芹菜素通過NF-κB和MAPK抑制脂多糖誘導(dǎo)的

1、PAGE 8食品安全質(zhì)量檢測學(xué)報(bào)第8卷第3期孫亞賽, 等: 芹菜素通過NF-B和MAPK抑制脂多糖誘導(dǎo)的骨髓來源的巨噬細(xì)胞炎性PAGE 7第8卷 第3期食品安全質(zhì)量檢測學(xué)報(bào)Vol. 8 No. 32017年3月Journal of Food Safety and QualityMar. , 2017 基金項(xiàng)目: 安徽省自然科學(xué)基金項(xiàng)目(1508085MC58)Fund: Supported by the Anhui Provincial Natural Science Foundation (1508085MC58)*通訊作者: 屈瑋, 副教授, 碩士生導(dǎo)師, 主要研究方向?yàn)樘烊划a(chǎn)物與肥胖相關(guān)

2、代謝疾病。E-mail: quweiok*Corresponding author: QU Wei, Associate Professor, Hefei University of Technology, No.193, Tunxi Road, Baohe District, Hefei 230009, China. E-mail: quweiok芹菜素通過NF-B和MAPK抑制脂多糖誘導(dǎo)的骨髓來源的巨噬細(xì)胞炎性孫亞賽1, 賀 云1, 劉 健2, 屈 瑋2*(1. 合肥工業(yè)大學(xué)食品科學(xué)與工程學(xué)院, 合肥 230009; 2. 合肥工業(yè)大學(xué)生物與醫(yī)學(xué)工程學(xué)院, 合肥 230009)摘 要: 目

3、的 研究芹菜素在脂多糖(lipopolysaccharide, LPS)誘導(dǎo)的骨髓來源的巨噬細(xì)胞(bone-marrow- derived macrophages, BMDMs)炎性的作用。方法 用MTT方法篩選出芹菜素起作用的濃度范圍。選擇適當(dāng)?shù)那鄄怂貪舛忍幚鞡MDMs, 用實(shí)時(shí)定量PCR方法檢測炎性基因的表達(dá), 用Western-blot方法檢測炎性信號通路蛋白的表達(dá), 以及熒光免疫檢驗(yàn)法考察NF-B P65蛋白的核轉(zhuǎn)位。結(jié)果 芹菜素的有效作用濃度范圍為030 mol/L, 選用10, 30 mol/L濃度的芹菜素培養(yǎng)BMDMs后, 炎性因子的mRNA水平明顯降低, 巨噬細(xì)胞從促炎性M1型

4、向抗炎性M2型轉(zhuǎn)化, 并且呈現(xiàn)劑量依賴的趨勢。NF-B和MAPK信號通路的激活也受到了明顯的抑制。結(jié)論 芹菜素能通過抑制NF-B和MAPK信號通路降低LPS誘導(dǎo)的BMDMs的炎性。關(guān)鍵詞: 芹菜素; 骨髓來源的巨噬細(xì)胞; 脂多糖; NF-B; MAPKInhibition of apigenin on lipopolysaccharide-induced bone-marrow-derived macrophages inflammation through NF-B and MAPKSUN Ya-Sai1, HE Yun1, LIU Jian2, QU Wei2*(1. School of

5、Food Science and Engineering, Hefei University of Technology, Hefei 230009, China;2. School of Biological and Medical Engineering, Hefei University of Technology, Hefei 230009, China)ABSTRACT: Objective To investigate the effect of apigenin on lipopolysaccharide (LPS) induced inflammation and in bon

6、e-marrow-derived macrophages (BMDMs). Methods The apigenin dosage range that did not influence cell viability was detected by MTT assay. After choosing the suitable dose of apigenin to co-culture with BMDMs, the inflammatory genes expression was detected by RT-PCR, proteins expression which particip

7、ated in inflammatory signaling pathways was detected by Western-blot, and NF-B P65 nuclear translocation was investigated by immunofluorescence. Results The effective dose of apigenin was between 030 mol/L. By choosing 10 and 30 mol/L apigenin to co-culture with BMDMs, inflammatory cytokines were re

8、duced in mRNA level, BMDMs transformed from M1 inflammatory macrophages to anti-inflammatory M2 macrophages and all these results showed in a dose-dependent manner. Apigenin also inhibited NF-B and MAPK signaling pathways significantly. Conclusion Apigenin can reduce LPS-induced macrophages inflamma

