N-甲基-N-顺-苯乙烯-肉桂酰胺衍生物体外抗炎作用探究

摘要本文建立了LPS诱导RAW264.7炎症细胞模型，并通过RT-qPCR和NO等多种检测方法对合成的N-甲基-N-顺-苯乙烯-肉桂酰胺（LB）衍生物的抗炎效果进行了检测。以下是本论文研究的主要内容：1.在10μM的给药浓度下，除了11c外，其它LB衍生物对Raw264.7细胞没有明显毒性。2.大多数化合物表现出较好的抑制NO、IL-6、IL-1β作用，其中11a的抗炎效果最为优秀。3.11a梯度降低了炎症因子IL-6、IL-1β和TNF-α的转录水平和IL-6蛋白表达水平。4.11a促进了Nrf2和HO-1的转录水平。5.11a促进了P62和HO-1的蛋白质表达水平，通过激活Nrf2/HO-1信号通路达到抗炎效果。综上所述，本课题对合成的LB衍生物进行了细胞毒性、抗炎活性以及免疫印迹实验测试，发现了11a具有很好的抗炎效果，为后续的深入研究提供了一定的理论基础。关键词：抗炎；LansiumamideB；Nrf2/HO-1；Raw264.7细胞 AbstractInthispaper,acellularmodelofLPS-inducedRAW264.7inflammationwasestablishedandtheanti-inflammatoryeffectofthesynthesisedN-methyl-N-cis-styrene-cinnamamide(LB)derivativeswasexaminedbyavarietyofmethodsincludingRT-qPCRandNO.Thefollowingarethemainpointsofthisthesisstudy:1.Attheadministeredconcentrationof10μM,allLBderivativesexcept11cshowednosignificanttoxicitytoRaw264.7cells.2.MostofthecompoundsshowedbetterinhibitionofNO,IL-6,IL-1β,amongwhich11ashowedthebestanti-inflammatoryeffect.3.11agradientreducedthetranscriptlevelsofinflammatoryfactorsIL-6,IL-1βandTNF-αandtheexpressionlevelofIL-6protein.4.11apromotedthetranscriptlevelsofNrf2andHO-1.5.11apromotedtheproteinexpressionlevelsofP62andHO-1,andachievedanti-inflammatoryeffectsbyactivatingtheNrf2/HO-1signallingpathway.Insummary,thesynthesisedLBderivativesweretestedforcytotoxicity,anti-inflammatoryactivityandimmunoblottingexperiments,and11awasfoundtohaveagoodanti-inflammatoryeffect,whichprovidedacertaintheoreticalbasisforsubsequentin-depthresearch.Keywords:Anti-inflammatory;LansiumamideB;Nrf2/HO-1;Raw264.7cells中英文缩略词对照表英文缩写英文全称中文全称COPDChronicObstructivePulmonaryDisease慢性阻塞性肺病ARDSAcuteRespiratoryDistressSyndrome急性呼吸窘迫综合征PAHPolycyclicAromaticHydrocarbon多环芳烃FRAPPlatelet-ActivatingFactorReceptor血小板活化因子受体IL-6Interleukin-6白细胞介素-6IL-1βInterleukin-1β白细胞介素-1βTNF-αTumornecrosisfactor-α肿瘤坏死因子-αLBLansiumamideBN-甲基-N-顺-苯乙烯-肉桂酰胺LPSLipopolysaccharide脂多糖CCK-8CellCountingKit-8细胞计数试剂盒-8DEXDexamethasone地塞米松ELISAEnzyme-linkedimmunosorbentassay酶联免疫吸附测试法RT-qPCRReal-timeRT-PCR实时荧光定量逆转录PCRWBWesternBlotting免疫印迹法Nrf2Nuclearfactorerythroidderived2-like2核因子-E2相关因子2HO-1HemeOxygenase-1血红素氧合酶1Keap-1Kelch-likeECH-associatedprotein1Kelch样ECH相关蛋白1DMEMDulbecco'sModifiedEagleMedium杜尔贝科改良伊格尔培养基FBSFoetalBovineSerum胎牛血清DMSODimethylSulfoxide二甲基亚砜前言由于全球范围内工业化进程的持续的、快速的推进，伴随着急剧增加的能源消耗和不断扩大的工业生产活动，日益加剧的严重空气污染，成为了一个不容忽视且亟待解决的重大环境问题，对人类健康、生态系统以及全球气候产生了严重的负面影响。在常见的空气污染物中，直径小于或等于2.5微米的颗粒物（即PM2.5）因其极小的体积而显得尤为危险，它们能够轻易地穿透人体的呼吸系统屏障。这些微小的颗粒物不仅能够深入到肺部组织，引发肺部的直接损伤，还进一步加剧了多种呼吸系统疾病的症状和进程，例如哮喘、支气管炎、过敏性鼻炎等常见的呼吸系统疾病。哮喘是一种常见的气道慢性炎症性疾病，虽然非病毒性哮喘恶化的分子机制尚不清楚，但当空气中的PM2.5浓度超过美国国家标准时，即使PM2.5浓度只是高而不是很高，城市中的哮喘易感人群也可能出现哮喘症状加重和发病率增加的情况。PM2.5浓度很可能导致哮喘症状加重和发病率上升ADDINEN.CITE<EndNote><Cite><Author>Altman</Author><Year>2023</Year><RecNum>1</RecNum><DisplayText><styleface="superscript">[1]</style></DisplayText><record><rec-number>1</rec-number><foreign-keys><keyapp="EN"db-id="95t2az5fcrvrtxexvamxexfhr2xfrd95pr52"timestamp="1741860506">1</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Altman,MatthewC.</author><author>Kattan,Meyer</author><author>O'Connor,GeorgeT.</author><author>Murphy,RyanC.</author><author>Whalen,Elizabeth</author><author>LeBeau,Petra</author><author>Calatroni,Agustin</author><author>Gill,MichelleA.