Objective: To investigate the mechanism by which amentoflavone inhibits polarization of THP-1-derived foam cells to M1 phenotype.
Objective: 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-α with RT-qPCR. THP-1-derived foam cells treated with 5 or 10 μmol/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.
Objective: 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-β mRNA (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.
Objective: Amentoflavone can inhibit the differentiation of macrophages into M1 type by activating PPAR-α/γ and restoring the expressions of the gene Arg-1 and Fizz1.
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、10 μmol/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。
Keywords: PPAR-α/γ; THP-1; amentoflavone; atherosclerosis; macrophage polarization.