9、tion and partly through NF-B and MAPK signaling pathways.KEY WORDS: apigenin; bone-marrow-derived macrophages; lipopolysaccharide; NF-B; MAPK1 引 言炎癥被認(rèn)為是慢性病發(fā)病機(jī)制的主要因素之一。巨噬細(xì)胞作為重要的免疫細(xì)胞, 可以通過分泌趨化因子募集其他的免疫細(xì)胞進(jìn)入炎性位點(diǎn)或者分泌炎性因子極化或者活化其他免疫細(xì)胞從而增加炎性 ADDIN EN.CITE Sica2012409140940917Sica, A.Mantovani, A.Istituto Cl

10、inico Humanitas IRCCS, Rozzano, Italy. antonio.sicahumanitasresearch.itMacrophage plasticity and polarization: in vivo veritasJ Clin InvestThe Journal of clinical investigationJ Clin InvestThe Journal of clinical investigationJ Clin InvestThe Journal of clinical investigation787-951223AnimalsCell Li

11、neageHumansInflammationMacrophage ActivationMacrophages/*cytologyMiceModels, BiologicalOxygen/chemistryPhenotypeSignal TransductionTranscription Factors/metabolism2012Mar1558-8238 (Electronic)0021-9738 (Linking)22378047/pubmed/22378047328722310.1172/JCI59643HYPERLINK l _ENREF_1 o Sica, 2012 #4091。因?yàn)?/p>

12、長期的促炎性的狀態(tài)會形成慢性炎癥疾病, 如類風(fēng)濕性關(guān)節(jié)炎、牛皮癬、炎癥性腸病 ADDIN EN.CITE Valledor2010422242242217Valledor, A. F.Comalada, M.Santamaria-Babi, L. F.Lloberas, J.Celada, A.Nuclear Receptors Group, Department of Physiology, School of Biology, Barcelona, Spain.Macrophage proinflammatory activation and deactivation: a questio

13、n of balanceAdv ImmunolAdvances in immunologyAdv ImmunolAdvances in immunologyAdv ImmunolAdvances in immunology1-20108AnimalsHumansInflammation/*physiopathologyInflammation Mediators/immunologyMacrophage ActivationMacrophages/*cytology/*immunology20101557-8445 (Electronic)0065-2776 (Linking)21056727

14、/pubmed/2105672710.1016/B978-0-12-380995-7.00001-XHYPERLINK l _ENREF_2 o Valledor, 2010 #4222, 所以巨噬細(xì)胞是慢性炎性相關(guān)疾病的重要研究對象。炎性狀態(tài)往往伴隨著許多信號通路的參與, 并轉(zhuǎn)錄調(diào)控炎性基因的表達(dá), 其中MAPK和NF-B信號通路在炎性過程中起到了十分重要的調(diào)節(jié)作用 ADDIN EN.CITE ADDIN EN.CITE.DATA HYPERLINK l _ENREF_3 o Neacsu, 2015 #4273。脂多糖(lipopolysaccharide, LPS)作為革蘭氏陰性菌膜上的

15、重要組成部分之一, 是一種能夠引起炎性反應(yīng)的誘導(dǎo)劑 ADDIN EN.CITE ADDIN EN.CITE.DATA HYPERLINK l _ENREF_4 o Park, 2013 #4314,HYPERLINK l _ENREF_5 o Abarikwu, 2014 #4395。在LPS的處理下, 會誘導(dǎo)巨噬細(xì)胞向M1型的轉(zhuǎn)變, NF-B和MAPK信號通路被激活, 并且分泌很多炎性因子, 如IL-1、IL-6、MCP-1是基因還是蛋白?基因需要斜體，蛋白不用，請核實(shí)等 ADDIN EN.CITE ADDIN EN.CITE.DATA HYPERLINK l _ENREF_3 o Neac

16、su, 2015 #4273。因此本研究采用此種處理方式進(jìn)行研究。芹菜素, 又名4,5,7-HYPERLINK /subview/6662826/6786174.htm t _blank三羥基黃酮, 廣泛存在于水果和蔬菜中 ADDIN EN.CITE ADDIN EN.CITE.DATA HYPERLINK l _ENREF_6 o Shukla, 2010 #4426,HYPERLINK l _ENREF_7 o Lee, 2015 #4457。近年來, 已經(jīng)有一些報(bào)道證明芹菜素有抗炎、抗氧化、抗癌的效果 ADDIN EN.CITE Shukla2010447644744717Shukla,