</author><author>Gruchalla,RebeccaS.</author><author>Liu,AndrewH.</author><author>Lovinsky-Desir,Stephanie</author><author>Pongracic,JacquelineA.</author><author>Kercsmar,CarolynM.</author><author>KhuranaHershey,GurjitK.</author><author>Zoratti,EdwardM.</author><author>Teach,StephenJ.</author><author>Bacharier,LeonardB.</author><author>Wheatley,LisaM.</author><author>Sigelman,SteveM.</author><author>Gergen,PeterJ.</author><author>Togias,Alkis</author><author>Busse,WilliamW.</author><author>Gern,JamesE.</author><author>Jackson,DanielJ.</author></authors></contributors><titles><title>Associationsbetweenoutdoorairpollutantsandnon-viralasthmaexacerbationsandairwayinflammatoryresponsesinchildrenandadolescentslivinginurbanareasintheUSA:aretrospectivesecondaryanalysis</title><secondary-title>TheLancetPlanetaryHealth</secondary-title></titles><periodical><full-title>TheLancetPlanetaryHealth</full-title></periodical><pages>e33-e44</pages><volume>7</volume><number>1</number><dates><year>2023</year><pub-dates><date>2023/01/01/</date></pub-dates></dates><isbn>2542-5196</isbn><urls><related-urls><url>/science/article/pii/S2542519622003023</url></related-urls></urls><electronic-resource-num>/10.1016/S2542-5196(22)00302-3</electronic-resource-num></record></Cite></EndNote>[1]。空气污染会对人体的免疫系统造成伤害，并且降低免疫系统（尤其是尚未具备成熟免疫系统的幼儿）的工作能力，使得其清除肺部细菌和其他病原体的能力降低ADDINEN.CITEADDINEN.CITE.DATA[2]。过敏性鼻炎是一种发生在鼻粘膜的、和哮喘一样的患病率极高的呼吸系统疾病，过敏性鼻炎导致的明显的流涕、打喷嚏和鼻塞是影响患者生活的主要原因ADDINEN.CITE<EndNote><Cite><Author>Jiang</Author><Year>2023</Year><RecNum>10</RecNum><DisplayText><styleface="superscript">[3]</style></DisplayText><record><rec-number>10</rec-number><foreign-keys><keyapp="EN"db-id="2vxp29wv5p2esdexz025f5tvzxdxrfadva92"timestamp="1741602111">10</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Jiang,Yuna</author><author>Nguyen,ThiVan</author><author>Jin,Juan</author><author>Yu,ZhenNan</author><author>Song,ChangHo</author><author>Chai,OkHee</author></authors></contributors><titles><title>BergaptenamelioratescombinedallergicrhinitisandasthmasyndromeafterPM2.5exposurebybalancingTreg/Th17expressionandsuppressingSTAT3andMAPKactivationinamousemodel</title><secondary-title>Biomedicine&Pharmacotherapy</secondary-title></titles><periodical><full-title>Biomedicine&Pharmacotherapy</full-title></periodical><pages>114959</pages><volume>164</volume><keywords><keyword>CARAS</keyword><keyword>PM2.5</keyword><keyword>Bergapten</keyword><keyword>Treg/Th17balance</keyword><keyword>STAT3</keyword><keyword>MAPKpathway</keyword></keywords><dates><year>2023</year><pub-dates><date>2023/08/01/</date></pub-dates></dates><isbn>0753-3322</isbn><urls><related-urls><url>/science/article/pii/S0753332223007497</url></related-urls></urls><electronic-resource-num>/10.1016/j.biopha.2023.114959</electronic-resource-num></record></Cite></EndNote>[3]。尤为值得关注的是，PM2.5的长期暴露还与慢性阻塞性肺病（ChronicObstructivePulmonaryDisease,COPD）的发展密切相关。这是一种以持续气流受限为特征的阻塞性肺疾病，严重影响患者的生活质量。