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.

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.

Stimulative Effects of Hominis Placental Pharmacopuncture Solution Combined with Zinc-oxide Nanoparticles on RAW 264.7 Cells - ZnO HPPS more easily stimulates RAW 264.7 cells -

Hong, Tae-Keun,Kim, Jee-Hye,Woo, Ju-Youn,Ha, Ki-Tae,Joo, Myung-Soo,Hahn, Yoon-Bong,Jeong, Han-Sol KOREAN PHARMACOPUNCTURE INSTITUTE 2012 Journal of pharmacopuncture Vol.15 No.3

Objectives: The purpose of this study is to examine whether Hominis Placental pharmacopuncture solution (HPPS) combined with zinc-oxide nanoparticles (ZnO NP) activates RAW 264.7 cells. Methods: We soaked ZnO nanoparticles in the Hominis Placenta pharmacopuncture solution, thereby making a combined form (ZnO NP HPPS). The effect of ZnO NP HPPS on the intracellular reactive oxygen species (ROS) production was measured by 2', 7'-dichlorofluorescin diacetate (DCFH-DA) assay. The effect of ZnO NP HPPS on NF-${\kappa}B$ was measured by using a luciferase assay. The effect of ZnO NP HPPS on the cytokine expression was assessed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The cellular uptake of ZnO NP HPPS was measured by using a flow cytometric analysis, and cellular structural alterations were analyzed by using transmission electron microscopy (TEM). Results: Neither the HPPS nor the ZnO NPs induced intracellular ROS production in RAW 264.7 cells. Neither of the materials activated NF-${\kappa}B$ or it's dependent genes, such as TNF-${\alpha}$, IL-1, and MCP-1. However, ZnO NP HPPS, the combined form of ZnO NPs and HPPS, did induce the intracellular ROS production, as well as prominently activating NF-${\kappa}B$ and it's dependent genes. Also, compared to ZnO NPs, it effectively increa-sed the uptake by RAW 264.7 cells. In addition, cellular structural alterations were observed in groups treated with ZnO NP HPPS. Conclusions: Neither ZnO NP nor HPPS activated RAW 264.7 cells, which is likely due to a low cellular uptake. The ZnO NP HPPS, however, significantly activated NF-${\kappa}B$ and up-regulated its dependent genes such as TNF-${\alpha}$, IL-1, and MCP-1. ZnO NP HPPS was also more easily taken into the RAW 264.7 cells than either ZnO NP or HPPS.

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.

RAW264.7세포주와 염증생쥐모델에서 항염증(抗炎症) 작용(作用)에 대한 청열활혈탕가계혈등(淸熱活血湯加鷄血藤)의 효과(效果)

한충희 ( Choong Hee Han ),유동열 ( Dong Youl Yoo ) 대한한방부인과학회 2005 大韓韓方婦人科學會誌 Vol.18 No.3

Purpose: The Purpose of this research was to investigate the effects of Cheongyeolhawlhyeoltanggagyehyeoldeung (CYHHT) on anti-inflammatory effects. Methods: As for the parameters of inflammation, levels of several inflammatory cytokines and chemical mediators were determined in mouse lung fibroblast cells (mLFC) and RAW264.7 cells. Also, changes in pathological features by drug treatment were investigated in the in vivo edema-induced rats by carrageenin /arachidonic acid or in the colitis-induced mice by DSS treatment. Results: The cytotoxicity of CYHHT on mLFC and RAW264.7 cells was not observed at 100, 50, 10, and 1㎍/㎖ of CYHHT treatments. IL-1β, IL-6 and NOS-II mRNA expression of RAW264.7 cells was inhibited by CYHHT treatments in a dose- dependent manner. CYHHT treatment of RAW264.7 cells inhibited TNF-α and COX-2 mRNA expression. CYHHT treatment of RAW264.7 cells significantly inhibited IL-6 and NO production. CYHHT treatment of RAW264.7 cells inhibited ROS production. CYHHT inhibited rat`s paw edema induced by carrageenin or arachidonate treatment in all concentrations examined. The body weight and colon length of colitis-induced mice were recovered to a normal level by DSS treatment. Clinical disease levels were significantly improved compared to the control animals. CYHHT treatment of colitis-induced mice significantly increased hematological values such as WBC and RBC counts, Hgb and HCT levels, but decreased PLT values. CYHHT treatment of colitis-induced mice decreased IL-6 and TNF-α production significantly CYHHT treatment of colitis-induced mice significantly increased CD3+(T) cell counts. In contrast, CYHHT treatment decreased CD19+ B cell counts and CD3+/CD69+ significantly, and also decreased B/T ratio (%) though not significant. Conclusion: These results indicated that CYHHT could be used for treating diverse female diseases caused by the inflammation.