17、 S.Gupta, S.Department of Urology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.Apigenin: a promising molecule for cancer preventionPharm ResPharmaceutical researchPharm ResPharmaceutical researchPharm ResPharmaceutical research962-78276AnimalsAnticarcinogenic Age

18、nts/*therapeutic useApigenin/*therapeutic useHumansNeoplasms/diet therapy/*prevention & control2010Jun1573-904X (Electronic)0724-8741 (Linking)20306120/pubmed/20306120287446210.1007/s11095-010-0089-7HYPERLINK l _ENREF_6 o Shukla, 2010 #4426。但芹菜素對LPS誘導(dǎo)的骨髓來源的巨噬細(xì)胞的作用的報(bào)道很少。因此, 本研究重點(diǎn)考察了芹菜素對小鼠骨髓來源的巨噬細(xì)胞炎性的

19、作用。2 材料與方法2.1 材料與試劑C57BL/6小鼠(北京維通利華實(shí)驗(yàn)動物技術(shù)有限公司); L929細(xì)胞(北京協(xié)和醫(yī)院細(xì)胞資源中心); 二甲基亞砜(dimethyl sulfoxide, DMSO)、LPS、噻唑藍(lán)(methylthiazolyldiphenyl-tetrazolium bromide, MTT)(美國Sigma公司); 細(xì)胞培養(yǎng)基(cell culture media, DMEM)、胎牛血清(fetal bovine serum, FBS)、雙抗(美國GIBCO公司); RNAiso Plus試劑、PrimeScriptTM RT Reagent試劑盒和SYBRPremi

20、x Ex TaqTM試劑盒(日本TaKaRa公司); 逆轉(zhuǎn)錄試劑盒(美國Invitrogen公司); Apigenin(南京春秋生物工程有限公司); 胰酶(美國Hyclone公司); 化學(xué)發(fā)光底物ECL(美國Thermo Scientific公司); NF-B抗體試劑盒、MAPK抗體試劑盒、-actin抗體(美國Cell Signaling Technology公司); 羊抗兔lgG(美國KPL公司); 異硫氰酸HYPERLINK /view/2062542.htm t _blank熒光素(fluorescein isothiocyanate, FITC)、4,6-二脒基-2-苯基吲哚(4,6

21、-diamidino-2-phenylindole, DAPI)(美國Biolegend公司); 三氯甲烷、氯仿、異丙醇(分析純, 國藥集團(tuán)化學(xué)試劑有限公司)。2.2 實(shí)驗(yàn)儀器與設(shè)備細(xì)胞培養(yǎng)箱(美國Thermo Scientific公司); 超凈工作臺(上海智誠公司); 漩渦混合器(琪特儀器公司); 低速離心機(jī)(美國Sigma公司); 高速冷凍離心機(jī)(上海天美生化儀器公司); 酶標(biāo)儀、定量PCR儀、蛋白電泳槽(美國Bio-Red公司); 搖床(Kylin-Bell公司); 分光光度計(jì)(德國Eppendorf公司); 生物顯微鏡(奧特光學(xué)公司); 熒光顯微鏡(Nikon公司); 熒光呈像系統(tǒng)(瑞

22、典GE Healthcare 公司); 臺式吸引器(紹興衛(wèi)星醫(yī)療設(shè)備公司); 純水儀(英國ELGA公司); 高壓滅菌鍋(上海申安醫(yī)療器械公司)。2.3 實(shí)驗(yàn)方法2.3.1 細(xì)胞培養(yǎng)(1) L929細(xì)胞培養(yǎng)用DMEM+10%胎牛血清(FBS)的培養(yǎng)基培養(yǎng)L929細(xì)胞, 每2 d更換1次培養(yǎng)基, 到細(xì)胞生長到單層融合后, 更換新的細(xì)胞液, 3 d后收集上含 HYPERLINK /view/3848155.htm t _blank 巨噬細(xì)胞集落刺激因子(M-CSF)的細(xì)胞培養(yǎng)基。(2) 骨髓來源的巨噬細(xì)胞(BMDMs)培養(yǎng)用CO2將C57BL/6小鼠處死, 取完整后肢并且浸泡在75%乙醇中。盡快將脛