长期暴露在PM2.5超标的空气中会导致对COPD的易感性增加、氧化还原失衡，让患者的抗氧化能力下降ADDINEN.CITE<EndNote><Cite><Author>Fan</Author><Year>2023</Year><RecNum>12</RecNum><DisplayText><styleface="superscript">[4]</style></DisplayText><record><rec-number>12</rec-number><foreign-keys><keyapp="EN"db-id="2vxp29wv5p2esdexz025f5tvzxdxrfadva92"timestamp="1741602283">12</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Fan,Xiaoye</author><author>Dong,Tingting</author><author>Yan,Kun</author><author>Ci,Xinxin</author><author>Peng,Liping</author></authors></contributors><titles><title>PM2.5increasessusceptibilitytoacuteexacerbationofCOPDviaNOX4/Nrf2redoximbalance-mediatedmitophagy</title><secondary-title>RedoxBiology</secondary-title></titles><periodical><full-title>RedoxBiology</full-title></periodical><pages>102587</pages><volume>59</volume><keywords><keyword>PM2.5</keyword><keyword>NOX4/Nrf2redoximbalance</keyword><keyword>Mitophagy</keyword><keyword>AcuteexacerbationofCOPD</keyword></keywords><dates><year>2023</year><pub-dates><date>2023/02/01/</date></pub-dates></dates><isbn>2213-2317</isbn><urls><related-urls><url>/science/article/pii/S2213231722003597</url></related-urls></urls><electronic-resource-num>/10.1016/j.redox.2022.102587</electronic-resource-num></record></Cite></EndNote>[4]。因此空气污染，尤其是PM2.5污染，已成为一个全球性的健康负担，对人类社会的整体福祉构成了严峻挑战。据全球健康统计数据显示，每年有高达700万人因空气污染引发的健康问题而失去生命，这一数字触目惊心。更令人担忧的是，即使是长期处于较低浓度的空气污染之下，人们的健康也会受到显著且持久的影响，包括但不限于呼吸系统疾病发病率的上升、心血管系统功能（如缺血性心脏病和冠心病）的损害，以及整体健康状况的下滑ADDINEN.CITE<EndNote><Cite><Author>Carvalho</Author><Year>2021</Year><RecNum>13</RecNum><DisplayText><styleface="superscript">[5]</style></DisplayText><record><rec-number>13</rec-number><foreign-keys><keyapp="EN"db-id="2vxp29wv5p2esdexz025f5tvzxdxrfadva92"timestamp="1741603076">13</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Carvalho,Helotonio</author></authors></contributors><titles><title>NewWHOglobalairqualityguidelines:morepressureonnationstoreduceairpollutionlevels</title><secondary-title>TheLancetPlanetaryHealth</secondary-title></titles><periodical><full-title>TheLancetPlanetaryHealth</full-title></periodical><pages>e760-e761</pages><volume>5</volume><number>11</number><dates><year>2021</year><pub-dates><date>2021/11/01/</date></pub-dates></dates><isbn>2542-5196</isbn><urls><related-urls><url>/science/article/pii/S2542519621002874</url></related-urls></urls><electronic-resource-num>/10.1016/S2542-5196(21)00287-4</electronic-resource-num></record></Cite></EndNote>[5]。据报道，神经肌肉阻断剂、皮质类固醇和中性粒细胞弹性蛋白酶抑制剂可用于肺保护性通气和液体管理，从而减轻肺损伤ADDINEN.CITEADDINEN.CITE.DATA[6,7]。然而，目前对于许多常见的呼吸系统疾病，具体的发病机制仍不明确，有些疾病例如亚急性或急性肺损伤的患者，由于目前尚缺乏有效的药物治疗手段，往往预后不佳；并且能够用于治疗的药物也十分有限。同时其他疾病例如急性呼吸窘迫综合征（AcuteRespiratoryDistressSyndrome,ARDS）因为患者的病情极为复杂多变，使得想要找到一种能够对所有ARDS患者都产生有效治疗效果的单一药物，变得极为困难。PM2.5颗粒物主要含有离子、元素、多环芳烃（PolycyclicAromaticHydrocarbon,PAH）和内毒素，可引发氧化应激和强烈的气道炎症反应ADDINEN.CITEADDINEN.CITE.DATA[8,9]。据报道，颗粒物可激活气道上皮细胞的多种受体，例如血小板活化因子受体（Platelet-ActivatingFactorReceptor,FRAP），从而导致各种炎症细胞的聚集，产生强烈的炎症反应，最终导致组织的损伤ADDINEN.CITEADDINEN.CITE.DATA[10,11]。作为组织常驻细胞，肺泡巨噬细胞与随后被招募的巨噬细胞在先天性免疫系统中发挥着关键作用，对维持肺部环境的平衡至关重要。其中，肺巨噬细胞主要分布在肺泡和肺间质，当先天性免疫系统无法进行吞噬时，肺巨噬细胞就会分泌大量炎性细胞因子，产生炎症ADDINEN.CITE<EndNote><Cite><Author>Aegerter</Author><Year>2022</Year><RecNum>15</RecNum><DisplayText><styleface="superscript">[12]</style></DisplayText><record><rec-number>15</rec-number><foreign-keys><keyapp="EN"db-id="2vxp29wv5p2esdexz025f5tvzxdxrfadva92"timestamp="1741753066">15</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Aegerter,Helena</author><author>Lambrecht,BartN.