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。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)、IL-1β、精氨酸酶1(Arg-1)、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。Western blot检测磷酸化信号传导及转录激活因子3表达。结果:牡荆素在25、50、100μmol/L浓度时对小鼠巨噬细胞无明显细胞毒性,确定25、50、100μmol/L浓度进行后续实验。RT-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。Griess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。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。Western blot结果显示牡荆素能够降低p-STAT3蛋白水平。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。
Objective:To explore the mechanisms of the fraction of vitexin on the M1/M2 phenotype of macrophages.Methods:RAW264.7 cell lines were induced by LPS and IL-4 in order to derive macrophages to be M1 and M2 phenotypes,respectively.The optimal concentration of vitexin was determined by checking the effect of vitexin with CCK8 on 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 M2 macrophage induced by IL-4 as well as the intervention of vitexin on the migration of non-small cell lung cancer(NSCLC)A549.The effect of vitexin on the M2 macrophage CD206 was detected by flow cytometry assay.Phosphorylation signal transduction and the expression of transcription activator 3 were 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 M1 macrophages,Arg-1 and MR in M2 macrophages in a dose dependent manner.Further study of Griess reaction showed the inhibition of vitexin in a concentration dependent manner on NO contents in M1 macrophages as well as the migration ability of the lung adenocarcinoma A549 cell line induced by M2 macrophages.Flow cytometry also illustrated that vitexin suppressed the expression of the M2 macrophages CD206 in 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/M2 macrophages and the expression of landmark products via regulation of STAT signalling pathway,then inhibited the macrophage polarization to M2,which eventually decre
作者
赵蓓
殷亦男
王程燕
毕凌
许玲
焦丽静(指导)
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)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2020年第20期2456-2461,共6页
Chinese Journal of Immunology
基金
国家自然基金项目(81704035,81973810,81904163)
中华中医药学会青年人才托举工程(QNRC2-C15)
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)
上海中医药大学2020年“研究生创新培养项目”(2020038)
上海市科学技术委员会“扬帆计划”(19YF1450000)资助项目。
关键词
牡荆素
巨噬细胞
侵袭
肺腺癌
Vitexin
Macrophages
Migration
Lung adenocarcinoma