23、骨和腓骨與肌肉組織分離, 用無菌注射器吸取DMEM培養(yǎng)基將骨髓吸出到培養(yǎng)皿中, 用移液槍移入50 mL離心管中, 1500 r/min離心5 min。棄上清之后, 用含有20%FBS和30%L929細(xì)胞培養(yǎng)基的DMEM培養(yǎng)基, 37 5%CO2條件下培養(yǎng), 每3 d換1次液。大約培養(yǎng)6 d骨髓細(xì)胞分化成BMDMs, 用10、30 mol/L的芹菜素, 對照組加入等量的二甲基亞砜(DMSO), 處理12 h。之后加入100 ng/mL LPS(處理30 min用于Western-blot實(shí)驗(yàn), 處理4 h用于定量PCR實(shí)驗(yàn)), 最后用PBS洗2遍并收集細(xì)胞。2.3.2 MTT實(shí)驗(yàn)將已經(jīng)分化成的B

24、MDMs細(xì)胞用胰酶消化后, 接到96孔板上, 大約每孔103104細(xì)胞, 20%FBS和30%L929細(xì)胞培養(yǎng)基的DMEM培養(yǎng)基, 37 5%CO2條件下培養(yǎng)。待細(xì)胞長到單層鋪滿并融合后, 用0、10、20、30、40、50 mol/L芹菜素處理BMDMs 24 h。用PBS清洗2遍, 每孔加入150 L培養(yǎng)基和20 L MTT, 培養(yǎng)4 h。用PBS清洗2遍, 每孔加入150 L DMSO并低速振蕩10 min, 紫色結(jié)晶溶解完全后用酶標(biāo)儀測定OD490值。2.3.3 定量PCR檢測基因表達(dá)實(shí)驗(yàn)(1)細(xì)胞總RNA提取用于提取RNA的BMDMs細(xì)胞培養(yǎng)于24孔板, 用PBS洗1遍后, 每孔加入

25、300 L RNAiso Plus, 放平培養(yǎng)板使其與BMDMs充分接觸, 然后用移液槍反復(fù)吹打, 待細(xì)胞裂解充分后, 轉(zhuǎn)入0.5 mL EP管中, 室溫靜置5 min。向細(xì)胞裂解液中加入60 L的氯仿, 充分振蕩并靜置5 min, 之后12000 g 4 離心15 min。此時(shí)分為3層: 上層澄清溶液, 中間白色蛋白層和下層有機(jī)層。將上層澄清溶液轉(zhuǎn)入新的0.5 mL EP管中, 加入與上清等體積的異丙醇, 充分震蕩后, 室溫靜置10 min, 在將其在12000 g 4 離心10 min離心, 有白色沉淀產(chǎn)生。將上清倒掉后, 加入1 mL 75%乙醇清洗沉淀中殘留的氯仿, 與上次離心放置方向

26、一致, 再次離心, 以防止沉淀丟失。棄去乙醇保留沉淀, 室溫放置510 min, 加入適量的RNAase-free 水溶解沉淀, 存于-80 。(2) RNA的反轉(zhuǎn)錄取2 L RNA加入到198 L去離子水中, 利用分光光度計(jì)測定提取RNA的濃度。根據(jù)PrimeScriptTMRT Master Mix試劑盒說明書進(jìn)添加逆轉(zhuǎn)錄試劑, 37 反應(yīng)30 min后再用85 水浴處理5 s后終止逆轉(zhuǎn)錄, 即得到了cDNA。(3)定量PCR每個(gè)樣品建立20 L反應(yīng)體系: TaKaRaSYBRPremix Ex TaqTM 10 L; 上游引物0.4 L、下游引物0.4 L、雙蒸水8.2 L、cDNA 1