</author><author>Jakubzick,ClaudiaV.</author></authors></contributors><titles><title>Biologyoflungmacrophagesinhealthanddisease</title><secondary-title>Immunity</secondary-title></titles><periodical><full-title>Immunity</full-title></periodical><pages>1564-1580</pages><volume>55</volume><number>9</number><keywords><keyword>alveolarmacrophages</keyword><keyword>interstitialmacrophages</keyword><keyword>lungdisease</keyword><keyword>recruitedmacrophages</keyword><keyword>pulmonary</keyword></keywords><dates><year>2022</year><pub-dates><date>2022/09/13/</date></pub-dates></dates><isbn>1074-7613</isbn><urls><related-urls><url>/science/article/pii/S1074761322004034</url></related-urls></urls><electronic-resource-num>/10.1016/j.immuni.2022.08.010</electronic-resource-num></record></Cite></EndNote>[12]。以下三种常见的炎症因子在本研究中被作为抗炎活性测试的参考指标。白细胞介素-6（Interleukin-6,IL-6）是一种常用的临床急性炎症反应标志物，产生于单核细胞、上皮细胞、内皮细胞，是一种在COPD患者体内炎症反应程度紧密相关的细胞因子，可用于监测治疗效果及病情转归ADDINEN.CITEADDINEN.CITE.DATA[13]。既往研究显示，COPD患者体内血清、肺部组织活检样本、痰液及支气管肺泡灌洗液中的白细胞介素-1β（Interleukin-1β,IL-1β）水平呈现上升趋势。在小鼠模型中，肺上皮细胞过度表达IL-1β可诱发类似COPD的病理特征，涵盖炎症细胞渗透、肺泡扩大（肺气肿）及气道结构改变，这凸显了其在COPD病理机制中的核心作用ADDINEN.CITEADDINEN.CITE.DATA[14]。肿瘤坏死因子-α（Tumornecrosisfactor-α,TNF-α）主要由处于激活状态的巨噬细胞等多种免疫细胞产生和分泌。当机体发生炎症时，这种重要的细胞因子就像一位经验丰富的“指挥官”，能够快速启动免疫防御机制，通过复杂的信号传导途径促使中性粒细胞、单核细胞等各类炎症细胞向病灶部位定向迁移，并显著增强这些效应细胞的活化状态和粘附能力。这一调控过程在有效提升白细胞吞噬清除病原体功能的同时，也会导致呼吸道局部炎症反应的持续加重。并且，TNF-α还能够引起全身性的炎症放大效应，如同推倒"多米诺骨牌"一般，借由通过NF-κB等信号传导，触发多种炎性介质的连锁分泌，致使系统性免疫应激反应增强ADDINEN.CITE<EndNote><Cite><Author>高岩</Author><Year>2024</Year><RecNum>2</RecNum><DisplayText><styleface="superscript">[15]</style></DisplayText><record><rec-number>2</rec-number><foreign-keys><keyapp="EN"db-id="95t2az5fcrvrtxexvamxexfhr2xfrd95pr52"timestamp="1741860960">2</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>高岩</author><author>张楠</author><author>徐丹丹</author><author>杨晓婷</author><author>王艳</author></authors></contributors><auth-address>张家口市第一医院检验科;衡水市第七人民医院检验科;衡水市第七人民医院药剂科;张家口市第一医院呼吸重症科;衡水市第六人民医院内科;</auth-address><titles><title>慢性阻塞性肺疾病急性加重期患者SAA、TNF-α、LTB4水平及相关性分析</title><secondary-title>临床误诊误治</secondary-title></titles><periodical><full-title>临床误诊误治</full-title></periodical><pages>31-35</pages><volume>37</volume><number>20</number><keywords><keyword>肺疾病</keyword><keyword>慢性阻塞性</keyword><keyword>急性加重期</keyword><keyword>血清淀粉样蛋白A</keyword><keyword>肿瘤坏死因子-α</keyword><keyword>白三烯B4</keyword><keyword>诊断价值</keyword><keyword>相关性</keyword></keywords><dates><year>2024</year></dates><isbn>1002-3429</isbn><call-num>10-1956/R</call-num><urls><related-urls><url>/kcms2/article/abstract?v=LY1OVaQjltyyJADnCOTReY3SHZtVA79G5ZtaujzbCCC-RQtCPfpEJooux0XEWgpxTdU5xpPUsh_RwO79ALpKf2HbR17M4c4bJSIL-bdc66KNsgrY2ZK70p9Y_GsVn2IW-yuAL7OJ3AKwmYSTyN4zFx0YUJZyJz0jnSwPyH8qpy4-l8qZ7k5iEUigcvnejG3jHB2NXUYOOyQ=&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[15]。由此可见，炎症调节机制被视为导致肺部疾病发生的关键因素之一。鉴于此，研发新型且高效的抗炎药物，用以治疗由PM2.5颗粒物所引发的肺损伤，具有极其重要的意义和价值。近年来，天然产品因其显著的药理作用和相对较低的副作用，在治疗各种疾病方面受到了广泛关注。在肺部疾病的治疗领域，各种天然化合物因其显著的抗炎作用而崭露头角。