된 macrophage RAW264.7 세포에서 ascochlorin에 대한 단백질체 분석

장영채(Young-Chae Chang) 한국생명과학회 2008 생명과학회지 Vol.18 No.6

아스코크로린(Ascochlorin, ASC)은 Ascochyta viciae로부터 추출된 프레닐페놀 물질로, 혈청 콜레스테롤과 트리글리세라이드 수치를 감소시키고 종양 성장을 억제한다는 연구 결과가 보고되어 있다. 본 논문에서는 아스코크로린이 생리학적 혹은 병리학적인 작용과 염증반응에서 약리학적으로 유도되는 반응을 어떻게 조절하며, 이러한 메커니즘에 대해 이해하기 위해 mouse macrophage Raw264.7 세포에 아스코크로린을 처리하여 이에 대한 프로테옴의 특이적인 발현에 대해 분석하였다. 따라서 본 연구는 LPS를 처리한 mouse macrophage Raw264.7 세포에 아스코크로린을 처리하여 염증과정에 관련된 단백질의 발현 양상을 확인하기 위해 프로테오믹스를 시행하였다. Mouse macrophage RAW264.7 세포에 아스코크로린을 처리한 조건과 무처리한 조건으로 나누어 two-dimensional electrophoresis (2-D SDS-PAGE), matrix-assisted laser de-sorption/ionization mass spectrometry (MALDI-TOF-MS) 와 bioinformatics 방법으로 아스코크로린을 처리한 mouse macrophage Raw264.6 세포의 프로테옴을 분석하였다. 그 결과 mouse macrophage Raw264.7 세포에 아스코크로린 처리 시 Calreticulin이 4배 감소, β-actin도 4배 감소 그리고 vimentin이 1.5배 감소함을 확인 할 수 있었다. 그러나 rabaptin 아스코크로린 처리에 의해 3배 증가함을 확인 할 수 있었다. 이러한 단백질 발현은 RT-PCR을 수행하여 결과에 대해 재확인 하였으며, 프로테오믹스와 동일한 결과를 얻을 수 있었다. 따라서 본 연구를 통해 LPS 처리에 의해 활성화된 mouse macrophage RAW264.7 세포에 ASC를 처리한 후 이차원 전기영동법을 이용하여, 단백질의 발현 변화 및 양상을 규명하고 단백질 지도를 확립 하였으며, RAW264.7 세포를 이용한 면역세포 모델에서 ASC의 항염증 작용을 중심으로 생리활성 조절기능을 확인 할 수 있었다. 향후 분자 기능 조절 연구와 전 임상 연구를 통해 ASC의 생리활성 조절 기능을 규명한다면 ASC는 항염증 및 항암활성을 갖는 약물로 개발될 것으로 기대된다. Ascochlorin (ASC) is prenyl-phenol compound that was isolated from the fungus Ascochyta viciae. ASC reduces serum cholesterol and triglyceride levels, and suppresses hypertension, tumor development, ameliorates type I and II diabetes. Here, to better understand the mechanisms by which ASC regulates physiological or pathological events and induces responses in the pharmacological treatment of inflammation, we performed differential analysis of the proteome of the mouse macrophage RAW264.7 cells in response to ASC. In this study, we used a proteomic analysis of LPS-induced RAW264.7 cells treated by ASC, to identify proteins potentially involved in inflammatory processes. The RAW264.7 cell proteomes with and without treatment with ASC were compared using two-dimensional electrophoresis (2-D SDS-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) and bioinformatics. The largest differences in expression were observed for the calreticulin (4-fold decrease), β-actin (4-fold decrease) and vimentin (1.5-fold decrease). In addition, rabaptin was increased 3-fold in RAW264.7 cells treated with ASC. The expression of some selected proteins was confirmed by RT-PCR analysis.