27、 L。充分振蕩混勻體系并短暫離心后放在Bio-rad IQ2定量PCR儀器中進(jìn)行反應(yīng)。用CT閾值循環(huán)方法進(jìn)行數(shù)據(jù)處理, 用目的基因相對于GAPDH的水平表示目的基因的表達(dá)水平。用于實(shí)時(shí)定量PCR引物序列詳見表1。2.3.4 Western-blot 實(shí)驗(yàn)(1) 細(xì)胞的總蛋白提取用于提取蛋白的BMDMs細(xì)胞培養(yǎng)于6孔板, 每孔加入200 L預(yù)冷RIPA裂解液(含蛋白酶抑制劑和胰酶抑制劑)后將細(xì)胞刮下, 轉(zhuǎn)移到EP管中, 每孔再加入300 L裂解液后轉(zhuǎn)移到EP管中。將蛋白裂解液放于冰上, 搖床上搖晃30 min, 以便于細(xì)胞充分裂解。裂解過程結(jié)束后, 將EP管12000 g 4 離心30 min,

28、 吸取上清轉(zhuǎn)移到新的EP管中, 即得到BMDMs細(xì)胞的總蛋白。以BSA為標(biāo)準(zhǔn)品, 檢測所提蛋白的濃度。每個(gè)樣品加入其1/4體積的上樣緩沖液, 100 煮沸10 min后, 分裝并凍存于-80 。(2) Western-blot電泳: 根據(jù)分子克隆實(shí)驗(yàn)指南的配方配制膠。裝配好電泳槽, 向電泳槽中加入電泳緩沖液。取出凍存于-80 蛋白樣品, 融化, 混勻后上樣。80 V電壓電泳30 min后, 將電壓調(diào)為120 V。等到藍(lán)色上樣緩沖液完全跑出膠之后終止電泳。轉(zhuǎn)膜: 200 mA恒流轉(zhuǎn)膜2 h。封閉: 轉(zhuǎn)膜結(jié)束后, 取出PVDF膜, 用脫脂牛奶室溫封閉2 h。一抗二抗: 用TBST清洗PVDF膜上殘

29、留的牛奶后, 孵育一抗, 4 過夜。用TBST洗3次, 每次5 min。之后孵育二抗, 室溫2 h。再用TBST洗3次, 每次5 min。ECL顯影。用凝膠成像儀拍照并分析。2.3.5 P65熒光定位實(shí)驗(yàn)細(xì)胞固定: 將BMDMs細(xì)胞板放在冰上, 用遇冷的PBS清洗2遍。用甲醇固定, 5 min后吸走, 在空氣中晾干大概1015 min。封閉: 加入含有2%BSA的PBS溶液進(jìn)行封閉, 室溫條件下處理30 min。孵育一抗: 封閉完成之后, 棄去封閉液, 加入NF-B P65抗體(1:500), 室溫孵育1 h。之后用PBS清洗3次, 每次5 min。孵育二抗: 在孔板中加入BL-羊抗兔(1:2

30、00), 避光條件下, 室溫放置1 h。之后用PBS清洗3次, 每次5 min。孵育三抗: 在孔板中加入FITC (1:300)和DAPI(1:100), 室溫避光條件下放置2 h, 之后用PBS清洗3次, 每次5 min。每張片子隨機(jī)選取5個(gè)視野, 400倍數(shù)條件下進(jìn)行拍照并分析。2.3.6 統(tǒng)計(jì)學(xué)方法文章中的所有數(shù)據(jù)都是用平均值標(biāo)準(zhǔn)誤來表示, 細(xì)胞分析中不同組之間用t檢驗(yàn)。當(dāng)P0.05視為具有統(tǒng)計(jì)學(xué)顯著性, 用*表示。3 結(jié)果與分析3.1 芹菜素對LPS誘導(dǎo)BMDMs炎性和極性的影響為了探討芹菜素對LPS誘導(dǎo)的BMDM炎性的影響, 首先探討不同濃度芹菜素對BMDMs細(xì)胞活性的影響。如圖1A

31、所示, 與對照組相比, 40 mol/L芹菜素開始降低細(xì)表1 定量PCR的引物序列Table 1 Primers for quantitative real-time PCR基因正向引物序列(5 to 3)反向引物序列(5 to 3)GAPDHTGTCATACTTGGCAGGTTTCTCGTGTTCCTACCCCCAATGTIL-1GCAACTGTTCCTGAACTCAACTTCTTTTGGGGTCCGTCAACTIL-6CCTTCCTACCCCAATTTCCAAAGATGAATTGGATGGTCTTGGTCMCP-1CCCCAAGAAGGAATGGGTCCGGTTGTGGAAAAGGTAGT

