表沒(méi)食子兒茶素沒(méi)食子酸酯對(duì)IFN-γ/LPS刺激的小鼠髓源性巨噬細(xì)胞極化的影響①

王衛(wèi)芳 王 慧 張曉靜 崔夢(mèng)珂 李衛(wèi)國(guó) 王坤英

(河南師范大學(xué)生命科學(xué)學(xué)院，新鄉(xiāng) 453007)

表沒(méi)食子兒茶素沒(méi)食子酸酯對(duì)IFN-γ/LPS刺激的小鼠髓源性巨噬細(xì)胞極化的影響①

王衛(wèi)芳 王 慧 張曉靜 崔夢(mèng)珂 李衛(wèi)國(guó) 王坤英

(河南師范大學(xué)生命科學(xué)學(xué)院，新鄉(xiāng) 453007)

目的探討表沒(méi)食子兒茶素沒(méi)食子酸酯(EGCG)對(duì)IFN-γ/LPS刺激的小鼠髓源性巨噬細(xì)胞促炎細(xì)胞因子表達(dá)的影響。方法取6～8周齡C57BL/6小鼠骨髓細(xì)胞，經(jīng)100 ng/ml巨噬細(xì)胞集落刺激因子(M-CSF)體外誘導(dǎo)分化為骨髓巨噬細(xì)胞(BMDM)，加入50 ng/ml干擾素γ(IFN-γ)和1 μg/ml細(xì)菌脂多糖(LPS)刺激，并同時(shí)暴露于12.5～50 μmol/L EGCG處理48 h，利用實(shí)時(shí)熒光定量PCR(qRT-PCR)和酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)巨噬細(xì)胞促炎細(xì)胞因子IL-1β、TNF-α和iNOS的mRNA表達(dá)水平和蛋白含量。結(jié)果IFN-γ和LPS處理可刺激巨噬細(xì)胞IL-1β、TNF-α和iNOS表達(dá)顯著上調(diào)，而EGCG則能抑制IFN-γ和LPS刺激的IL-1β、TNF-α和iNOS表達(dá)，且存在劑量依賴(lài)性。結(jié)論EGCG能夠減弱IFN-γ和LPS刺激的髓源性巨噬細(xì)胞的促炎作用。

髓源性巨噬細(xì)胞；表沒(méi)食子兒茶素沒(méi)食子酸酯；巨噬細(xì)胞極化

巨噬細(xì)胞是一類(lèi)具有高度異質(zhì)性和可塑性的細(xì)胞群體，浸潤(rùn)于機(jī)體的大多數(shù)組織中，發(fā)揮著不同的作用[1]。起源于骨髓的單核細(xì)胞，經(jīng)血液循環(huán)進(jìn)入組織分化為巨噬細(xì)胞，在所處微環(huán)境因子的刺激下進(jìn)一步分化[2]。巨噬細(xì)胞極化分為經(jīng)典活化型巨噬細(xì)胞(M1)和替代活化型巨噬細(xì)胞(M2)。M1巨噬細(xì)胞由γ干擾素(Interferon-γ,IFN-γ)、細(xì)菌脂多糖(Lipopolysaccharide,LPS)誘導(dǎo)產(chǎn)生，可分泌多種促炎細(xì)胞因子和炎性介質(zhì)，如腫瘤壞死因子(Tumor necrosis factor α,TNF-α)、白介素-1β(Interleukin-1β,IL-1β)、誘導(dǎo)型一氧化氮合酶(inducible nitric oxide synthesis,iNOS)等，通過(guò)清除病原體、壞死組織和激活其他免疫細(xì)胞以利于機(jī)體抵御感染。M2巨噬細(xì)胞由IL-4、IL-13誘導(dǎo)產(chǎn)生，與炎癥消退、腫瘤生長(zhǎng)維持、抗腸道寄生蟲(chóng)感染有關(guān)，并參與組織修復(fù)等[3]。M1/M2型巨噬細(xì)胞的分化與許多疾病的發(fā)生發(fā)展都密切相關(guān)，如Ⅱ型糖尿病、胰島素抵抗、癌癥、心血管疾病和神經(jīng)退行性疾病等[4]。

