牡荆素调控M1M2型巨噬细胞极化抗肺腺癌转移的作用研究

doi:10.3969/j.issn.1000⁃484X.2020.20.006
㊃中医中药与免疫㊃
牡荆素调控M1/M2型巨噬细胞极化抗肺腺癌转移的作用研究①
赵　蓓　殷亦男　王程燕　毕　凌　许　玲　焦丽静②
(上海中医药大学附属岳阳中西医结合医院肿瘤一科,上海200437)
中图分类号　Q291 文献标志码　A 文章编号　1000⁃484X (2020)20⁃2456⁃06
①本文为国家自然基金项目(81704035,81973810,81904163)㊁中华中医药学会青年人才托举工程(QNRC2⁃C15)㊁上海市进一步加快中医药事业发展三年行动计划(ZY(2018⁃2020)⁃CCCX⁃2004⁃09)㊁上海中医药大学2020年 研究生创新培养项目”(2020038)和上海市科学技术委员会 扬帆计划”(19YF1450000)资助项目㊂
②上海中医药大学附属岳阳中西医结合医院临床免疫研究所,上海200437㊂
作者简介:赵　蓓,女,在读硕士,主要从事恶性肿瘤的中医药防治方面的研究,E⁃mail:zhaobei0207@㊂
通讯作者及指导教师:焦丽静,女,在读博士,助理研究员,主要从事恶性肿瘤的中医药防治方面的研究,E⁃mail:jli969@㊂
[摘　要]　目的:探讨牡荆素对M1/M2型巨噬细胞的调控作用和机制㊂方法:利用脂多糖(LPS)和IL⁃4诱导M1/M2型鼠源巨噬细胞RAW264.7,通过CCK8法检测不同浓度牡荆素对RAW264.7细胞的活力影响来确定牡荆素的最适浓度;实时荧光定量聚合酶链式反应(Real⁃time RT⁃PCR)法检测牡荆素对巨噬细胞内诱导型一氧化氮合酶(iNOS)㊁IL⁃1β㊁精氨酸酶1(Arg⁃1)㊁甘露糖受体(MR)基因表达的影响;通过Griess 反应法检测牡荆素对巨噬细胞上清液中NO 含量的影响;Transwell 小室迁移实验检测由IL⁃4诱导的M2型巨噬细胞对非小细胞肺癌A549的体外迁移及牡荆素干预的影响;流式实验检测牡荆素对M2型巨噬细胞表型CD206的影响㊂Western blot 检测磷酸化信号传导及转录激活因子3表达㊂结果:牡荆素在25㊁50㊁
100μmol /L 浓度时对小鼠巨噬细胞无明显细胞毒性,确定25㊁50㊁100μmol /L 浓度进行后续实验㊂RT⁃PCR 实验表明牡荆素呈剂量依赖性抑制M1表型标记物iNOS 和IL⁃1β基因的mRNA 表达,抑制M2型巨噬细胞表型中Arg⁃1和MR 基因的mRNA 表达㊂Griess 反应法表明牡荆素呈浓度依赖抑制巨噬细胞M1表型中NO 含量,同时抑制由M2型巨噬细胞诱发肺腺癌A549细胞的迁移能力的加剧㊂流式细胞术结果显示牡荆素能够抑制M2表型CD206的表达且呈浓度依赖性㊂Western blot 结果显示牡荆素能够降低p⁃STAT3蛋白水平㊂结论:牡荆素通过调控STAT 信号通路,调节巨噬细胞M1/M2的表型极化和标志性产物的表达,抑制巨噬细胞向M2极化,从而降低肺腺癌细胞A549细胞体外迁移力㊂
[关键词]　牡荆素;巨噬细胞;侵袭;肺腺癌
Mechanism of vitexin on regulation of M1/M2macrophages polarization against metastasis of lung adenocarcinoma
ZHAO Bei ,YIN Yi⁃Nan ,WANG Cheng⁃Yan ,BI Ling ,XU Ling ,JIAO Li⁃Jing .Department of Oncology ,Yueyang Integrated Chinese and Western Medicine Hospital ,Affiliated to Shanghai University of Traditional Chinese Medicine ,Shanghai 200437,China
[Abstract ]　Objective :To explore the mechanisms of the fraction of vitexin on the M1/M2phenotype of macrophages.