니켈 및 코발트의 세포독성 기전에서 Nitric Oxide의 역할

염정호,오경재,유영천 대한산업의학회 2001 대한직업환경의학회지 Vol.13 No.3

목적 : 이 연구는 RAW 264.7 cell의 배양조건에 니켈, 코발트 또는 iNOS의 competitive inhibitor인 NMLA를 여러 조건으로 처리하여 NO 생성의 변조유무 및 이와 관련된 ATP 생성과 세포생존율의 변조양상을 관찰하므로써 니켈 및 코발트가 염증반응을 일으키는 세포독성 과정에서의 NO 역할에 대해 알아보고자 실시하였다. 방법 : Balb/c 마우스의 복강내에 Abelson leukemia virus(A-MuLV)를 주입하여 발생시킨 대식세포주 RAW 264.7 세포의 배양조건에 니켈, 코발트 또는 iNOS의 특이억제체인 NMLA를 여러 조건으로 처리하여 NO 생성의 변조유무 및 이와 관련된 ATP 생성과 세포생존율의 변조양상을 관찰하였다. 세포생존율은 trypan-blue dye exclusion 방법을 이용하였으며 NO는 Hibbs 등(1987)의 방법에 따라 그 대사물질인 nitrite(NO2-)의 측정을 통해 간접 측정하였다. 또한, ATP는 세포막을 파괴한 후 luciferase와 ATP의 반응에 따른 발광정도를 luminometer로 측정하였다. 한편, 각 실험조건에서의 세포의 형태학적 변화는 inverted microscope(×400)를 이용하여 관찰하였다. 결과 : 기본배양조건에 cytokines과 여러 농도의 니켈 또는 코발트의 단독 및 동시첨가의 경우 두 금속 모두 50 μM, 48시간을 기점으로 NO 생성량은 첨가농도의 증가에 따라 용량의존적으로 증가하다가 그 이상의 농도 및 시간에서는 현저히 감소하였으며, 동일 조건에서의 세포생존율은 저 농도에서는 대조군과 차이가 없었으나 전반적으로 첨가한 금속의 농도증가에 따라 용량 및 시간 경과에 따라서 감소하는 경향을 보였다. 두 금속 모두 세포생존율 및 NO의 생성율이 높게 유지되는 50 μM, 48시간 배양 조건에서의 결과 또한, 니켈 및 코발트 첨가는 두 금속 모두 cytokines만의 첨가시보다 NO 생성을 더욱 증가시켰으며 ATP 생성정도는 NO 생성 양상과는 반대로 니켈 및 코발트 모두 대조군보다 현저히 감소하였다. 한편 동일 조건에서의 세포생존율은 ATP 감소양상과 비슷하였으며, 이러한 결과들은 두 금속의 동시첨가시 상가(additive)작용으로 나타났다. 한편, 니켈 및 코발트를 단독 또는 동시 첨가한 경우 모두에서 나타났던 NO의 증가와 ATP 감소 및 세포생존율의 감소는 iNOS 억제제인 NMLA를 전처리로 NO 생성은 감소하고 ATP 및 세포생존율은 증가하여 모든 경우에서 대조군 수준으로 완전히 회복되었다. 또한, 이러한 경향은 RAW 264.7 세포를 여러 실험 조건으로 배양한 후 세포상태를 inverted microscope로 관찰한 결과에서도 동일한 결과를 나타내어 니켈 또는 코발트의 첨가로 나타났던 전반적인 세포의 위축과 불규칙한 외형들은 NMLA의 전처리에서는 나타나지 않았다. 결론 : 이상의 결과에서, 니켈 및 코발트는 대식세포의 NO 생성을 증가시키며 이들 금속에 의한 ATP 생성 억제는 그 간 알려졌던 NO의 궁극적인 독성양상인 ATP 생성억제와 동일한 결과로서, 니켈 및 코발트는 대식세포를 활성화시켜 NO 생성을 증가시키고 NO는 ATP 생성을 억제하여 생존율을 감소시키는 것으로 사료된다. 한편, 니켈 및 코발트의 독성효과들은 NMLA를 전처리로 완전하게 억제되고 있어 니켈 및 코발트의 독성은 대부분 NO에 의해 매개됨을 알 수 있었다. 따라서 NO는 니켈 및 코발트의 염증유발 과정에서 중요한 매개역할을 수행하리라 사료된다. Objectives : The nickel and cobalt present in many industrial working environments and consumer products. They are two of the leading causes of allergic contact dermatitis, which is a typical delayed(type IV) hypersensitivity reaction. However, the mechanism by which nickel and cobalt causes this pathology is not well known. The nickel and cobalt induced contact dermatitis is mediated primarily through macrophages. This mechanism is