表沒(méi)食子兒茶素沒(méi)食子酸酯(Epigallocatechin-3-gallate,EGCG)是綠茶多酚的主要活性成分，具有抗氧化、抗炎、抗癌等多種生物學(xué)效應(yīng)[5-7]。Jang等[8]研究發(fā)現(xiàn)經(jīng)EGCG處理的小鼠乳腺癌細(xì)胞系4T1能下調(diào)M2巨噬細(xì)胞的TGF-β分泌，表明EGCG能影響腫瘤相關(guān)巨噬細(xì)胞(Tumor-associated macrophages,TAM)的極化。本實(shí)驗(yàn)室的研究表明EGCG抑制LPS誘導(dǎo)的小鼠RAW264.7細(xì)胞促炎因子TNF-α和IL-1β基因表達(dá)和生成，并能影響IL-4刺激的小鼠腹腔巨噬細(xì)胞精氨酸酶-1(Arginase,Arg-1)和誘導(dǎo)性一氧化氮合酶(iNOS)表達(dá)[9，10]。但是，EGCG能否影響小鼠髓源性巨噬細(xì)胞(Myeloid macrophages)極化，未見(jiàn)研究報(bào)道。本研究將通過(guò)實(shí)時(shí)熒光定量PCR(quantitative real-time PCR,qRT-PCR)和酶聯(lián)免疫吸附試驗(yàn)(Enzyme linked immunosorbent assay,ELISA)檢測(cè)巨噬細(xì)胞促炎因子TNF-α、IL-1β和iNOS的mRNA水平和蛋白生成，探討EGCG對(duì)髓源性巨噬細(xì)胞極化的影響作用。

1 材料與方法

1.1材料

1.1.1實(shí)驗(yàn)動(dòng)物 實(shí)驗(yàn)選用6～8周齡、體重(20±3)g的清潔級(jí)C57BL/6小鼠，雌雄比例1∶1，購(gòu)自濟(jì)南朋悅實(shí)驗(yàn)動(dòng)物繁育有限公司。正常膳食喂養(yǎng)，光照∶黑暗為12 h∶12 h。

1.1.2主要試劑 實(shí)驗(yàn)使用的RPMI1640培養(yǎng)液為HyClone公司產(chǎn)品，血清為四季青公司產(chǎn)品，紅細(xì)胞裂解液為Solarbio公司產(chǎn)品，重組小鼠M-CSF為R&D公司產(chǎn)品，IFN-γ為PeproTech公司產(chǎn)品，RNAiso Plus和cDNA第一合成試劑盒為T(mén)aKaRa公司產(chǎn)品，qRT-PCR試劑盒為Vazyme Biotech公司產(chǎn)品，qRT-PCR引物由上海捷瑞生物工程有限公司合成，IL-1β、TNF-α和iNOS的ELISA試劑盒為南京森貝伽生物科技有限公司產(chǎn)品。

1.2實(shí)驗(yàn)方法

1.2.1小鼠骨髓細(xì)胞分離培養(yǎng) 將小鼠頸椎脫臼處死后，無(wú)菌條件下分離股骨和脛骨，用完全培養(yǎng)基將骨髓內(nèi)容物吹入無(wú)菌平皿內(nèi)分散均勻。離心、棄上清后，加入紅細(xì)胞裂解液裂解紅細(xì)胞，30 s后用完全培養(yǎng)基終止裂解。經(jīng)離心、重懸后，置于37℃、5%CO2培養(yǎng)箱中孵育2 h，收集未貼壁的骨髓細(xì)胞。