32、GGINOSCCAAGCCCTCACCTACTTCCCTCTGAGGGCTGACACAAGGARG1CTCCAAGCCAAAGTCCTTAGAGAGGAGCTGTCATTAGGGACATC圖1 A: 不同濃度的芹菜素對BMDMs細(xì)胞存活率的影響; BD: 芹菜素對BMDMs炎性基因IL-6、MCP-1和IL-1表達(dá)的影響; E: 芹菜素對BMDMs中M1型巨噬細(xì)胞標(biāo)記基因INOS表達(dá)的影響; F: 芹菜素對BMDMs中M2型巨噬細(xì)胞標(biāo)記基因ARG1表達(dá)的影響Fig. 1 A: The effect of different concentrations of apigenin on BMDMs

33、 cell viability; BD: The effect of apigenin on BMDMs inflammatory genes expression: IL-6, MCP-1 and IL-1; E: The effect of apigenin on M1 macrophages marker gene INOS expression in BMDMs; F: The effect of apigenin on M2 macrophages marker gene ARG1 expression in BMDMs胞存活率近50%。為了避免過量的芹菜素本身對細(xì)胞活性的影響, 選

34、擇10、30 mol/L芹菜素培養(yǎng)細(xì)胞, 從圖1B、圖1C中可以看出, 芹菜素可以降低LPS誘導(dǎo)的BMDMs中炎性基因IL-6、MCP-1和IL-1的表達(dá), 并且隨著劑量的增加抑制效果更顯著。因?yàn)樵贚PS的刺激下從M2抗炎性巨噬細(xì)胞向M1促炎性巨噬細(xì)胞轉(zhuǎn)變 ADDIN EN.CITE Lee2016452845245217Lee, J. W.Park, S.Han, H. K.Um, S. H.Moon, E. Y.Department of Bioscience and Biotechnology, Sejong University, Seoul, 05006, Republic of K

35、orea.Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi-do, 16419, Republic of Korea.Department of Health Sciences and Technology, SAIHST, Samsung Medical Center, Sungkyunkwan University, Seoul, 06351, Republic of

36、Korea.Polarized macrophages treated with nonylphenol differently regulate lipopolysaccharide-induced sepsisEnviron ToxicolEnvironmental toxicologyEnviron ToxicolEnvironmental toxicologyEnviron ToxicolEnvironmental toxicology2016Aug 291522-7278 (Electronic)1520-4081 (Linking)27570978/pubmed/275709781

37、0.1002/tox.22340HYPERLINK l _ENREF_8 o Lee, 2016 #4528。圖1E、圖1F顯示, 在芹菜素處理下, M1型促炎性巨噬細(xì)胞的標(biāo)記基因INOS明顯降低, M2型抗炎性巨噬細(xì)胞的標(biāo)記基因ARG1顯著升高。這些結(jié)果也證明芹菜素可以明顯降低LPS誘導(dǎo)的BMDMs的炎性, 抑制BMDMs向促炎性的轉(zhuǎn)變, 且這個(gè)現(xiàn)象呈現(xiàn)劑量依賴的趨勢。3.2 芹菜素抑制LPS誘導(dǎo)的BMDMs中NF-B信號通路的激活因?yàn)镹F-B轉(zhuǎn)錄調(diào)控的炎性因子在芹菜素的處理下有所改善, 所以進(jìn)一步考察芹菜素是否影響到NF-B信號通路。從圖2A圖2E可以看出, LPS導(dǎo)致的p-IKK/激活在

38、芹菜素處理下降低了60%左右, IKK呈現(xiàn)出了降低的趨勢, IKK總蛋白也明顯降低。在IKK的影響下, IB磷酸化和降解都有明顯的改善。因?yàn)檠仔曰虻霓D(zhuǎn)錄是發(fā)生在NF-B P65進(jìn)入細(xì)胞核之后, 所以用熒光定位實(shí)驗(yàn)檢測芹菜素是否影響了NF-B P65進(jìn)入細(xì)胞核。從圖1F中可以看出, 在加入芹菜素之后, NF-B P65進(jìn)入細(xì)胞核明顯降低。這些結(jié)果也就說明, 芹菜素可以抑制LPS誘導(dǎo)的BMDMs中NF-B信號通路的激活, 從而降低對炎性因子的轉(zhuǎn)錄表達(dá)。3.3 芹菜素抑制LPS誘導(dǎo)的BMDMs中MAPK信號通路的激活因?yàn)镸APK作為另外一條重要的調(diào)控炎性因子表達(dá)的信號通路, 在這里也做了相應(yīng)的考察

