扶正抗癌方通过miR155-C/EBP-β途径逆转M2型巨噬细胞极化  

Fuzheng Kangai Formula Reverse M2 Macrophage Polarization by miR155-C/EBP-β Pathway

作　　者：李龙妹 王苏美 唐青 廖桂雅 吴万垠 LI Long-mei;WANG Su-mei;TANG Qing;LIAO Gui-ya;WU Wan-yin(The Second Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510370,China)

机构地区：[1]广州中医药大学第二附属医院,广州510370

出　　处：《中国中医基础医学杂志》2022年第5期768-773,779,共7页JOURNAL OF BASIC CHINESE MEDICINE

基　　金：国家自然科学基金资助项目(81803919)-基于肿瘤与微环境的交叉对话研究扶正抗癌方逆转EMT的分子机制;国家自然科学基金资助项目(81974543)-扶正抗癌方通过调控铁蛋白促进铁死亡逆转非小细胞肺癌对吉非替尼耐药的机制研究;广东省自然科学基金博士启动项目(2018A030310601)-扶正抗癌方通过外泌体途径介导肿瘤相关巨噬细胞极化抑制NSCLC转移的研究。

摘　　要：目的:探讨扶正抗癌方(fuzheng kangai formula,FZKA)对小鼠M2型巨噬细胞RAW264.7极化的影响及其作用机制。方法:采用白细胞介素-4(interleukin-4,IL-4)作用RAW264.7细胞48 h建立巨噬细胞M2型极化模型,利用流式细胞术、实时荧光定量PCR、蛋白质印迹法检测M2型标志物巨噬细胞甘露糖受体(CD)206、CD163和转化生长因子-β(transforming growth factor-β,TGF-β)以及M1型标志物一氧化氮合成酶(inductible nitric oxide synthase,iNOS)的表达,将扶正抗癌方作用于M2型巨噬细胞后,检测M1及M2型巨噬细胞标志物、转录因子CCAAT增强子结合蛋白β(CCAAT enhancer-binding proteinβ,C/EBP-β)和miR155的表达,利用瞬时转染法将miRNA模拟物(mimics)和抑制剂(inhibitors)转入细胞后检测C/EBP-β及巨噬细胞极性的变化。结果:在IL-4(20 ng•mL^(-1))作用下,CD206、CD163、TGF-β的表达增加(P<0.01),iNOS表达降低(P<0.01),IL-4成功诱导巨噬细胞向M2型极化;加入扶正抗癌方后CD206、CD163和TGF-β的表达降低(P<0.01),iNOS表达增加(P<0.01),扶正抗癌方可逆转M2型巨噬细胞的极化,同时C/EBP-βmRNA和蛋白的表达降低(P<0.01),miR155的表达增加(P<0.01);将miRNA的mimics和inhibitors转染至M2型巨噬细胞后miR155的表达分别增加和降低(P<0.01),miR155 mimics和inhibitors转染成功;在M2型巨噬细胞中转入miR155 inhibitors后,C/EBP-βmRNA的表达增加(P<0.01),miR155可靶向抑制C/EBP-βmRNA的表达;M2型巨噬细胞中转入miR155 inhibitors后加入扶正抗癌方,CD206和CD163表达增加(P<0.01,P<0.05),miR155 inhibitors抑制了扶正抗癌方对M2型巨噬细胞极化的逆转。结论:扶正抗癌方可通过上调miR155的表达,进而靶向抑制C/EBP-β基因的表达,并进一步通过下调M2型巨噬细胞标志物CD206、CD163和TGF-β,上调M1型巨噬细胞标志物iNOS的表达,逆转M2型巨噬细胞的极化。Objective:To discuss the effects and mechanism of Fuzheng Kangai Formula(FZKA)on the polarization of mouse M2-type macrophage RAW264.7 cells.Methods:IL-4 was used to stimulate RAW264.7 cells for 48 hours to establish theM2-type macrophage model.The expression of the M2-type markers CD206,CD163 and TGF-β,and the M1-type marker iNOS were detected by flow cytometry,Quantitative Real-time PCR and western blot assay.The expression of M1 and M2-type macrophage markers,C/EBP-βand miR155 were detected after FZKA was applied to M2-type macrophages.The transient transfection was used to transfect miRNA mimics and inhibitors into cells,and then detected the changes of C/EBP-βexpression and macrophage polarity.Results:It showed that IL-4(20 ng•mL^(-1))could induce macrophages polarized to M2,which resulting that the expression of CD206,CD163 and TGF-βwere increased(P<0.01),while the expression of iNOS was decreased(P<0.01).Under the intervention of FZKA,the expression of CD206,CD163 and TGF-βwere decreased(P<0.01),while the expression of iNOS was increased(P<0.01).The polarization of M2-type macrophages could be reversed by FZKA.Meanwhile,the mRNA and protein expression of C/EBP-βwas decreased(P<0.01),while the expression of miR155 was increased(P<0.01).When transfecting the mimics and inhibitors of miRNA into M2-type macrophages,the expression of miR155 was increased or decreased,respectively(P<0.01).The miR155 mimics and inhibitors were transfected into the cells successfully.The mRNA expression of C/EBP-βwas increased when the miR155 inhibitors were transfected into the M2-type macrophages(P<0.01).The miR155 could inhibit the mRNA expression of C/EBP-βspecifically.When the FZKA was added to M2-type macrophages which were transfected into miR155 inhibitors already,the expression of CD206 and CD163 were increased(P<0.01,P<0.05).The miR155 inhibitors could inhibit the reversal of M2-type macrophage polarization by FZKA.Conclusion:FZKA can reverse the polarization of M2-type macrophage by up-regulating the expressio

关 键 词：扶正抗癌方 miR155 转录因子CCAAT增强子结合蛋白β M2极化 

分 类 号：R273[医药卫生—中西医结合]