1.2.2髓源性巨噬細(xì)胞的誘導(dǎo)和分組處理 將收集的骨髓細(xì)胞鋪于6孔細(xì)胞培養(yǎng)板中，1×106個(gè)細(xì)胞/孔。按實(shí)驗(yàn)設(shè)計(jì)將骨髓細(xì)胞分為5組，即對(duì)照組、LPS/IFN-γ處理組以及3個(gè)LPS/IFN-γ與EGCG共處理組。依Ying等[11]的方法，各組骨髓細(xì)胞經(jīng)100 ng/ml M-CSF誘導(dǎo)7 d分化為巨噬細(xì)胞，第3天時(shí)更換含M-CSF的新鮮培養(yǎng)液一次。在M-CSF誘導(dǎo)第5天時(shí)，LPS/IFN-γ處理組加入1 μg/ml LPS和50 ng/ml IFN-γ培養(yǎng)48 h，而3個(gè)LPS/IFN-γ與EGCG共處理組則在加入1 μg/ml LPS和50 ng/ml IFN-γ的同時(shí)分別加入12.5 μmol/L、25 μmol/L和50 μmol/L的EGCG培養(yǎng)48 h。

1.2.3巨噬細(xì)胞促炎細(xì)胞因子mRNA水平的qRT-PCR分析 收集誘導(dǎo)處理7 d后的細(xì)胞，提取RNA，并按反轉(zhuǎn)錄試劑盒操作說(shuō)明，去除基因組DNA，進(jìn)行cDNA第一鏈的合成，并置于4℃保存。按照標(biāo)準(zhǔn)qRT-PCR引物設(shè)計(jì)原則，運(yùn)用primer 5.0軟件設(shè)計(jì)IL-1β、TNF-α和iNOS的引物序列(表1)。

qRT-PCR反應(yīng)按兩步法標(biāo)準(zhǔn)程序設(shè)置：95℃預(yù)變性5 min，95℃變性10 s、退火60 s、延伸30 s，共40個(gè)循環(huán)。然后依照qRT-PCR試劑盒操作說(shuō)明在羅氏Light Cycler?96熒光定量PCR儀上完成實(shí)驗(yàn)。實(shí)驗(yàn)所得數(shù)據(jù)則按照2-ΔΔCt法進(jìn)行計(jì)算，并進(jìn)行統(tǒng)計(jì)學(xué)分析。

1.2.4巨噬細(xì)胞促炎細(xì)胞因子含量的ELISA檢測(cè) 收集誘導(dǎo)處理7 d后的細(xì)胞培養(yǎng)液放入-80℃冰箱，以備ELISA法檢測(cè)細(xì)胞IL-1β和TNF-α的分泌量。然后用預(yù)熱PBS清洗后，按照6孔板每孔加入200 μl細(xì)胞裂解液，混勻后，10 000～14 000 g離心3～5 min，取上清用ELISA試劑盒檢測(cè)iNOS含量。

2 結(jié)果

2.1EGCG抑制IFN-γ/LPS刺激的髓源性巨噬細(xì)胞IL-1β基因表達(dá)和蛋白生成 小鼠骨髓細(xì)胞經(jīng)100 ng/ml M-CSF誘導(dǎo)分化為髓源性巨噬細(xì)胞。運(yùn)用qRT-PCR分析表明，與對(duì)照組巨噬細(xì)胞相比，經(jīng)50 ng/ml IFN-γ和1 μg/ml LPS聯(lián)合處理的巨噬細(xì)胞IL-1β mRNA表達(dá)水平極顯著地上調(diào)，存在極顯著性差異；與50 ng/ml IFN-γ和1 μg/ml LPS聯(lián)合處理組細(xì)胞相比，12.5～50 μmol/L EGCG處理可抑制IFN-γ/LPS誘導(dǎo)的巨噬細(xì)胞IL-1β基因表達(dá)上調(diào)，12.5 μmol/L EGCG處理組細(xì)胞的IL-1β mRNA水平顯著下調(diào)(P<0.05)，而25 μmol/L和50 μmol/L EGCG處理組細(xì)胞的IL-1β mRNA水平呈極顯著下調(diào)(P<0.01)(圖1A)。運(yùn)用ELISA技術(shù)檢測(cè)顯示，與對(duì)照組巨噬細(xì)胞相比，50 ng/ml IFN-γ和1 μg/ml LPS聯(lián)合刺激可使髓源性巨噬細(xì)胞的IL-1β蛋白生成極顯著性增加(P<0.01)，而12.5～50 μmol/LEGCG處理可極顯著性地抑制IFN-γ和LPS聯(lián)合刺激的IL-1β生成(P<0.01)(圖1B)。這些結(jié)果提示EGCG能夠抑制IFN-γ和LPS聯(lián)合刺激的小鼠髓源性M1巨噬細(xì)胞的IL-1β表達(dá)和生成，且存在劑量依賴(lài)效應(yīng)。