39、, 并且MAPK是由3個(gè)主要的信號通路組成: ERK、JNK、P38 MAPK ADDIN EN.CITE Bost20055701357057017Bost, F.Aouadi, M.Caron, L.Binetruy, B.Inserm U568, Universite de Nice Sophia-Antipolis, Faculte de Medecine, Avenue de Valombrose, 06107 Nice cedex, France. bostunice.frThe role of MAPKs in adipocyte differentiation and obes

40、ityBiochimieBiochimieBiochimieBiochimieBiochimieBiochimie51-6871Adipocytes/*cytologyAnimals*Cell DifferentiationExtracellular Signal-Regulated MAP Kinases/physiologyJNK Mitogen-Activated Protein Kinases/physiologyMAP Kinase Signaling System/*physiologyMiceMice, KnockoutMitogen-Activated Protein Kina

41、ses/*physiologyModels, BiologicalObesity/etiology/*physiopathologyp38 Mitogen-Activated Protein Kinases/physiology2005Jan0300-9084 (Print)0300-9084 (Linking)15733737/pubmed/1573373710.1016/j.biochi.2004.10.0189。由于JNK和P38 MAPK主要是受到代謝壓力如滲透休克, 厭氧, 熱休克等的影響, 特別是在LPS、TNFs刺激下產(chǎn)生大量的促炎性因子 ADDIN EN.CITE ADDIN

42、EN.CITE.DATA 10,11。所以, 這里重點(diǎn)考察對JNK和P38 MAPK的影響。在圖3A圖3E中, 芹菜素可以明顯降低p-JNK(約40%)和p-P38 MAPK(約50%)?？傊? 芹菜素可以通過MAPK信號通路調(diào)節(jié)LPS誘導(dǎo)BMDMs的炎性。4 討 論巨噬細(xì)胞作為各個(gè)組織中很重要的炎性細(xì)胞, 經(jīng)歷了幾個(gè)重要的分化階段, 從骨髓造血干細(xì)胞到血液中的單核細(xì)胞, 再分化成為各個(gè)組織的巨噬細(xì)胞 ADDIN EN.CITE Rickard20095711657157117Rickard, A. J.Young, M. J.Prince Henry's Institute of

43、Medical Research, Victoria, Australia. amanda.rickardCorticosteroid receptors, macrophages and cardiovascular diseaseJ Mol EndocrinolJournal of molecular endocrinologyJ Mol EndocrinolJournal of molecular endocrinologyJ Mol EndocrinolJournal of molecular endocrinology449-59426Cardiovascular Diseases/

44、*physiopathologyHumansMacrophages/*physiologyReceptors, Glucocorticoid/*physiologyReceptors, Mineralocorticoid/*physiology2009Jun1479-6813 (Electronic)0952-5041 (Linking)19158233/pubmed/1915823310.1677/JME-08-014412。目前雖然已經(jīng)有芹菜素對于抑制LPS誘導(dǎo)的巨噬細(xì)胞炎性的報(bào)道, 但是, 都是采用單一的細(xì)胞系, 比如RAW264.7 ADDIN EN.CITE ADDIN EN.CIT

45、E.DATA 13、小鼠ANA-1巨噬細(xì)胞 ADDIN EN.CITE ADDIN EN.CITE.DATA 14、人的THP-1誘導(dǎo)的巨噬細(xì)胞和小鼠J774A.1巨噬細(xì)胞 ADDIN EN.CITE ADDIN EN.CITE.DATA 15的細(xì)胞系。為了模擬巨噬細(xì)胞在體內(nèi)的情況, 本研究選取小鼠的骨髓細(xì)胞, 通過誘導(dǎo)得到巨噬細(xì)胞 ADDIN EN.CITE ADDIN EN.CITE.DATA 16并做一系列的考察。芹菜素是重要的黃酮類物質(zhì), 廣泛存在于自然界當(dāng)中, 有些報(bào)道已經(jīng)證明了它在抗炎、抗癌等方面發(fā)揮著巨大的作用 ADDIN EN.CITE ADDIN EN.CITE.DATA 1