Methods :RAW264.7cell lines were induced by LPS and IL⁃4in order to derive macrophages to be M1and M2phenotypes,respective⁃
ly.The optimal concentration of vitexin was determined by checking the effect of vitexin with CCK8on the vigour of RAW264.7.The expression of inducible nitric oxide synthase (iNOS),IL⁃1β,arginase 1(Arg⁃1)and mannose receptor (MR)in macrophages were detected by real⁃time fluorescence quantitative polymerase chain reaction (RT⁃PCR).The effect of vitexin on NO content in macrophage supernatant was determined by Griess reaction.Transwell assay showed that the effect of M2macrophage induced by IL⁃4as well as the intervention of vitexin on the migration of non⁃small cell lung cancer (NSCLC)A549.The effect of vitexin on the M2macrophage CD206was detected by flow cytometry assay.Phosphorylation signal transduction and the expression of transcription activator 3were detected by Western blot.Results :There was no obvious cytotoxicity on macrophages of vitexin with the concentrations of 25,50,100μmol /L.This was the concentration basis for our future experiments.RT⁃PCR showed that vitexin inhibited the mRNA expression of iNOS and IL⁃1βin M1macrophages,Arg⁃1and MR in M2macrophages in a dose dependent manner.Further study of Griess reaction showed the inhibition of vitexin in a concentration dependent manner on NO contents in M1macrophages as well as the migration ability of the lung adenocarcinoma A549cell line induced by M2macrophages.Flow cytometry also illustrated that vitexin suppressed the
expression of the M2macrophages CD206in a concentration⁃dependent manner.Western blot results demonstrated that vitexin could down⁃regulate the protein expression of p⁃STAT3.Conclusion:Vitexin regulated the phenotypic polarization of M1/M2macrophages and the expression of landmark products via regulation of STAT signalling pathway,then inhibited the macrophage polarization to M2,which eventually decreased the migration ability of A549lung adenocarcinoma cell in vitro.