表1IL-1β、TNF-α和iNOS基因的引物序列

Tab.1PrimersequencesofIL-1β,TNF-αandiNOSgene

GenePrimers5'→3'IL-1βForward:CAGGCAGTATCACTCATT-GTGGCIL-1βReverse:CAGCAGGTTATCATCAT-CATCCCTNF-αForward:GAGTGACAAGCCTGTAGCCTNF-αReverse:CTCCTGGTATGAGATAG-CAAAiNOSForward:CACCAAGCTGAACTT-GAGCGiNOSReverse:GTGGCTTTGGGCTCCTCβ-actinForward:CGTCAGGCAGCTCAT-AGCTCTTCTβ-actinReverse:TGGCTACAGCTTCACCAC-CACAG

圖1 EGCG抑制IFN-γ/LPS刺激的小鼠髓源性巨噬細(xì)胞IL-1β基因表達(dá)和生成Fig.1 EGCG inhibits IL-1β gene expression and IL-1β production stimulated by IFN-γ/LPS in murine myeloid macrophagesNote: The myeloid cells were isolated from bone marrow,cultured with RPMI1640 medium contained 10% FBS.After induced with M-CSF(100 ng/ml)for 7 days,the myeloid cells differentiated into the myeloid macrophages.At 5th day,the myeloid macrophages were induced to M1 macrophage stimulated with 50 ng/ml IFN-γ and 1 μg/ml LPS,and at the same time,these cells were treated with 12.5 μmol/L,25 μmol/L,and 50 μmol/L EGCG for 48 h,respectively.After macrophages were harvested,the IL-1β mRNA level was analyzed with qRT-PCR(A),and the IL-1β level was detected with ELISA(B).**.P<0.01,compared with control;#.P<0.05 and ##.P<0.01,compared with IFN-γ+LPS group.

2.2EGCG抑制IFN-γ/LPS刺激的小鼠髓源性巨噬細(xì)胞TNF-α基因表達(dá)及生成 體外分離培養(yǎng)的小鼠骨髓細(xì)胞經(jīng)M-CSF誘導(dǎo)分化為髓源性巨噬細(xì)胞，進(jìn)一步在IFN-γ和LPS刺激下，分化為M1型巨噬細(xì)胞，并分泌細(xì)胞因子TNF-α。運(yùn)用qRT-PCR分析表明，M-CSF單獨(dú)誘導(dǎo)的對(duì)照組髓源性巨噬細(xì)胞TNF-α mRNA幾乎不表達(dá)，經(jīng)IFN-γ和LPS聯(lián)合刺激后，髓源性巨噬細(xì)胞的TNF-α mRNA水平極顯著性的增加(P<0.01)；與IFN-γ和LPS聯(lián)合刺激組的髓源性巨噬細(xì)胞相比，12.5～50 μmol/L EGCG處理后，巨噬細(xì)胞的TNF-α mRNA水平出現(xiàn)下降，且在50 μmol/L EGCG處理時(shí)下降最為明顯，呈現(xiàn)劑量依賴(lài)效應(yīng)(圖2A)。運(yùn)用ELISA檢測(cè)顯示，與對(duì)照組巨噬細(xì)胞相比，IFN-γ和LPS聯(lián)合刺激組的巨噬細(xì)胞TNF-α生成量極顯著性增加(P<0.01)；而經(jīng)12.5～50 μmol/L EGCG 處理后，巨噬細(xì)胞TNF-α生成量出現(xiàn)極顯著性下降(P<0.01)(圖2B)。這些結(jié)果提示EGCG能夠抑制IFN-γ和LPS聯(lián)合刺激的小鼠髓源性M1巨噬細(xì)胞的TNF-α表達(dá)和生成，且存在劑量依賴(lài)效應(yīng)。