46、7-21。本研究重點(diǎn)考察了芹菜素與BMDMs之間的關(guān)系。考慮到劑量過高會影響到細(xì)胞正常的生命活動, 通過MTT實(shí)驗(yàn)得到最大使用濃度為30 mol/L, 并且增設(shè)了10 mol/L劑量去考察芹菜素的劑量和改善炎性效應(yīng)和細(xì)胞極性的關(guān)系。BMDMs在LPS處理?xiàng)l件下通過TLR4會激活NF-B, MAPK等信號通路后會調(diào)控轉(zhuǎn)錄調(diào)節(jié)炎性因子的表達(dá), 例如IL-6、IL-1等 ADDIN EN.CITE ADDIN EN.CITE.DATA 22, 并且巨噬細(xì)胞在募集之后會自身分泌MCP-1, 募集更多的巨噬細(xì)胞啟動炎性過程 ADDIN EN.CITE Sica2012584158458417Sica,

47、A.Mantovani, A.Istituto Clinico Humanitas IRCCS, Rozzano, Italy. antonio.sicahumanitasresearch.itMacrophage plasticity and polarization: in vivo veritasJ Clin InvestThe Journal of clinical investigationJ Clin InvestThe Journal of clinical investigationJ Clin InvestThe Journal of clinical investigati

48、on787-951223AnimalsCell LineageHumansInflammationMacrophage ActivationMacrophages/*cytologyMiceModels, BiologicalOxygen/chemistryPhenotypeSignal TransductionTranscription Factors/metabolism2012Mar1558-8238 (Electronic)0021-9738 (Linking)22378047/pubmed/22378047328722310.1172/JCI59643HYPERLINK l _ENR

49、EF_1 o Sica, 2012 #4091。所以, 在研究芹菜素對BMDMs炎性的作用時(shí)檢測了MCP-1, IL-6和IL-1, 得知芹菜素能夠明顯降低BMDMs炎性因子的表達(dá)。芹菜素還能夠明顯降低INOS, 升高ARG1, 說明芹菜素促進(jìn)BMDMs向M2型巨噬細(xì)胞轉(zhuǎn)化。進(jìn)一步對調(diào)控炎圖2 A: 芹菜素對NF-B信號通路蛋白表達(dá)的影響(-actin作為基準(zhǔn)); BE: NF-B信號通路中p-IKK/、IKK、IKK和p-IB/IB蛋白表達(dá)的定量結(jié)果; F: 芹菜素對NF-B P65蛋白核轉(zhuǎn)移的熒光定位結(jié)果Fig.2 A: The effect of apigenin on NF-B sig

50、naling pathway protein expression (-actin as control); BE: The quantitative results of p-IKK/, IKK, IKK and p-IB/IB protein expression; F: The effect of apigenin on NF-B P65 nucleus traslocation圖3 A: 芹菜素對MAPK信號通路蛋白表達(dá)的影響(-actin作為基準(zhǔn)); BE: MAPK信號通路中p-JNK、JNK、p-P38 MAPK和P38 MAPK蛋白表達(dá)的定量結(jié)果Fig. 3 A: The ef

51、fect of apigenin on MAPK signaling pathway protein expression (-actin as control); BE: The quantitative results of p-JNK, JNK, p-P38 MAPK and P38 MAPK protein expression性基因的NF-B和MAPK炎性信號通路分別進(jìn)行考察, 對于NF-B信號通路, 芹菜素能夠明顯降低p-IKK/和p-IB, NF-B p-65的核轉(zhuǎn)位明顯降低, 這一結(jié)果和之前的芹菜素降低PPAR和P65復(fù)合物進(jìn)入核的結(jié)果相似 ADDIN EN.CITE ADDI

52、N EN.CITE.DATA 23。因?yàn)樵贚PS刺激條件下, 激活的P38 MAPK會調(diào)控到NF-B的激活, 且TLR4的激活通過TAK1調(diào)控JNK和P38 MAPK的激活10, 芹菜素會明顯改善p-P38 MAPK和p-JNK的激活。綜上所述, 芹菜素可以降低LPS誘導(dǎo)的BMDMs的炎性, 促進(jìn)巨噬細(xì)胞向M2型轉(zhuǎn)變, 而且這一抗炎的現(xiàn)象可以通過調(diào)控NF-B和MAPK信號通路實(shí)現(xiàn)。參考文獻(xiàn)Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas J. J Clin Invest, 2012, 122

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