삼채(三菜) 물추출물이 RAW 264.7 세포의 항산화 및 염증반응에 미치는 영향

이상수,한효상,유자연,남명수,김기광 대한본초학회 2020 大韓本草學會誌 Vol.35 No.4

Objectives : Allium hookeri is a well-known traditional herbal remedy and its root used for treatment of inflammation and tumor. However, the mechanism of anti-inflammatory effect of Allium hookeri is still unknown. This study aims to examine the mechanism of anti-inflammatory effect of Allium hookeri on mouse macrophage cell line, RAW 264.7 cells. Methods : Anti-oxidant effect of water extract of Allium hookeri (WEAH) was measured by 2,2'-azino-bis-3- ethylbenzothiazoline-6-sulphonic acid (ABTS) assay. 3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed to determine the effect of WEAH on cell viability in RAW 264.7 cells. In addition, anti-inflammatory effect of WEAH was investigated in RAW 264.7 cells. Inflammation of RAW 264.7 cells induced by lipopolysarccharide (LPS) treatment and expression levels of inflammatory cytokine interleukin 1 β (IL-1β) and interleukin 6 (IL-6) gene were analyzed using quantitative reverse transcription PCR (qRT-PCR) analysis. Furthermore, the phosphorylation of inhibitor of nuclear factor kappa B (IκBα) after LPS treatment with WEAH-treated RAW 264.7 cells was confirmed by immunoblot analysis. Results : WEAH showed a strong anti-oxidant effect and no cytotoxicity to RAW 264.7 cells up to 2 ㎎/㎖concentration. The LPS-induced mRNA expression levels of IL-1β and IL-6 were decreased by WEAH treatment. Furthermore, the LPS-induced phosphorylation of IκBα is attenuated by WEAH treatment. Conclusions : Through experimental demonstration of anti-oxidant and anti-inflammatory effects of WEAH, we suggest that Allium hookeri is a valuable material for prevention and treatment of various inflammatory diseases.

Conditioned Media of RAW 264.7 Cells Stimulated with Phellinus linteus Extract Regulates the Epithelial-mesenchymal Transition in Prostate Cancer Cells

Taewoo Kang(강태우),Hyun-Hee An(안현희),Sul-Gi Park(박슬기),Sun-Nyoung Yu(유선녕),You-Lim Hwang(황유림),Ji-Won Kim(김지원),Soon-Cheol Ahn(안순철) 한국생명과학회 2019 생명과학회지 Vol.29 No.8

전립선암은 전이성 종양 중의 하나로 치료를 위해 호르몬 요법이나 외과 적 거세 방법이 주로 수행되지만 많은 부작용을 나타내었다. 최근 많은 연구자들이 이러한 상황을 해결하기 위해 종양 미세 환경을 연구하고 있으며 그 중 면역 세포, 특히 대식세포는 종양 미세 환경의 중요한 구성요소이다. 정상적인 조건에서 대식세포는 여러 암세포에 대해 약한 종양 살균 활성을 갖으나 interferon-γ 또는 lipopolysaccharide에 의해 활성화되면, 염증성 사이토카인 및 케모카인을 분비함으로써 암세포를 직접 또는 간접적으로 사멸 시키게 된다. 본 연구에서는, 마우스 대식세포인 RAW 264.7 세포에 Phellinus linteus 추출물을 처리하여 산화질소의 방출과 pro-inflammatory cytokine들을 real-time PCR과 ELISA 방법으로 분석하였다. RAW 264.7의 조정 배지는 48시간 동안 전립선 암세포처리하여 상피간엽세포전이 관련 유전자의 발현을 측정 하였다. 그 때에 mesenchymal 관련 유전자들인 N-cadherin, snail, twist, slug 및 cadherin 11이 감소했을 뿐만 아니라 epithelial 관련 유전자인 E-cadherin은 증가하였다. 또한 암 전이 및 신생 혈관 형성에 관여하는 vimentin, ccl2 및 vegfa가 감소되었는데, 이는 EMT가 암세포의 이동과 침범에 밀접한 관련이 있기 때문이다. 따라서 Phellinus linteu에 의해 자극된 RAW 264.7 세포의 조정 배지는 인간 전립선 암세포주인 PC-3 세포의 이동과 전이를 억제하고 EMT 경로를 조절한다는 것을 나타낸다. Prostate cancer (PCa) is one of the most metastatic tumor. Although hormone therapy or surgical castration is mostly conducted to treat PCa, it has a lot of side effects. Recently, many researchers have been exploring the tumor microenvironment to remedy these circumstances. Immune cells, especially macrophages, are an important composition of the tumor microenvironment. Under normal conditions, macrophages exhibit mild tumoricidal activity against tumors. However, once activated by interferon gamma or lipopolysaccharides, macrophages can kill cancer cells directly or indirectly by secreting cytokines and chemokines. In this study, murine macrophage RAW 264.7 cells were treated with Phellinus linteus extract. To analyze their pro-inflammatory phenotype, we were used several assays such as a real-time polymerase chain reaction, an enzyme-linked immunosorbent and nitric oxide assay. Prostate cancer cells were treated with the RAW 264.7-conditioned media, which was identified as a pro-inflammatory nature, for 48 h, and the expression of epithelial-mesenchymal transition (EMT)-related genes was determined. Not only N-cadherin, Snail, Twist, Slug, and Cadherin 11, which are mechenchymal-related proteins, were decrease, but epithelial marker of E-cadherin was increased. In addition, the mRNA level of vimentin, ccl2, and vegfa were decreased, as the EMT is closely related to the migration and invasion of cancer cells. In conclusion, the RAW 264.7-conditioned media stimulated with P. linteus extract inhibited migration and invasion and regulated the EMT pathway in human prostate cancer cells.