黄芩素是从传统中药黄芩中提取的一种有效成分，可通过减少炎症细胞因子的表达来减轻肺部炎症，并通过减少丙二醛、保护超氧化物歧化酶和过氧化氢酶来抑制肺部氧化损伤ADDINEN.CITE<EndNote><Cite><Author>He</Author><Year>2021</Year><RecNum>3</RecNum><DisplayText><styleface="superscript">[16]</style></DisplayText><record><rec-number>3</rec-number><foreign-keys><keyapp="EN"db-id="95t2az5fcrvrtxexvamxexfhr2xfrd95pr52"timestamp="1741878748">3</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>He,Yu-Qiong</author><author>Zhou,Can-Can</author><author>Yu,Lu-Yao</author><author>Wang,Liang</author><author>Deng,Jiu-ling</author><author>Tao,Yu-Long</author><author>Zhang,Feng</author><author>Chen,Wan-Sheng</author></authors></contributors><titles><title>Naturalproductderivedphytochemicalsinmanagingacutelunginjurybymultiplemechanisms</title><secondary-title>PharmacologicalResearch</secondary-title></titles><periodical><full-title>PharmacologicalResearch</full-title></periodical><pages>105224</pages><volume>163</volume><keywords><keyword>Naturalcompounds</keyword><keyword>Acutelunginjury</keyword><keyword>Acuterespiratorydistresssyndrome</keyword><keyword>Chemicalstructures</keyword><keyword>Mechanisms</keyword></keywords><dates><year>2021</year><pub-dates><date>2021/01/01/</date></pub-dates></dates><isbn>1043-6618</isbn><urls><related-urls><url>/science/article/pii/S1043661820315322</url></related-urls></urls><electronic-resource-num>/10.1016/j.phrs.2020.105224</electronic-resource-num></record></Cite></EndNote>[16]；丹参酮IIA可通过上调B淋巴细胞瘤-2(B-celllymphoma-2,Bcl-2)家族蛋白、抑制半胱天冬氨酸蛋白酶级联反应（caspasecascade,Caspase级联反应）、减少氧化损伤和抑制炎症反应来抑制肺氧化损伤。在急性呼吸衰竭大鼠模型中，它还通过多种机制（包括抗氧化应激和抗细胞凋亡）发挥抗细胞凋亡作用ADDINEN.CITE<EndNote><Cite><Author>高月彩</Author><Year>2025</Year><RecNum>4</RecNum><DisplayText><styleface="superscript">[17]</style></DisplayText><record><rec-number>4</rec-number><foreign-keys><keyapp="EN"db-id="95t2az5fcrvrtxexvamxexfhr2xfrd95pr52"timestamp="1741879294">4</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>高月彩</author><author>苏明</author><author>刘荣格</author></authors></contributors><auth-address>邢台医学院第二附属医院呼吸内科;</auth-address><titles><title>丹参酮ⅡA调控NF-κB信号通路对急性呼吸衰竭大鼠炎症因子及细胞凋亡的影响</title><secondary-title>现代药物与临床</secondary-title></titles><periodical><full-title>现代药物与临床</full-title></periodical><pages>37-43</pages><volume>40</volume><number>01</number><keywords><keyword>丹参酮ⅡA</keyword><keyword>急性呼吸衰竭</keyword><keyword>核因子-κB</keyword><keyword>氧化应激</keyword><keyword>细胞凋亡</keyword></keywords><dates><year>2025</year></dates><isbn>1674-5515</isbn><call-num>12-1407/R</call-num><urls><related-urls><url>/kcms2/article/abstract?v=LY1OVaQjltz0hdvt0cMzoz4ASmwGpn1InNgH6puhU8N2kWXIpQ588s9CNXc9MmVSPfUgU1RzcN06oTYIW5NZMkc3y0TGYm5Ag187Q880NSIe5o4vAbhHRsa0TlnmkQ2dmfe7L80yLJQF1gSwe8KXoxLPORCDpO2X8PIlS0mmaDfQtSev-g3nAiCwOYif6-fAReZCpIQz6Ko=&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[17]；槲皮素是一种研究较多的类黄酮，因其强大的抗炎和抗氧化特性（包括抑制炎症因子的产生、调节过氧化氢酶和超氧化物歧化酶的活性）而成为一种有效的抗炎剂。抑制炎症因子的产生、调节免疫细胞浸润和抑制氧化应激）ADDINEN.CITEADDINEN.CITE.DATA[18]。