similar to the autotoxicity procedure for NO. Therefore, this study was designed to examine whether the metals could modulate NO productihb and how the metals may affect ATP production and cell viability. In summary, the purpose of this study was to elucidate the role of NO in the nickel and cobalt induced cytotoxicity. Methods : This study is based on observations of cultures of RAW 264.7 cells which are originated from a tumor of Balb/c mouse that was induced by Abelson murine leukemia virus. RAW 264.7 cells were treated with either Ni, Co, Ni plus Co, or N-monomethyl-L- arginine(NMLA) for 24-72 h. The cytotoxicity of the nickel and cobalt was measured by cell viability and NO2-, and mitochondrial function was evaluated by adenosine triphosphate(ATP) production in RAW 264.7 cells. In addition, the morphology of cells was observed using an inverted microsope. Results : The NO2- synthesis of RAW 264.7 cells increased with increasing concentrations of Ni and Co up to 50 μM after 24 and 48 h of exposure to Ni and Co but then decreased if the concentration was greater than 50 μM and the time period was greater than 48 h. However, the viability of cells was decreased by Ni and Co exposure in a dose and time dependent manner. Therefore, 50 μM Ni or Co and 48 h of treatment were used in this study. A complete inhibition of NO2- synthesis by Ni or/and Co occurred when iNOS inhibitor, NMLA, were pretreated prior to addition of Ni or/and Co, whereas Ni or/and Co induced decrease of synthesis of ATP and viability completely recovered when NMLA were pretreated prior to addition of Ni or/and Co. Ni or/and Co(50 μM) induced the characteristic morphological features of cytotoxicity which is characterized by a shrinkage of cytoplasm and irregular shape of the cells, but the pretreatment of NMLA resulted in a recovered morphological change of the cells to their normal appearance. Conclusions : These results suggest that NO plays an important role in the pathogenesis of the cytotoxicity of nickel and cobalt, and nickel and cobalt may exert their toxicities by means of modulation of NO prduction. The results from this study may facilitate further understanding the role of NO of nickel and cobalt induced immune and inflammatory processes.