[Key words]　Vitexin;Macrophages;Migration;Lung adenocarcinoma
肺癌是世界上最常见的癌症,在男性中占所有癌症死亡人数24%,女性占23%[1]㊂随着手术㊁化疗㊁靶向治疗等各种治疗方案的应用,肺癌5年生存率有了很大的提高,但近90%的肺癌患者因癌细胞发生转移而死亡[1,2]㊂不可避免的癌细胞转移依然是肺癌整体治疗所面临的主要难题[3]㊂免疫应答是人体防御肿瘤的天然屏障,而免疫效应的失衡是肿瘤发生和进展的重要原因㊂巨噬细胞是一种非特异性免疫效应细胞,在人体免疫系统中发挥重要的作用,具有免疫监视㊁免疫防御㊁免疫调节以及抗原呈递等多种免疫功能[4]㊂肿瘤相关巨噬细胞(tumor⁃associated macrophage,TAMs),是浸润在肿瘤组织中的巨噬细胞,是肿瘤微环境中最多的免疫细胞[5]㊂有研究发现,TAMs能够分泌细胞因子如肿瘤坏死因子α和血管内皮生长因子A等促进肿瘤形成新的血管,从而促进肿瘤的转移[6]㊂
牡荆素是一种广泛存在于植物中的天然黄酮类化合物,具有抗炎㊁抗氧化㊁止痛等生物活性,并且对食管癌㊁淋巴瘤等有着明显的增殖抑制效果,在肿瘤治疗上有着较好的应用前景[7⁃9]㊂在本项目组的前期研究中,牡荆素能够抑制非小细胞肺癌(non⁃small cell lung cancer,NSCLC)A549细胞增殖㊁迁移及侵袭,并且能够上调Caspase3㊁Caspase9㊁Bcl⁃2和Bax 基因表达,具有多靶点的抗肺癌效应㊂在此基础上,本研究进一步探讨牡荆素多靶点抗肺癌的机制,推动牡荆素在NSCLC中的应用㊂
1　材料与方法
1.1　材料　小鼠单核巨噬细胞株(RAW264.7细胞),A549细胞株购自中国科学院上海细胞库,支原体检测阴性㊂中药单体牡荆素(诗丹德生物技术公司);脂多糖(LPS,Sigma);IL⁃4(PeproTech);PE anti⁃mouseCD206(Biolegend);pSTAT3(Abcam); RNA提取试剂,结晶紫染料(美国Sigma公司); CCK8试剂盒(同仁化学研究所);Transwell小室(美国Corning公司);Perfect real time⁃PCR试剂盒(日本TaKaRa公司);SYBR Green RCR Kit(QuantiNova 公司);PCR引物设计和合成(生工生物工程有限公司);DMEM培养基,胎牛血清(美国Gibco公司); Griess试剂盒(上海碧云天)㊂1.2　方法
1.2.1　RAW264.7细胞的培养　用完全培养基(含10%FBS的高糖DMEM培养基)在37℃㊁5%CO2的培养箱中培养RAW264.7细胞,每日更换培养基,待细胞生长到80%左右传代㊂
1.2.2　A549细胞的培养　用完全培养基(含10% FBS的高糖DMEM培养基)在37℃㊁5%CO2的培养箱中培养A549细胞,隔日更换培养基,待细胞生长到80%左右传代㊂
1.2.3　CCK8检测牡荆素对RAW264.7细胞活力的影响　取对数生长期的RAW264.7细胞,按照每孔100μl(密度为5×104个/ml)接种到96孔板,每组3个复孔㊂除空白对照组外,设置不同的给药浓度组(200㊁100㊁50㊁25㊁12.5μmol/L),培养24h㊂随后,每孔加入10μl的CCK8试剂避光孵育1.5h,利用酶标仪于OD450nm波长处检测各孔的吸光度A㊂细胞活力计算公式为:细胞活力(%)=各浓度给药组的A/空白组的平均A×100%㊂
1.2.4　Real⁃time PCR法观察牡荆素对细胞内iNOS㊁IL⁃1β㊁Arg⁃1㊁MR基因表达的影响　RAW264.7细胞培养于6孔板,设置空白对照组㊁M1模型组㊁M2模型组㊁M1模型牡荆素干预组㊁M2模型牡荆素干预组(25㊁50㊁100μmol/L),贴壁生长过夜㊂M1模型组和M1模型牡荆素组加入LPS,处理24h后收集各组RNA样品㊂M2模型组和M2牡荆素干预组加入IL⁃4(20ng/ml),协同刺激细胞,收集24h各种的RNA样品㊂用Trizol提取方法细胞总的RNA,用PerfectRealTime试剂盒逆转录提取表1　检测基因的引物序列
Tab.1　Primer sequence for detection gene
Primer Sequence(5′⁃3′)
iNOS F:GGAGCGAGTTGTGGATTGTC
R:GTGAGGGCTTGGCTGAGTGAG IL⁃1βF:TGGGAAACAACAGTGGTCAGG
R:CTGCTCATTCACGAAAAGGGA Arg⁃1F:TGAGAGACCACGGGGACCTG
R:GCACCACACTGACTCTTCCATTC MR F:GCTGAATCCCAGAAATTCCGC
R:ATCACAGGCATACAGGGTGAC