许多的天然化合物（例如上文提到的黄芩苷、丹参酮IIA、槲皮素，还有山奈酚、木犀草素）都因为其抗炎和抗氧化的特性被认为是可以治疗肺病的天然药物，不同种类的化合物可能具有相似的保护作用和相似的靶标ADDINEN.CITE<EndNote><Cite><Author>He</Author><Year>2021</Year><RecNum>6</RecNum><DisplayText><styleface="superscript">[16]</style></DisplayText><record><rec-number>6</rec-number><foreign-keys><keyapp="EN"db-id="95t2az5fcrvrtxexvamxexfhr2xfrd95pr52"timestamp="1741881138">6</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>He,Yu-Qiong</author><author>Zhou,Can-Can</author><author>Yu,Lu-Yao</author><author>Wang,Liang</author><author>Deng,Jiu-ling</author><author>Tao,Yu-Long</author><author>Zhang,Feng</author><author>Chen,Wan-Sheng</author></authors></contributors><titles><title>Naturalproductderivedphytochemicalsinmanagingacutelunginjurybymultiplemechanisms</title><secondary-title>PharmacologicalResearch</secondary-title></titles><periodical><full-title>PharmacologicalResearch</full-title></periodical><pages>105224</pages><volume>163</volume><keywords><keyword>Naturalcompounds</keyword><keyword>Acutelunginjury</keyword><keyword>Acuterespiratorydistresssyndrome</keyword><keyword>Chemicalstructures</keyword><keyword>Mechanisms</keyword></keywords><dates><year>2021</year><pub-dates><date>2021/01/01/</date></pub-dates></dates><isbn>1043-6618</isbn><urls><related-urls><url>/science/article/pii/S1043661820315322</url></related-urls></urls><electronic-resource-num>/10.1016/j.phrs.2020.105224</electronic-resource-num></record></Cite></EndNote>[16]。N-甲基-N-顺-苯乙烯-肉桂酰胺衍生物(LansiumamideB,LB)是从黄皮种子中分离出来的一种天然化合物ADDINEN.CITE<EndNote><Cite><Author>Lin</Author><Year>1989</Year><RecNum>7</RecNum><DisplayText><styleface="superscript">[19]</style></DisplayText><record><rec-number>7</rec-number><foreign-keys><keyapp="EN"db-id="95t2az5fcrvrtxexvamxexfhr2xfrd95pr52"timestamp="1741882530">7</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Lin,Jer-Huei</author></authors></contributors><titles><title>CinnamamidederivativesfromClausenalansium</title><secondary-title>Phytochemistry</secondary-title></titles><periodical><full-title>Phytochemistry</full-title></periodical><pages>621-622</pages><volume>28</volume><number>2</number><keywords><keyword>Rutaceae</keyword><keyword>cinnamamide</keyword><keyword>lansiumamides</keyword><keyword>lansamide-I</keyword><keyword>cinnamicacidderivative.</keyword></keywords><dates><year>1989</year><pub-dates><date>1989/01/01/</date></pub-dates></dates><isbn>0031-9422</isbn><urls><related-urls><url>/science/article/pii/0031942289800639</url></related-urls></urls><electronic-resource-num>/10.1016/0031-9422(89)80063-9</electronic-resource-num></record></Cite></EndNote>[19]，根据研究表明，LB可以显著降低组胺释放、降低TNF-α的蛋白质和/或mRNA水平，减轻肥大细胞诱导的炎症ADDINEN.CITE<EndNote><Cite><Author>Lin</Author><Year>1989</Year><RecNum>8</RecNum><DisplayText><styleface="superscript">[19]</style></DisplayText><record><rec-number>8</rec-number><foreign-keys><keyapp="EN"db-id="95t2az5fcrvrtxexvamxexfhr2xfrd95pr52"timestamp="1741883173">8</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Lin,Jer-Huei</author></authors></contributors><titles><title>CinnamamidederivativesfromClausenalansium</title><secondary-title>Phytochemistry</secondary-title></titles><periodical><full-title>Phytochemistry</full-title></periodical><pages>621-622</pages><volume>28</volume><number>2</number><keywords><keyword>Rutaceae</keyword><keyword>cinnamamide</keyword><keyword>lansiumamides</keyword><keyword>lansamide-I</keyword><keyword>cinnamicacidderivative.