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2020, v.45(09) 2158-2164

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丹皮酚通过下调miR-155/JAK1-STAT1通路抑制巨噬细胞M1极化
Paeonol inhibits macrophage M1 polarization by down-regulating miR-155/JAK1-STAT1 pathway

孙颖;刘玲;施晓艳;何海;黄翰文;戴敏;
SUN Ying;LIU Ling;SHI Xiao-yan;HE Hai;HUANG Han-wen;DAI Min;College of Pharmacy, Anhui University of Chinese Medicine;Key Laboratory of Xin′an Medicine, Ministry of Education;

摘要(Abstract):

探究丹皮酚(paeonol)对小鼠腹腔巨噬细胞RAW264.7 M1型极化的影响,并从基因层面探讨该干预作用是否与miR-155的下调及下游JAK1-STAT1通路受抑制有关,进一步为丹皮酚抗动脉粥样硬化(atherosclerosis,AS)的分子机制提供新的思路。实验采用脂多糖(lipopolysaccharide,LPS)和干扰素-γ(interferon-γ,IFN-γ)共刺激24 h建立巨噬细胞M1型极化的模型,丹皮酚在刺激前24 h给予细胞起到预保护作用。CCK-8法检测LPS和IFN-γ共刺激对细胞的损伤作用,流式细胞术检测M1型表面标志物F4/80和CD86的表达,ELISA法检测细胞上清液中炎症因子白介素-6(interleukin 6,IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的分泌,RT-qPCR法检测各组细胞miR-155的表达,Western blot法检测JAK1-STAT1-SOCS1通路上的蛋白表达。结果显示,LPS和IFN-γ在最适浓度下对细胞没有明显损伤作用,但可诱导巨噬细胞向M1极化,使得细胞表面的M1型标志性因子F4/80和CD86高表达,同时导致细胞表面炎症因子IL-6和TNF-α的分泌增多(P<0.05或P<0.01)。丹皮酚可显著降低RAW264.7细胞由LPS和IFN-γ诱导的表面M1型标志物F4/80和CD86的高表达,同时减少了炎症因子IL-6和TNF-α的分泌(P<0.05或P<0.01),降低了miR-155的表达水平,显著下调了JAK1-STAT1的蛋白磷酸化水平,上调SOCS1蛋白表达(P<0.01)。结果表明,丹皮酚通过下调细胞表面标志性因子及细胞分泌的炎症因子,达到抑制巨噬细胞M1型极化的作用,这种作用可能与丹皮酚下调miR-155的表达,抑制JAK1-STAT1通路的活化有关。
The aim of this paper was to investigate the effect and mechanism of paeonol on peritoneal macrophage M1 polarization in mice, explore whether the intervention action is related to the down-regulation of miR-155 and the inhibition of downstream JAK1-STAT1 pathway, and provide a new idea for the molecular mechanism of paeonol against atherosclerosis(AS). Lipopolysaccharide(LPS) and interferon-γ(IFN-γ) were used to stimulate macrophages for 24 hours to establish the M1 polarization model, and paeonol was given 24 hours before co-stimulation to provide a pre-protective effect on cells. CCK-8 assay was used to detect the cells damage induced by LPS and IFN-γ co-stimulation; flow cytometry was used to detect the expression of M1 surface markers F4/80 and CD86. ELISA was used to detect the secretion of interleukin 6(IL-6) and tumor necrosis factor-α(TNF-α) in supernatant. RT-qPCR was used to detect the expression of miR-155, and Western blot was used to detect the protein expression at JAK1-STAT1-SOCS1 pathway. The results showed that LPS and IFN-γ had no obvious damage to the cells at the optimal concentration, but they induced macrophages polarized to M1, resulted in high expression of M1 type marker factors F4/80 and CD86 on the cell surface, and increased secretion of IL-6 and TNF-α on the cell surface(P<0.05 or P<0.01). Paeonol significantly reduced the LPS and IFN-γ-induced high expression of F4/80 and CD86, the secretion of inflammatory factors IL-6 and TNF-α(P<0.05 or P<0.01), decreased the expression level of miR-155, significantly down-regulated the protein phosphorylation level of JAK1-STAT1 and up-regulated the protein expression of SOCS1(P<0.01) in RAW264.7 cells. The results showed that paeonol could inhibit M1 polarization of macrophages by down-regulating cell surface marker factors and inflammatory factors secreted by cells, which may be related to the down-regulation of miR-155 expression and the inhibition JAK1-STAT1 pathway activation.

关键词(KeyWords): 丹皮酚;miR-155;JAK1-STAT1通路;M1极化
paeonol;miR-155;JAK1-STAT1 pathway;M1 type polarization

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81773937)

作者(Authors): 孙颖;刘玲;施晓艳;何海;黄翰文;戴敏;
SUN Ying;LIU Ling;SHI Xiao-yan;HE Hai;HUANG Han-wen;DAI Min;College of Pharmacy, Anhui University of Chinese Medicine;Key Laboratory of Xin′an Medicine, Ministry of Education;

DOI: 10.19540/j.cnki.cjcmm.20200210.409

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