圖2 EGCG抑制IFN-γ/LPS刺激的小鼠髓源性巨噬細(xì)胞TNF-α基因表達(dá)和生成Fig.2 EGCG represses TNF-α gene expression and TNF-α production stimulated by IFN-γ/LPS in mlurine myeloid macrophagesNote: The experimental method is same as Fig 1.After macrophages were harvested,the TNF-α mRNA level was analyzed with qRT-PCR(A),and the TNF-α level was detected with ELISA(B).**.P<0.01,compared with control;##.P<0.01,compared with IFN-γ+LPS group.

圖3 EGCG抑制IFN-γ/LPS刺激的小鼠髓源性巨噬細(xì)胞iNOS基因表達(dá)和生成Fig.3 EGCG decreases iNOS gene expression and iNOS production stimulated by IFN-γ/LPS in murine myeloid macrophagesNote: The experimental method is same as Fig 1.After macrophages were harvested,the iNOS mRNA level was analyzed with qRT-PCR(A),and the iNOS level was detected with ELISA(B).**.P<0.01,compared with control;##.P<0.01,compared with IFN-γ+LPS group.

2.3EGCG抑制IFN-γ/LPS刺激的小鼠髓源性巨噬細(xì)胞iNOS基因表達(dá)及生成 巨噬細(xì)胞在IFN-γ或LPS的刺激下極化為M1型巨噬細(xì)胞，高表達(dá)iNOS，并產(chǎn)生NO，因而iNOS是M1型巨噬細(xì)胞的重要標(biāo)志物。運(yùn)用qRT-PCR分析表明，經(jīng)M-CSF單獨(dú)誘導(dǎo)的對(duì)照組髓源性巨噬細(xì)胞iNOS mRNA幾乎不表達(dá)，經(jīng)IFN-γ和LPS聯(lián)合刺激后，髓源性巨噬細(xì)胞的iNOS mRNA水平極顯著性增加(P<0.01)；與IFN-γ和LPS聯(lián)合刺激組的髓源性巨噬細(xì)胞相比，12.5～50 μmol/L EGCG處理后，巨噬細(xì)胞的iNOS mRNA水平出現(xiàn)下降，并隨著EGCG濃度增加，下降幅度越大，呈現(xiàn)出明顯的劑量依賴(lài)效應(yīng)(圖3A)。運(yùn)用ELISA檢測(cè)顯示，與對(duì)照組巨噬細(xì)胞相比，IFN-γ和LPS聯(lián)合刺激組的巨噬細(xì)胞iNOS生成量極顯著性增加(P<0.01)；而經(jīng)12.5～50 μmol/L EGCG 處理后，巨噬細(xì)胞iNOS生成量出現(xiàn)極顯著性下降(P<0.01)(圖3B)。這些結(jié)果提示EGCG能夠抑制IFN-γ和LPS聯(lián)合刺激的小鼠髓源性M1巨噬細(xì)胞的iNOS表達(dá)和生成，且存在劑量依賴(lài)效應(yīng)。