</keyword></keywords><dates><year>1989</year><pub-dates><date>1989/01/01/</date></pub-dates></dates><isbn>0031-9422</isbn><urls><related-urls><url>/science/article/pii/0031942289800639</url></related-urls></urls><electronic-resource-num>/10.1016/0031-9422(89)80063-9</electronic-resource-num></record></Cite></EndNote>[19]；还具有抗坏死ADDINEN.CITE<EndNote><Cite><Author>Liu</Author><Year>2015</Year><RecNum>9</RecNum><DisplayText><styleface="superscript">[20]</style></DisplayText><record><rec-number>9</rec-number><foreign-keys><keyapp="EN"db-id="95t2az5fcrvrtxexvamxexfhr2xfrd95pr52"timestamp="1741935089">9</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Liu,Yueqiu</author><author>Staerk,Dan</author><author>Nielsen,MiaN.</author><author>Nyberg,Nils</author><author>Jäger,AnnaK.</author></authors></contributors><titles><title>High-resolutionhyaluronidaseinhibitionprofilingcombinedwithHPLC–HRMS–SPE–NMRforidentificationofanti-necrosisconstituentsinChineseplantsusedtotreatsnakebite</title><secondary-title>Phytochemistry</secondary-title></titles><periodical><full-title>Phytochemistry</full-title></periodical><pages>62-69</pages><volume>119</volume><keywords><keyword>Snakebite</keyword><keyword>Hyaluronidaseinhibition</keyword><keyword>High-resolutionprofiling</keyword><keyword>HPLC–HRMS–SPE–NMR</keyword></keywords><dates><year>2015</year><pub-dates><date>2015/11/01/</date></pub-dates></dates><isbn>0031-9422</isbn><urls><related-urls><url>/science/article/pii/S0031942215300844</url></related-urls></urls><electronic-resource-num>/10.1016/j.phytochem.2015.09.005</electronic-resource-num></record></Cite></EndNote>[20]、降血脂ADDINEN.CITEADDINEN.CITE.DATA[21,22]和抗肥胖ADDINEN.CITE<EndNote><Cite><Author>Huang</Author><Year>2017</Year><RecNum>37</RecNum><DisplayText><styleface="superscript">[23]</style></DisplayText><record><rec-number>37</rec-number><foreign-keys><keyapp="EN"db-id="2vxp29wv5p2esdexz025f5tvzxdxrfadva92"timestamp="1741783063">37</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Huang,Li</author><author>Li,Dan</author><author>Xu,Yun-Shao</author><author>Feng,Zhe-Ling</author><author>Meng,Fan-Chen</author><author>Zhang,Qing-Wen</author><author>Gan,Li-She</author><author>Lin,Li-Gen</author></authors></contributors><titles><title>Clausoxamine,analkaloidpossessinga1,3-oxazine-4-oneringfromtheseedsofClausenalansiumandtheanti-obesityeffectoflansiumamideB</title><secondary-title>RSCAdvances</secondary-title></titles><periodical><full-title>RSCAdvances</full-title></periodical><pages>46900-46905</pages><volume>7</volume><number>74</number><dates><year>2017</year></dates><publisher>TheRoyalSocietyofChemistry</publisher><work-type>10.1039/C7RA09793J</work-type><urls><related-urls><url>/10.1039/C7RA09793J</url></related-urls></urls><electronic-resource-num>10.1039/C7RA09793J</electronic-resource-num></record></Cite></EndNote>[23]等药理活性。