신경교세포 및 RAW 264.7 세포에서 Protein kinase의 활성에 의한 유도성 Nitric oxide synthase의 발현

박상철,노삼길,배소현,박지선,이충재,허강민,석정호,이재흔 충남대학교 의학연구소 2003 충남의대잡지 Vol.30 No.2

NO(nitric oxide) plays an important role as neurotransmitter or cytokine, and pathologic factor for some diseases by the large amount production with iNOS(inducible NO synthase) expression in macrophages or glial cells. The expression of iNOS is regulated by various cytokines, protein kinases and transcription factors. In this experiment, to investigate the roles of progein kinase and NF-kB for iNOS expression, the effects of PMA(phorbol 12-myristate 13-acetate), cAMP, and various protein kinase inhibitors on LPS(lipopolysaccharide)-induced iNOS mRNAN expression and nuclear NF-kB binding complex were examined in C6 glial cells and RAW 264.7 cells. In C6 glial cells, iNOS mRNA expression by LPS was induced from 1 hour and peak at 3 hour after treatment. In RAW 264.7 cells, the mRNA was observed from 3 hour and peak at 6 hour. PMA enhanced markedly LPS-induced iNOS mRNA expression and NF-kB binding complex in C6 glial cells, but did not much influence on LPS-induced iNOS mRNA expression in RAW 264.7 cells, in spite of increased LPS-induced NF-kB binding complex at 30 min. cAMP(dibutyryl cAMP) did not much influence on LPS-induced iNOS mRNA expression, by increased LPS-induced NF-kB binding complex in C6 glial cells. However, in RAW 264.7 cells, cAMP increased slightly LPS-induced iNOS mRNA expression without change of NF-kB binding complex. Staurosporine did not influence on LPS-induced iNOS mRNA expression and NF-kB binding complex in C6 glial cells, but in RAW 264.7 cells, decreased LPS-induced iNOS mRNA expression and NF-kB binding complex. Ro-31-8220 did not much influence on LPS-induced iNOS mRNA expression and NF-kB binding complex in C6 glial cells, but in RAW 264.7 cells, decreased significantly LPS-induced iNOS mRNA expression in spite of increased LPS-induced NF-kB binding complex for 3hours. G 6976 did not much influence on LPS-induced iNOS mRNA expression with decreased NF-kB binding complex in C6 glial cells, but in RAW 264.7 cells, decreased iNOS mRNA expression without influence on LPS-induced NF-kB binding complex. Genistein did not influence on LPS-induced iNOS mRNA expression and NF-kB binding complex in C6 glial cells, but in RAW 264.7 cells, decreased LPS-induced iNOS mRNA expression inspite of increased NF-kB binding complex. These results suggest that LPS-induced regulation of iNOS expression or NF-kB activity in C6 glial cells, might be different from RAW 264.7 cells through various protein kinases or other factors.

JAK/STAT 신호전달 경로를 통한 LPS 유도 RAW 264.7 세포의 염증에 대한 노니의 항염증 효과

조범길,방인석 한국생명과학회 2022 생명과학회지 Vol.32 No.2

본 연구는 노니(Morinda citrifolia)에 함유된 주요 생리활성물질이 RAW 264.7 세포에서 JAK/STAT 신호전달 경로를 통하여 항염증 작용에 관여하는 것을 조사하였다. 건조된 M. citrifolia 열매의 MeOH 추출에 의한 유기용매의 순차 분획에서 얻은 EtOAc 분획물(Mc-EtOAc)에서 가장 높은 항산화 활성을 확인하였고, H2O2로 유도된 RAW 264.7 세포의 산화적 스트레스에 대한 세포보호 효과는 처리 농도의존적으로 세포독성을 차단하였다. LPS 처리로 71.6%의 RAW 264.7 세포 생존율에서 240 μg/ml의 Mc-EtOAc를 전처리한 군에서 84.5%의 세포 생존율 증가를 보였다. LPS로 유도된 RAW 264.7 세포의 NO 생성 저해활성은 240 μg/ml의 Mc-EtOAc에서 양성 대조군의 절반 수준으로 NO의 생성양을 감소시켰다. Mc-EtOAc 처리로 iNOS의 발현량은 농도의존적으로 유의하게 감소하였고, COX-2의 발현은 LPS 유도로 3배 정도로 증가되었으나, 120, 240 μg/ml의 Mc-EtOAc를 전처리시 농도의존적으로 유의하게 감소시켰다. 또한 pro-inflammatory cytokine IL-1β와 TNF-α의 mRNA 발현도 억제하였다. 이러한 Mc-EtOAc의 항염증 작용이 상위 신호전달 경로인 JAK/STAT 신호전달 경로 통해 일어나지를 조사한 결과, Mc-EtOAc의 전처리로 LPS로 유도된 RAW 264.7 세포에서 pJAK1과 pSTAT3의 인산화 정도가 유의성 있게 감소하였다. 따라서 RAW 264.7 세포에서 Mc-EtOAc의 항염증 효과는 JAK1/STAT3 신호전달 경로를 통해 염증반응을 억제하는 것으로 사료된다. This study investigated whether or not the major bioactive compounds of Noni (Morinda citrifolia) are involved in anti-inflammatory activity through the JAK/STAT upper signaling pathway in RAW 264.7 cells. The experimental results show that the M. citrifolia ethyl acetate fraction (Mc-EtOAc) obtained by sequential fractionation with organic solvents from the plant’s dried fruits exhibits the highest antioxidant activity. In addition, the cytoprotective effects of Mc-EtOAc against H2O2-induced oxidative stress in the RAW 264.7 cells suppressed cytotoxicity in a dose-dependent manner. The group pretreated with Mc-EtOAc at a concentration of 240 μg/ml showed higher cell viability of 84.5%, compared to 71.6% in the LPS-treated group, and LPS-induced NO production decreased to half the amount in the positive control group. Mc-EtOAc treatment also led to a significant dose-dependent reduction in iNOS expression. Although COX-2 expression was increased by 300% following LPS induction, it was significantly decreased in a dose-dependent manner by pretreatment with Mc-EtOAc at concentrations of 120 and 240 μg/ml. An inhibition of the mRNA expression of pro-inflammatory cytokines IL-1β and TNF-α was observed. The investigation also revealed that the phosphorylation levels of pJAK1 and pSTAT3 in LPS-induced RAW 264.7 cells were significantly reduced by Mc-EtOAc treatment.