总cDNA,反应体系37℃15min,85℃5s,4℃保存,后用RT⁃PCR进行检测㊂PCR反应条件:95℃
2min,95℃5s,60℃10s,共40个循环㊂重复3次,β⁃肌动蛋白(β⁃actin)作为内标基因㊂使用2-ΔΔCt法计算mRNA含量㊂
1.2.5　Griess法测定RAW264.7细胞中NO含量　RAW264.7细胞以5×103个/孔接种于96孔板中,贴壁生长过夜,无血清培养24h㊂分别设置空白对照组㊁M1模型组㊁给药组,空白对照组不给予任何处理,M1模型组和给药组采用LPS(500μg/L)协同刺激细胞,同时牡荆素以25㊁50㊁100μmol/L剂量处理给药组㊂24h后吸取上清液50μl加入新的96孔板中,加入等量的Griess反应试剂,反应时间为10min,在540nm下读数,计算细胞上清液中NO 含量㊂
1.2.6　Transwell小室迁移实验观察巨噬细胞诱导M2型巨噬细胞前后对肺腺癌A549细胞的迁移能力及牡荆素干预的影响　处于对数生长期的RAW264.7细胞以1×106个/孔培养于24孔板中,分别设置M2模型组和牡荆素组采用IL⁃4(20μg/L)刺激细胞,同时牡荆素组以25㊁50㊁100μmol/L剂量处理细胞,24h后收集上清液,各取600μl置于24孔板下室,放入Transwell小室,在Transwell小室上室加入无血清培养基调整密度为5×105个/ml的A549细胞,体积均为100μl,作用24h后,用4%多聚甲醛固定30min,0.5%结晶紫染色30min,水漂洗后用棉签轻轻拭去Transwell小室上层未迁移的细胞,200倍显微镜下随机5个视野观察细胞并计数㊂
1.2.7　流式细胞术检测牡荆素对于巨噬细胞M2型的抑制作用　RAW264.7细胞分组同1.2.4,24h后收集细胞,用预冷的PBS冲洗2遍,PBS重悬细胞,调整细胞浓度为1×107个/ml,取100μl到流式管中,加入PE抗鼠CD206抗体以及IgG2a的同型对照组,4℃避光孵育20min;1000r/min离心5min,收集细胞,弃上清,再加入200μl PBS,Cytoflex流式细胞仪检测㊂用Cytoflex软件分析结果㊂
1.2.8　Western blot和RT⁃PCR法考察牡荆素对蛋白的影响　细胞分组同1.2.4,24h后,用冰PBS洗涤,加入含蛋白酶抑制剂和磷酸酶抑制剂的裂解液后收集细胞,放置冰上30min,离心(12000r/min, 15min,4℃),取蛋白上清液进行蛋白测定㊂每孔加入20μg的蛋白上样,80V恒压电泳30min,再转为120V恒压电泳分离蛋白,经200mA恒流转膜2h,用5%脱脂牛奶封闭液常温下封闭1h,PBS洗涤3次,每次10min,再加入p⁃STAT3和GAPDH抗体,置4℃孵育过夜(抗体稀释比例1∶1000),用TBST 洗涤3次,每次10min,再加入二抗(1∶10000)室温下孵育1.5h,再洗涤3次,曝光㊂
1.3　统计学处理　应用GraphPad Prism6统计软件分析,独立样本采用t检验分析,计量资料用x±s描述,组间比较采用单因素方差分析(ANOVA),以P<
0.05表示差异有统计学意义㊂
2　结果
2.1　牡荆素对RAW264.7细胞活力的影响　不同浓度牡荆素作用于巨噬细胞,CCK8结果显示,牡荆素25㊁50㊁100μmol/L浓度时对静息状态下的巨噬细胞无明显的细胞毒性,因此选用25㊁50㊁100μmol/L 用于后续的实验,如图1㊂
2.2　牡荆素对不同表型细胞内iNOS㊁IL⁃1β㊁Arg⁃1㊁MR基因表达的影响　正常状态下,iNOS㊁IL⁃1β㊁Arg⁃1㊁MR基因在巨噬细胞中呈低表达,经LPS诱导刺激后iNOS和IL⁃1β的表达均显著升高;与M1组相比,牡荆素在25㊁50㊁100μmol/L浓度下可明显抑制M1表型相关基因iNOS和IL⁃1β的表达,且呈浓度依赖性㊂而IL⁃4可诱导M2组中的Arg⁃1和MR 的表达上升,牡荆素可呈浓度依赖下调Arg⁃1和MR 的基因表达,如图2㊂
2.3　牡荆素对LPS诱导下RAW264.7细胞分泌NO的影响　与静息状态下的巨噬细胞相比,经LPS 干预后的RAW264.7细胞上清液中NO明显升高;与M1组相比,加入25㊁50㊁100μmol/L的牡荆素均可抑制NO的释放量,且随着浓度的增高,NO的释放量越低,如图3㊂
2.4　牡荆素对IL⁃4诱导M2型巨噬细胞对肺癌A549的迁移能力变化　与空白对照组比较,经IL⁃4诱导的M2型巨噬细胞上清液可显著增强
NSCLC
图1　牡荆素对巨噬细胞的细胞活力的影响
Fig.1　Effect of vitexin on cell viability of macrophages Note:Compared with control group,△△.P<0.01.