Anti-inflammatory Effects of Complex Extract including Eucommia ulmoides in LPS-induced RAW 264.7 Cells

류화연,이현,공해진,강재희 대한침구의학회 2019 대한침구의학회지 Vol.19 No.6

Background: The purpose of this study was to investigate the anti-inflammatory response of lipopolysaccharide (LPS) activated macrophages (RAW 264.7 murine cell line) to JCE003 which is an extract including Eucommia ulmoides, Juglans regia, Eleutherococcus senticosus, and Zingiber officinale. Methods: An MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed to analyze the survival rate of RAW 264.7 cells. The production of nitric oxide and proinflammatory cytokines (IFN-γ, TNF-α, IL-1β, IL-6) in LPS-induced RAW 264.7 cells was measured by enzyme-linked immunosorbent assay. mRNA expression levels of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1β, and IL-6) were analyzed by quantitative polymerase chain reaction analysis. Results: Exposure of LPS-activated RAW 264.7 cells to JCE003 was not cytotoxic up to 400 μg/mL, but cell survival was statistically significantly decreased at 800 μg/mL (p < 0.001). Nitric oxide production was not markedly lowered in LPS-activated RAW 264.7 cells by exposure to JCE003 (10, 50, 100, 200, 400, 800 μl/mL) compared with the Control group. In addition, JCE003 reduced the production of TNF-α in LPS-induced RAW 264.7 cells at 400 μg/mL (p < 0.05), but IFN-γ and TNF-α mRNA expression in LPS-induced RAW 264.7 cells was decreased at 100, 200, and 400 μg/mL JCE003 (p < 0.01). Conclusion: These results suggest that JCE003 inhibited the expression and production of pro-inflammatory cytokines in LPS-activated RAW 264.7 cells. The findings of this study provide basic data for the development of new Korean medicine anti-inflammatory drugs.

아까시 나무 고온추출물의 항염증 효과

노종현,강병만,정원석 한국자원식물학회 2018 한국자원식물학회지 Vol.31 No.4

This study was conducted to compare anti-inflammatory effect of Robinia pseudoacacia L. using different extraction methods (water extraction, ethanol extraction and high temperature extraction). We investigated anti-inflammatory effect of Robinia pseudoacacia L. extract (RP1, water extract; RP2, ethanol extract; RP3, high temperature extract) on lipopolysaccharide (LPS)-stimulated inflammation using Raw 264.7 cell. Cells were treated with various concentrations (12.5, 25, 50, 100 or 200 ㎍/㎖) of water extract, ethanol extract and high temperature extract. Cytotoxicity was not observed on Raw 264.7 cells, LPS-stimulated production of NO (nitric oxide), PGE2 (prostaglandin E2) and cytokines (TNF-α, IL-6 and IL-1β) was reduced by RP3 treatment more than RP1 and RP2. In conclusion, these results indicated that inflammation on Raw 264.7 cells was improved by RP3. Treatment of RP3 could be used to natural medicine for improving inflammatory response. However, further experiment is required to observe how the high temperature extraction at 500℃ for 48 h influences on alteration of active ingredient in Robinia pseudoacacia L., and conducts the inflammation signal pathway on Raw 264.7 cells. Key words - Extraction, Inflammation, Raw 264.7 cells, Robinia pseudoacacia L. 본 연구는 아까시나무의 물 추출물, 에탄올 추출물 및 고온추출물을 이용하여 마우스 대식세포주인 Raw 264.7 세포에 대해 염증억제 효과가 있는지 알아보고자 수행하였다. RP1(아까시 나무 물 추출물), RP2(아까시 나무 에탄올 추출물) 및 RP3(아까시 나무 고온 추출물)은 세포생존율 분석에서 200 ㎍/㎖의 농도까지 Raw 264.7 세포에 세포독성을 나타나지 않았다. NO 생성 억제효과를 분석하였을 때 LPS 처리군 과 비교하여 RP3는약 87% 정도의 억제효과를 나타내 RP1과 RP2에 비해 NO 억제활성이 가장 높았다. 뿐만 아니라 RP3는 RP1과 RP2와 비교하여염증매개인자의 억제율이 각각 PGE2 (86.3%), TNF-α (64.1%), IL-6 (65.1%) 및 IL-1β (63.3%)로 가장 높았다. 이는 RP3의 처리가 LPS에 의해 증가된 염증매개인자의 분비를 억제함을 통해항염증 효과가 있을 것으로 생각되며, 염증관련 신호전달경로에 직접적으로 작용할 가능성이 있는 것으로 판단된다. 하지만Raw 264.7 세포주는 염증조절복합체를 구성하는 ASC 단백질이 발현되지 않아 다른 신호전달 경로를 통해 염증매개인자를분비하기 때문에, 설치류의 대식세포를 직접 일차배양(primary culture)하여 이에 관련된 신호전달경로를 확인하는 추가 실험이 필요하다고 사료된다.