바이칼레인(baicalein)이 poly-IC와 lipoteichoic acid로 자극된 마우스 대식세포 RAW 264.7의 hydrogen peroxide 생성에 미치는 영향

박완수,Wansu Park 대한본초학회 2023 大韓本草學會誌 Vol.38 No.4

Objectives : The aim of this study was to investigate the effect of baicalein (BA) on the production of hydrogen peroxide and nitric oxide (NO) in RAW 264.7 mouse macrophages stimulated with polyinosinic-polycytidylic acid (poly-IC) and lipoteichoic acid. Methods : RAW 264.7 co-stimulated with poly-IC and lipoteichoic acid were incubated with baicalein at concentrations of 25 and 50 μM. Incubation time is 16 h, 18 h, 20 h, 22 h, and 24 h. After incubation, The production of hydrogen peroxide in RAW 264.7 was measured with dihydrorhodamine 123 assay. Chrysin was used as a comparative material. NO production was evaluated by griess assay. Results : For 16 h, 18 h, 20 h, 22 h, and 24 h incubation, BA at the concentration of 25 and 50 μM significantly inhibited the production of hydrogen peroxide in RAW 264.7 stimulated by poly-IC and lipoteichoic acid (p <0.001). In details, production of hydrogen peroxide in 'poly-IC and lipoteichoic acid'-stimulated RAW 264.7 treated for 16 h with BA at concentrations of 25 and 50 μM was 82.36% and 77.24% of the control group treated with poly-IC and lipoteichoic acid only, respectively; the production of hydrogen peroxide for 18 h was 83.15% and 77.91%, respectively;production of hydrogen peroxide for 20 h was 82.88% and 77.82%, respectively; production of hydrogen peroxide for 22 h was 83.27% and 78.17%, respectively; production of hydrogen peroxide for 24 h was 83.54% and 78.35%, respectively. Additionally, BA at the concentration of 50 and 100 μM significantly inhibited NO production in lipoteichoic acid-induced RAW 264.7 (p <0.001). Conclusions : BA might have anti-oxidative activity related to its inhibition of hydrogen peroxide production in 'poly-IC and lipoteichoic acid'-stimulated RAW 264.7 macrophages.