A549体外迁移能力;而牡荆素低㊁中㊁高三个剂量干预组的细胞上清液能够显著抑制M2型A549细胞迁移,图4㊂
2.5　牡荆素对IL⁃4诱导M2型巨噬细胞表型
CD206的变化　静息状态下的巨噬细胞CD206表型不明显,经过IL⁃4诱导的M2型巨噬细胞显著提升了巨噬细胞表型CD206的表达;而牡荆素可以明显抑制M2型巨噬细胞CD206的表达,并且存在浓度依赖性,图5㊂
2.6　
牡荆素对IL⁃4诱导M2型巨噬细胞IL⁃4/
STAT3通路的影响　在正常的巨噬细胞中
,STAT3
图2　牡荆素对M1/M2型巨噬细胞中IL⁃1β㊁iNOS ㊁MR
和Arg⁃1的mRNA 表达的影响
Fig.2　Effects of vitexin on mRNA expression of IL⁃1β,
iNOS ,MR and Arg⁃1in M1/M2macrophages
Note:Compared with control group,△△△.P <0.01;compared with
M1group,*.P <0.05,**.P <0.01,***.P <0.001;compared with M2group,#.P <0.05,##.P <0.01,###.P <0.
001.
图3　牡荆素对M1型巨噬细胞上清液中NO 含量的影响Fig.3　Effect of vitexin on NO content in supernatant of
M1macrophage
Note:Compared with M1group,**.P <0.01,***.P <0.001.
蛋白磷酸化水平低表达,经刺激剂IL⁃4激活后巨噬细胞磷酸化水平显著提高㊂牡荆素低剂量和高剂量均能够显著降低STAT3的磷酸化水平,呈剂量依赖性,图
6㊂
图4　M2型巨噬细胞对非小细胞肺癌A549细胞的体外迁
移及牡荆素干预的影响(×200)
Fig.4　Effects of M2macrophage on migration of non⁃
small cell lung cancer A549cells and vitexin intervention (×200)
Note:Compared with control group,△△△.P <0.001;compared with
M2group,##.P <0.01,###.P <0.
001.
图5　牡荆素对M2型巨噬细胞CD206表型的影响
Fig.5　Effect of vitexin on macrophage M2macrophage
phenotype CD206
Note:Compared with control group,△△△.P <0.001;compared with
M2group,###.P <0.001.
图6　牡荆素对M2型巨噬细胞STAT3蛋白磷酸化水平的影响
Fig.6　Effect of vitexin on phosphorylation of STAT3 protein in M2macrophage
Note:Compared with control group,△△△.P<0.001;compared with
M2group,##.P<0.01,###.P<0.001.