此外，LB的骨架与其他已知的抗炎药物不同，这意味着它有可能成为一种主要化合物和新的治疗途径ADDINEN.CITE<EndNote><Cite><Author>Song</Author><Year>2023</Year><RecNum>12</RecNum><DisplayText><styleface="superscript">[24]</style></DisplayText><record><rec-number>12</rec-number><foreign-keys><keyapp="EN"db-id="95t2az5fcrvrtxexvamxexfhr2xfrd95pr52"timestamp="1741936373">12</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Song,Liyan</author><author>Li,Gang</author><author>Guan,Wen</author><author>Zeng,Zhijun</author><author>Ou,Yanghui</author><author>Zhao,Tongchao</author><author>Li,Jiayu</author><author>He,Dengqin</author><author>Fang,Xiangxiang</author><author>Zhang,Yali</author><author>Wu,Jia-qiang</author><author>Tong,Rongbiao</author><author>Yao,Hongliang</author></authors></contributors><titles><title>Design,synthesisandanti-inflammatoryactivitystudyoflansiumamideanaloguesfortreatmentofacutelunginjury</title><secondary-title>Biomedicine&Pharmacotherapy</secondary-title></titles><periodical><full-title>Biomedicine&Pharmacotherapy</full-title></periodical><pages>115412</pages><volume>166</volume><keywords><keyword>Lansiumamideanalogues</keyword><keyword>Anti-inflammatory</keyword><keyword>Nrf2/HO-1pathway</keyword><keyword>Expectorant</keyword><keyword>ALI</keyword></keywords><dates><year>2023</year><pub-dates><date>2023/10/01/</date></pub-dates></dates><isbn>0753-3322</isbn><urls><related-urls><url>/science/article/pii/S0753332223012106</url></related-urls></urls><electronic-resource-num>/10.1016/j.biopha.2023.115412</electronic-resource-num></record></Cite></EndNote>[24]。由于LB本身的骨架含有共轭π电子系统，平坦且刚性的结构会影响化合物的活性、降低其抗炎的效果，所以本研究对化合物的结构进行了改进，将LB与吡唑骨架进行杂合，因为吡唑结构在抗炎药物中十分常见。通过采用骨架跃迁的策略，将肉桂酰胺部分中的共轭双键替换为吡唑环，以期提升药物的生物活性和理化性质（如图1）。因此，本研究设计出了31个新型的N-甲基-N-顺-苯乙烯-肉桂酰胺衍生物。其中一个N-甲基-N-顺-苯乙烯-肉桂酰胺的衍生物——化合物11a，经过评估，在对脂多糖（Lipopolysaccharide,LPS）诱导的小鼠单核巨噬细胞白血病细胞Raw264.7细胞模型上表现出了很强的抗炎活性，能以剂量依赖的方式抑制IL-6、IL-1β和TNF-α的表达，这可能与Kelch样ECH相关蛋白1(Kelch-likeECH-associatedprotein1,简称Keap1)/核因子-E2相关因子2(Nuclearfactorerythroidderived2-like2,简称Nrf2)/血红素氧合酶1(HemeOxygenase-1，简称HO-1)信号通路的激活有关。图1化合物设计与修改方案2实验材料2.1细胞来源、培养方法与收集样本方法小鼠单核巨噬细胞白血病细胞株Raw264.7和肺癌细胞株NCL-H292购自武汉普罗凯尔生命科学有限公司，使用DMEM培养基含10%胎牛血清及青/链霉素（100U/ml/μg/ml），37℃、5%CO₂培养，当显微镜下可见单层细胞占据约4/5培养区域时，进行传代。在本研究中，各细胞实验使用的细胞类型、使用的细胞培养皿、种板密度（个/孔）和培养时间和收集样品方法如表1所示。表1各细胞实验种板使用类型及详细培养条件实验类型使用细胞类型细胞培养皿种板密度（个/孔）培养时间样品收集方法CCK-8Raw264.796孔板1×10424小时不需要加入裂解液，直接加入CCK-8溶液NO表达量Raw264.796孔板1×105过夜用移液枪小心吸掉孔板里的旧培养基，接着用预冷的磷酸盐缓冲液(PhosphateBufferedSaline,PBS)轻柔冲洗两遍，把残留的血清成分洗干净，在每个孔里加入冰上预冷的裂解液，静置等待裂解充分/用细胞刮刀沿着孔底轻轻刮取，放进离心管，在冰上静置10分钟，将离心机预冷到四度，离心十分钟，收集细胞上清液酶联免疫吸附Raw264.712孔板2×105过夜免疫印迹实验Raw264.712孔板2×105等待细胞贴壁实时荧光定量逆转录PCRRaw264.712孔板4×105等待细胞贴壁弃培养基，冰冷PBS洗涤NCL-H2926孔板3×105等待细胞贴壁2.2实验仪器表1实验所需仪器及其生产厂家实验仪器所属公司/厂家电泳仪Biorad化学发光成像仪上海Tano显微镜尼康酶标仪PerkinElmer病理切片机Leica金属浴仪器Monad摇床海门市其林贝尔仪器制造有限公司RT-qPCR仪Bio-Rad电热恒温水浴锅上海一恒台式高速冷冻离心机艾本德小型冷冻离心机新加坡艺思高科技有限公司DLJJ-101自动洗板机迪乐嘉2.3实验试剂与材料表2实验试剂与材料实验材料所属公司/厂家磷酸盐缓冲液HyClone二甲基亚砜SIGMARAW264.7细胞专用培养基普诺赛Western及IP细胞裂解液（无抑制剂）Beyotime磷酸酶抑制剂cocktail（mini片剂）Biosharp蛋白酶抑制剂cocktail不含EDTA（mini片剂）BiosharpBCAProteinAssayKit弗德生物丙烯酰胺/甲叉双丙烯酰胺30%溶液（29：1）生工4×Tris-HClbuffer(0.5M,PH6.8)生工4×Tris-HClbuffer(1.5M,PH8.8)生工SDS生工Ammoniumpersulfatesubstitude(APSsubstitude)Beyotime四甲基乙二胺MacklinPVDF膜Millipore甘氨酸北京索莱宝Tris生工氯化钠General-reagent®HCl广州化学试剂厂甲醇General-reagent®5×LoadingBufferBiosharp180kDaPrestainedProteinMarker诺唯赞脱脂奶粉生工western一抗稀释液BeyotimeP62AntibodyAbmartHO-1AntibodyCellSingnalingKeap1Antibody武汉三鹰β-actin武汉三鹰IgG鼠抗，HRP偶联抗体武汉三鹰IgG兔抗，HRP偶联抗体武汉三鹰一氧化氮检测试剂盒BeyotimeCCK-8溶液Beyotime吐温20北京索莱宝脂多糖美国Sigma小鼠白细胞介素6(IL-6)