이삭물수세미(Myriophyllum spicatum L.) 에탄올 추출물의 항산화와 항염증 효과

김철환,이영경,김민진,최지수,황병수,조표연,김영준,정용태 한국자원식물학회 2023 한국자원식물학회지 Vol.36 No.1

Abstract - Myriophyllum spicatum L. has been used as an ornamental in ponds and aquariums, and as a folk remedy for inflammation and pus. Nevertheless, the biological activity and underlying mechanisms of anti-inflammatory effects are unclear. This study is aimed at investigating the antioxidative and anti-inflammatory activities of ethanol extract of Myriophyllum spicatum L. (EMS) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Antioxidant activity of EMS was assessed by radical-scavenging effects on ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. As inflammatory response parameters produced by LPS-stimulated RAW 264.7 cells were quantified to assess the anti-inflammatory activity of EMS. Our results showed that EMS increased FRAP and DPPH radicalscavenging activity. In EMS-treated RAW 264.7 cells, the production of NO, PGE2, TNF-α and IL-1β was significantly inhibited at the non-cytotoxic concentration. In addition, EMS significantly attenuated LPS-stimulated the toll-like receptor (TLR) 4/myeloid differentiation protein (MyD) 88 signaling pathway, and inhibited nuclear translocation of nuclear factor-kappa B(NF-κB). Positive correlations were noted between anti-inflammatory activity and antioxidant activity. In conclusion, it was indicated that EMS suppresses the transcription of inflammatory factors by inhibiting the TLR4/MyD88/NF-κB signaling pathway, thereby suppressing LPS-stimulated inflammation in RAW 264.7 cells. This study highlights the potential role of EMS against inflammation and associated diseases. 적 요이삭물수세미는 민간에서는 전초를 고름, 염증 등에 약용으로 사용하였으나, 염증에 대한 연구가 미비한 상황이다. 이에본 연구에서는 이삭물수세미 추출물(EMS)의 항산화 효능과 항염증 효능을 분석하였다. 항산화 효능은 DPPH 라디칼 소거능과 환원력을 통해 산화적 스트레스를 통해 염증을 유발시킬 수있는 ROS (Hong et al., 2020; Snezhkina et al., 2019)를 억제하는지 확인하였고, 항염증 효능은 염증 발현 인자인 LPS를 이용하여 RAW 264.7 대식세포에 염증을 유도한 뒤pro-inflammatory cytokine (TNF-α, IL-1β)과 염증 매개체(NO, PGE2)의억제 및 TLR4/Myd88/NF-κB signaling pathway 발현 억제를통해 확인하였다. 연구 결과, 항산화 효능에 있어서는 DPPH 라디칼 소거능과 Fe3+를 Fe2+로 환원시키는 환원력이 농도 의존적으로 증가함을 확인하였다. 무독성 상태에서 실험하기 위해LPS와 EMS를 처리한 RAW 264.7 대식세포에서 90% 이상의 생존율을 나타내는 조건에서 실험을 진행하였다. LPS로 염증이유도된 RAW 264.7 세포에서 EMS는 염증 매개 인자의 발현 및생성 억제(iNOS에 의한 NO 생성 및 COX-2에 의한 PGE2 생성억제)와 pro-inflammatory cytokine (TNF-α및 IL-1β)의 생성 또한 억제하였다. 특이적으로 COX-2에 의한 PGE2 생성 억제에서는 고농도에서 작용함을 확인하였고, IL-1β에서는 약한억제력을 보였다. 이후signaling pathway에서 염증 전사인자경로를 확인하기 위하여 TLR4/MyD88의 활성을 확인하였고, EMS 처리에 따라 농도 의존적으로 억제되는 것을 확인하였다. 이에 따라 염증 초기 단계에서 NF-κB p65가 nuclear로 들어가는 것을 억제하는지 확인하기 위해 early time (LPS 처리 후30, 60 min) 조건으로 nuclear에서 p65 인산화를 확인하였다. 그 결과, LPS 자극으로 인해 증가된 p65 인산화가 EMS에 의해 부분적으로 억제됨을 확인하였다. 이상의 결과를 통해 LPS로 염증이 유도된 RAW 264.7 대식세포에서 EMS가 COX-2에 의한PGE2 생성 억제와 IL-1β의 생성에 있어 낮은 억제력을 가진 반면, iNOS에 의한 NO과 TNF-α생성 및 TLR4/MyD88 singnaling pathway에 있어 강한 억제력을 가짐을 확인하였다. 결론적으로 EMS가 ROS를 제거하고 TLR4/MyD88/NF-κB signaling pathway를 억제함으로써 염증 인자들의 전사를 억제하고, 염증 인자 부분에서는 iNOS에 의한 NO 생성과 TNF-α생성을 강하게 억제하여 RAW 264.7 대식세포에서 LPS로 자극된 염증을억제하는 것으로 판단된다. 또한 TLR4/Myd88/NF-κB signaling pathway를 통한 pro-inflammatory cytokine과염증 매개체와의 연관성에 대한 기초자료로 활용할 수 있는 근거 자료가 될 수있을 것으로 생각된다.