3　讨论
肺癌作为全球人类死亡的重要原因,给人类健康造成沉重负担,并给临床医生和患者带来重大的挑战[1]㊂NSCLC是肺癌的一种亚型,约占肺癌的85%~90%,是癌症死亡最常见的类型[10]㊂目前在NSCLC治疗中已经引入一些新的治疗方法,如免疫治疗㊁靶向治疗[11]㊂巨噬细胞是炎症效应细胞和宿主防御肿瘤,巨噬细胞在肿瘤中具有丰富的表达[6]㊂但是与其他细胞不同,巨噬细胞存在着高度异质,可以通过局部微环境刺激从M0阶段极化到不同的亚型即M1和M2,显示完全相反的功能[12,13]㊂
巨噬细胞的极化现象在肿瘤发展中广泛存在, M1型巨噬细胞通过分泌促炎因子和趋化因子参与机体的炎症反应,M2型巨噬细胞则是分泌抑制细胞炎症的因子,抑制炎症,降低机体免疫反应,促进肿瘤的转移[14,15]㊂其极化调节主要是通过一些核内信号蛋白发挥作用[16]㊂M1型巨噬细胞被IFN⁃γ㊁LPS或TNF⁃α激活,使IL⁃1β和iNOS在M1型巨噬细胞中高表达㊂其中iNOS是一氧化氮合酶(NOS)的一种,细胞中的NOS以L⁃精氨酸为底物,生成大量的NO,诱导致炎信号通路,过度的炎症则对机体造成一定的损伤[17,18]㊂牡荆素浓度低于200μmol/L,可以下调M1型巨噬细胞iNOS和IL⁃1β细胞因子mRNA的水平,并且显著抑制M1型巨噬细胞中NO,抑制炎症反应㊂M2型巨噬细胞由IL⁃4和IL⁃13诱导,特异性表达Arg⁃1和甘露糖(MR,CD206)㊂而Arg⁃1是M2型巨噬细胞的标记物,它将L⁃精氨酸水解成尿素和鸟氨酸,并通过与iNOS竞争共同
底物L⁃精氨酸来抑制NO介导途径,给肿瘤细胞提
供营养,协助肿瘤的远端转移和免疫逃逸[19,20]㊂M2型巨噬细胞能够调节炎症反应和适应性Th2免疫,
主要发挥免疫抑制作用[21],通过调节免疫应答,驱动肿瘤的生长和促进肿瘤微血管系统的形成[22]㊂CD206是M2型巨噬细胞特异性标志物,高表达能够促进肿瘤细胞的转移[23]㊂TAMs大多是M2型巨噬细胞,促进肿瘤转移,侵袭和免疫逃逸,其数量与多种肿瘤预后呈负相关[24]㊂所以阻断巨噬细胞的M2型极化是抑制肿瘤进展的一个重要的策略㊂黄酮类药物具有抗炎的生物活性,能够显著抑制LPS诱导M1型巨噬细胞的炎症介质,能够调节M2型巨噬细胞的极化[25⁃27]㊂牡荆素具有低细胞毒性,能够通过LPS激活RAW264.7,降低TNF⁃α㊁IL⁃1β㊁NO㊁PGE2的释放,促进IL⁃10释放,同时还能够调节促炎介质的转录因子,降低LPS诱导细胞中pP38㊁p⁃ERK1/2和p⁃JNK的表达[28]㊂但是牡荆素对于M2型巨噬细胞的生物作用尚缺少深入研究,本研究通过研究牡荆素对小鼠巨噬细胞极化的影响发现,牡荆素可以抑制IL⁃4诱导的M2型标志物MR㊁Arg⁃1和CD206的表达,发挥M2型极化抑制的作用㊂据文献报道,JAK/STAT3信号通路在多种肿瘤细胞系中均有异常,并在肿瘤的发生发展有重要的作用㊂在正常组织中,STAT3信号处于失活状态,而在肿瘤细胞中过度活化[29]㊂在巨噬细胞静息的状态下,STAT3信号通路并不活化,IL⁃4能够激活STAT3信号的通路㊂文献报道,天然产物成分可以通过抑制STAT3的激活来抑制肿瘤细胞增殖和巨噬细胞向M2表型极化的进程[30]㊂本研究发现牡荆素可以降低STAT3蛋白的磷酸化水平,使得IL⁃4/STAT3信号通路失活,从而影响巨噬细胞M2型的极化㊂
综上,牡荆素调节M1型巨噬细胞iNOS㊁IL⁃1β
的基因表达;牡荆素通过抑制STAT3信号通路活
化,影响M2型巨噬细胞Arg⁃1㊁MR和CD206的表
达,减弱M2表型促非小细胞肺癌的体外转移的能
力,最终发挥抗肺癌的作用㊂
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[收稿2019⁃09⁃24　修回2019⁃10⁃11]
(编辑　苗　磊)。