3 討論

眾所周知，巨噬細(xì)胞極化是機(jī)體免疫系統(tǒng)維持免疫穩(wěn)態(tài)的一個(gè)動(dòng)態(tài)過(guò)程，某些特定疾病的發(fā)生條件與巨噬細(xì)胞極化密切相關(guān)，了解這些機(jī)制將有利于疾病的治療[12]，而骨髓來(lái)源的巨噬細(xì)胞(Bone marrow-derived macrophages,BMDM)是研究巨噬細(xì)胞極化的適宜細(xì)胞模型[11]。巨噬細(xì)胞的發(fā)育與分化依賴(lài)于巨噬細(xì)胞集落刺激因子(Macrophages colony-stimulating factor,M-CSF)的誘導(dǎo)，骨髓細(xì)胞在M-CSF誘導(dǎo)下激活ERK1/2通路，促使骨髓前體細(xì)胞向巨噬細(xì)胞分化[13]；巨噬細(xì)胞在IFN-γ和LPS刺激下極化成M1型巨噬細(xì)胞。LPS能與巨噬細(xì)胞表面的Toll樣受體4(Toll-like receptor 4,TLR4)結(jié)合，經(jīng)Akt1/2誘導(dǎo)巨噬細(xì)胞極化[14]，進(jìn)而增強(qiáng)促炎細(xì)胞因子的基因表達(dá)和分泌[15，16]；而IFN-γ可進(jìn)一步增強(qiáng)M1型巨噬細(xì)胞的活化。

在本實(shí)驗(yàn)中，M-CSF誘導(dǎo)小鼠骨髓細(xì)胞形成BMDM，后者在IFN-γ和LPS刺激下極化為M1型巨噬細(xì)胞，其分泌的IL-1β、TNF-α、iNOS在mRNA表達(dá)水平是增加的，并且具有極顯著性差異。這與Ying等[11]的實(shí)驗(yàn)結(jié)果一致的，他們將小鼠骨髓細(xì)胞經(jīng)M-CSF誘導(dǎo)形成髓源性巨噬細(xì)胞，IFN-γ和LPS刺激1周后，檢測(cè)顯示巨噬細(xì)胞的IL-1β、TNF-α表達(dá)水平顯著上升；流式細(xì)胞術(shù)檢測(cè)也顯示巨噬細(xì)胞的表面標(biāo)記物CD11b、F4/80、CD206表達(dá)增強(qiáng)；同時(shí)還發(fā)現(xiàn)細(xì)胞的p65可在IFN-γ和LPS刺激后活化，表明巨噬細(xì)胞極化與p65活化相關(guān)。本實(shí)驗(yàn)中，EGCG和LPS/IFN-γ共處理后，M1型巨噬細(xì)胞促炎細(xì)胞因子IL-1β、TNF-α和iNOS表達(dá)下降，表明EGCG能降低LPS/IFN-γ刺激的巨噬細(xì)胞促炎細(xì)胞因子表達(dá)。