Trans-10, cis-12 Conjugated Linoleic Acid Modulates Tumor Necrosis Factor-α Production and Nuclear Factor-κB Activation in RAW 264.7 Macrophages Through Formation of Reactive Oxygen Species

박소영,강병택,강지훈,양만표 한국임상수의학회 2014 한국임상수의학회지 Vol.31 No.6

The aims of this study were to explore the effects of conjugated linoleic acid (CLA) on reactive oxygenspecies (ROS) production in lipopolysaccharide (LPS)-naïve and LPS-stimulated RAW 264.7 macrophages and toexamine whether these effects affect the regulation of tumor necrosis factor-alpha (TNF-α) production, and nuclearfactor-kappa B (NF-κB) and peroxisome proliferator-activated receptor gamma (PPARγ) activation. Trans-10, cis-12(t10c12)-CLA increased the production of ROS, as well as TNF-α in LPS-naïve RAW 264.7 cells. The CLA-inducedTNF-α production was suppressed by treatment of diphenyleneiodonium chloride (DPI), a NADPH oxidase inhibitor. In addition, CLA enhanced the activities of NF-κB and PPARγ in LPS-naïve RAW 264.7 cells, and this effect wasabolished with DPI treatment. LPS treatment increased ROS production, whereas CLA reduced LPS-induced ROSproduction. LPS increased both TNF-α production and NF-κB activity, whereas t10c12-CLA reduced TNF-α productionand NF-κB activity in LPS-stimulated RAW 264.7 cells. DPI treatment suppressed LPS-induced ROS production andNF-κB activity. Moreover, DPI enhanced the inhibitory effects of t10c12-CLA on TNF-α production and NF-κBactivation in LPS-stimulated RAW 264.7 cells. However, neither t10c12-CLA nor DPI affected PPARγ activity in LPSstimulatedRAW 264.7 cells. Taken together, these data indicate that t10c12-CLA induces TNF-α production byincreasing ROS production in LPS-naïve RAW 264.7 cells, which is mediated by the enhancement of NF-κB activityvia PPARγ activation. By contrast, t10c12-CLA suppresses TNF-α production by inhibiting ROS production and NF-κB activation via a PPARγ-independent pathway in LPS-stimulated RAW 264.7 cells. These results suggest that t10c12-CLA can modulate TNF-α production and NF-κB activation through formation of ROS in RAW 264.7 macrophages

바이칼레인(baicalein)이 peptidoglycan으로 자극된 RAW 264.7 mouse macrophages의 hydrogen peroxide 생성에 미치는 영향

박완수 ( Wansu Park ) 대한본초학회 2023 大韓本草學會誌 Vol.38 No.1

Objectives : The aim of this study was to investigate the effect of baicalein (BA) on the production of hydrogen peroxide in peptidoglycan-stimulated RAW 264.7 mouse macrophages. Methods : Peptidoglycan-stimulated RAW 264.7 were incubated with baicalein at concentrations of 50 and 100 μM. Incubation time is 30 min, 2 h, 12 h, and 18 h. After incubation, The production of hydrogen peroxide in RAW 264.7 was measured with dihydrorhodamine 123 assay. Berberine and gallic acid were used as the comparative materials. Results : BA at the concentration of 50 and 100 μM did not show cytotoxicity on RAW 264.7 for 24 h incubation. For 30 min, 2 h, 12 h, and 18 h incubation, BA at the concentration of 50 and 100 μM significantly inhibited the production of hydrogen peroxide in RAW 264.7 stimulated by peptidoglycan (p < 0.05). In details, production of hydrogen peroxide in peptidoglycan-stimulated RAW 264.7 treated for 30 min with BA at concentrations of 50 and 100 μM was 93.91% and 93.52% of the control group treated with peptidoglycan only, respectively; the production of hydrogen peroxide for 2 h was 93.8% and 92.71%, respectively; production of hydrogen peroxide for 12 h was 94.86% and 95.93%, respectively; production of hydrogen peroxide for 18 h was 95.37% and 96.48%, respectively. Conclusions : BA might have anti-oxidative activity related to its inhibition of hydrogen peroxide production in peptidoglycan-stimulated RAW 264.7 macrophages.

