穗花杉双黄酮激活PPAR-α/γ抑制THP-1源性巨噬细胞向M1型极化  

Amentoflavone inhibits M1 polarization of THP-1-derived foam cells by activating PPAR-α/γ

作　　者：邱峰 张琳[2] 郑介婷 操龙斌 张子康 邓屹琪 QIU Feng;ZHANG Lin;ZHENG Jieting;CAO Longbin;ZHANG Zikang;DENG Yiqi(Department of Laboratory Medicine,Nanhai Hospital,Southern Medical University,Foshan 528244,China;School of Traditional Chinese Medicine,Southern Medical University,Guangzhou 510515,China;Department of Emergency,Second Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510120,China)

机构地区：[1]南方医科大学南海医院医学检验科,广东佛山528244 [2]南方医科大学中医药学院,广东广州510515 [3]广州中医药大学第二附属医院急诊科,广东广州510120

出　　处：《南方医科大学学报》2021年第3期344-351,共8页Journal of Southern Medical University

基　　金：广东省佛山市医学类科技攻关项目(2018AB001112);佛山市中医药领域科技攻关专项(2020001005585);佛山市“新型冠状病毒感染的肺炎”应急科技攻关专项(2020001000376);广东省医学科研基金项目(B2020144)。

摘　　要：目的探讨穗花杉双黄酮调节体外诱导的M1型巨噬细胞极化的相关机制。方法设实验组、溶媒对照组和无药对照组,实验组:不同浓度的(5、10μmol/L)穗花杉双黄酮干预用联合脂多糖及重组人干扰素-γ诱导的M1型巨噬细胞;溶媒对照组:溶媒3‰DMSO;无药对照组:不加穗花杉双黄酮处理。显微镜下观察细胞形态学;通过ELISA检测干预后细胞上清液L-6、IL-10、TNF-α、TGF-β各自的表达水平;通过CCK-8法检测计算出穗花杉双黄酮对细胞的安全浓度;通过分子对接建模确定穗花杉双黄酮的靶蛋白,利用RT-qPCR、Western blot检测L-6、IL-10、TNF-α、TGF-β、PPAR-α/γ、Arg-1、Fizz1基因和蛋白表达。结果穗花杉双黄酮干预后可以阻止被诱导引起的THP-1细胞向M1极化;穗花杉双黄酮可下调M1极化时高表达的IL-6、TNF-α的mRNA,上调M2型主要标志细胞因子IL-8和TGF-β的mRNA表达(P<0.05),同时上调M1极化状态下的细胞内Arg1和Fizz1蛋白的表达(P<0.05)。随着穗花杉双黄酮浓度的增加和作用时间的延长,其抑制细胞的增殖效果增强(P<0.05),且高浓度具有杀伤作用。穗花杉双黄酮可与PPAR-α/γ关键靶点蛋白活性位点非共价结合并激活该蛋白。结论穗花杉双黄酮可通激活PPAR-α/γ,恢复Arg-1、Fizz1基因表达,抑制巨噬细胞向M1型分化。Objective To investigate the mechanism by which amentoflavone inhibits polarization of THP-1-derived foam cells to M1 phenotype.Methods Human monocyte cell line THP-1 was stimulated to differentiate into M1-type macrophages using phorbol 12-myrislate13-acetate(PMA)combined with lipopolysaccharide(LPS)and recombinant human interferon-γ(rhlFN-γ).M1 polarization of THP-1-derived macrophages was confirmed by observing morphological changes of the cells and detecting the mRNA expression of L-6 and TNF-αwith RT-qPCR.THP-1-derived foam cells treated with 5 or 10μmol/L amentoflavone for 24 h were examined for cytokines using ELISA.The mRNA and protein expressions of IL-6,IL-10,TNF-α,TGF-β,PPAR-α/γ,Arg-1 and Fizz1 in the cells were detected using RT-qPCR and Western blotting.Results Amentoflavone prevented induced M1 polarization of THP-1 cells.Amentoflavone down-regulated the mRNA expressions of IL-6 and TNF-α,up-regulated mRNA expressions of IL-8 and TGF-βmRNA(P<0.05),and increased the protein expressions of PPAR-α/γ,Arg-1 and Fizz1.Molecular docking simulation showed that amentoflavone could bind to the surface of PPARα/γ.Conclusion Amentoflavone can inhibit the differentiation of macrophages into M1 type by activating PPAR-α/γand restoring the expressions of the gene Arg-1 and Fizz1.

关 键 词：穗花杉双黄酮 巨噬细胞极化 动脉粥样硬化 PPAR-α/γ THP-1 

分 类 号：R543.5[医药卫生—心血管疾病]