EGCG通過(guò)何種機(jī)制調(diào)控LPS/IFN-γ刺激的巨噬細(xì)胞促炎細(xì)胞因子表達(dá)和生成？有研究表明，EGCG可通過(guò)細(xì)胞膜表面的67 kD層黏連蛋白受體(67-kD laminin receptor,67 LR)機(jī)制介導(dǎo)促炎細(xì)胞因子的分泌[17]，EGCG不但通過(guò)67 LR抑制IKKβ活性進(jìn)而阻斷NF-κB通路的活化[18]，而且還能通過(guò)67 LR下調(diào)LPS刺激的巨噬細(xì)胞TLR4信號(hào)轉(zhuǎn)導(dǎo)作用，抑制絲裂原活化蛋白激酶(Mitogen activated protein kinase,MAPK)的激活，進(jìn)而減少促炎細(xì)胞因子的生成[19，20]。EGCG也可通過(guò)AMP激活的蛋白激酶(Adenosine 5′-monophosphate-activated protein kinase,AMPK)抑制LPS刺激的促炎細(xì)胞因子TNF-α、IL-6和iNOS的生成[21]。有學(xué)者發(fā)現(xiàn)EGCG抑制LPS誘導(dǎo)的巨噬細(xì)胞產(chǎn)生NO時(shí)，還能夠抑制Rab5與小凹蛋白-1(caveolin-1)的相互作用以及降低Rab5的活性，這些現(xiàn)象提示EGCG的這些作用是通過(guò)抑制LPS的內(nèi)吞作用進(jìn)而干擾Rab5與caveolin-1的相互作用和降低Rab5的活性實(shí)現(xiàn)的[22]。在THP-1巨噬細(xì)胞上的研究顯示，EGCG抑制LPS刺激的IL-1β和TNF-α表達(dá)的機(jī)制并不僅僅依賴(lài)于經(jīng)典的NF-κB、MAPK信號(hào)通路[23]，提示EGCG抗炎機(jī)制可能是通過(guò)多種信號(hào)通路組成的調(diào)控網(wǎng)絡(luò)實(shí)現(xiàn)的，但這還需要更多的實(shí)驗(yàn)證據(jù)予以支持。

本研究表明EGCG能夠下調(diào)IFN-γ和LPS刺激的髓源性巨噬細(xì)胞促炎細(xì)胞因子IL-1β、TNF-α和iNOS表達(dá)，抑制巨噬細(xì)胞向M1型巨噬細(xì)胞極化，降低其炎性反應(yīng)，這為EGCG在防治炎性疾病方面的潛在作用提供了一定的實(shí)驗(yàn)依據(jù)。

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Influenceofepigallocatechin-3-gallateonmyeloidmacrophagespolarizationinducedwithIFN-γ/LPSinmice

WANGWei-Fang,WANGHui,ZHANGXiao-Jing,CUIMeng-Ke,LIWei-Guo,WANGKun-Ying.

CollegeofLifeScience,HenanNormalUniversity,Xinxiang453007,China

Objective:To investigate the effect of epigallocatechin-3-gallate(EGCG)on the expression of pro-inflammatory cytokines in murine bone marrow-derived macrophages(BMDM)induced by IFN-γ/LPS.MethodsThe bone marrow cells were isolated from 6-8 weeks C57BL/6 mice,which cultured in RPMI1640 medium with 10% FBS and stimulated with 100 ng/ml M-CSFinvitro,and then exposed to 50 ng/ml IFN-γ and 1 μg/ml LPS with various concentrations of EGCG(12.5-50 μmol/L).The expression of pro-inflammatory factors,IL-1β,TNF-α and iNOS were detected,in murine myeloid macrophages stimulated by IFN-γ/LPS with qRT-PCR and ELISA.ResultsIFN-γ/LPS remarkably up-regulated the expression levels of inflammatory cytokines,IL-1β,TNF-α and iNOS,but EGCG effectively repressed these cytokines expression in IFN-γ/LPS-stimulated murine myeloid macrophages,in dose-dependent manner.ConclusionEGCG attenuates the pro-inflammatory phenotype of murine myeloid macrophages stimulated with IFN-γ/LPS.

Myeloid macrophages;Epigallocatechin-3-gallate;Macrophage polarization

10.3969/j.issn.1000-484X.2017.12.011

R392.12

A

1000-484X(2017)12-1810-05

①本文受河南省重點(diǎn)科技攻關(guān)計(jì)劃(No.122102310282)項(xiàng)目資助。

王衛(wèi)芳(1990年-), 女, 在讀碩士, 主要從事巨噬細(xì)胞極化方面的研究,E-mail:1475165901@qq.com。

及指導(dǎo)教師：李衛(wèi)國(guó)(1963年-), 男, 博士, 教授, 碩士生導(dǎo)師, 主要從事細(xì)胞免疫學(xué)方面的研究，E-mail:liwg0618@ htu.cn。

[收稿2017-04-20 修回2017-06-29]

(編輯 張曉舟)