Raw 264.7 대식세포에서 Chrysophanol의 LPS로 유도된 전염증성 사이토카인 억제 효과

반연예,박의성,홍근혜,임양이,박건영 한국식품영양과학회 2019 한국식품영양과학회지 Vol.48 No.12

Chrysophanol은 30~60 μM 범위에서 Raw 264.7 세포에서 세포증식을 억제하지 않았다. 또한 LPS와 함께 chrysophanol을 처리했을 때 60 μM 처리 시 가장 높은 세포 성장률을 보였다. Raw 264.7 세포에 LPS만 단독으로 처리했을 때 처리하지 않은 군보다 NO 생성이 증가하였으나 chrysophanol을 Low(30 μM), High(60 μM) 농도로 처리했을 때 NO의 생성이 유의적으로 감소하였다(P<0.05). 전염증성 사이토카인인 TNF-α, IL-1β, IL-6, IL-10은 LPS군에 비해 chrysophanol을 Low, High 농도로 처리했을 때 유의적으로 감소했으며, 마찬가지로 TNF-α, IL-1β, IFN-γ의 mRNA 수준도 감소하였다. 또한 염증관련 효소인 COX-2의 mRNA 수준도 억제하였다. 단백질 발현 수준에서도 전염증성 사이토카인인 TNF-α, IL-1β와 IL-6, 염증관련 효소인 iNOS와 COX-2의 발현이 LPS와 chrysophanol을 Low, High 농도로 함께 처리했을 때 LPS만 처리한 군에 비해 유의적으로 감소하였다(P<0.05). 이러한 결과를 종합해볼 때, 식물유래 화합물인 chrysophanol은 LPS로 유도된 염증반응을 억제하며, 특히 전염증성 사이토카인과 염증관련 효소(iNOS, COX-2)의 발현을 억제하여 염증반응을 조절하는 것으로 나타났다. The level of pro-inflammatory cytokines was markedly suppressed by chrysophanol in lipopolysaccharide (LPS)-treated Raw 264.7 macrophage cells. Chrysophanol had no toxic effect on the Raw 264.7 cells at the treatment concentrations ranging from 30 to 60 μM. Sixty μM (High) chrysophanol exhibited the highest protection against the LPS effect on the Raw 264.7 cells, as was determined by MTT assay. Thirty μM (Low) and 60 μM concentrations of chrysophanol significantly decreased the production of the pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-10 in the LPS-treated Raw 264.7 cells as compared to that of LPS treatment only. LPS+Low and LPS+High also significantly suppressed the mRNA expressions of the pro-inflammatory cytokines TNF-α, IL-1β, INF-γ, and COX-2 compared to that of LPS treatment only. Moreover, LPS+Low and LPS+High significantly decreased the protein expressions of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, and also the inflammation related proteins of iNOS and COX-2 compared to that of LPS treatment only in the cells. According to our results, chrysophanol, which is derived from plants and especially curly dock, showed suppressive effects by inhibiting the inflammatory effects induced by LPS in Raw 264.7 cells by regulating pro-inflammatory cytokines and inflammation related enzymes. These results indicated that chrysophanol may possibly be used to treat inflammatory diseases and it is perhaps a marker of anti-inflammatory functions in plants.