IRF5通过MyD88/TGF-β1/Smads信号通路介导AngⅡ诱导巨噬细胞的极化和炎症反应

IRF5 mediates AngⅡ-induced polarization and inflammatory response of macrophages through MyD88/TGF-β1/Smads signaling pathway

目的探讨转录因子干扰素调节因子5(IRF5)对RAW264.7巨噬细胞极化的调控机制。方法小鼠巨噬细胞(RAW264.7)被分为正常对照组、血管紧张素II(Ang II)处理组,IRF5 siRNA转染组,Ang II+IRF5 siRNA转染组,共4组。2’,7’-二氯荧光素二乙酸盐(DCHFDA)法测定巨噬细胞活性氧簇(ROS)的水平;免疫荧光染色方法检测细胞一氧化氮合酶(i NOS)和IRF5的表达;试剂盒检测巨噬细胞丙二醛(MDA)含量;qRT-PCR和Wetern blotting分别检测巨噬细胞iNOS、肿瘤坏死因子α(TNF-α)、IRF5 mRNA表达水平,以及IRF5、髓样分化因子88(MyD88)、转化生长因子(TGF-β1)、Smad家族蛋白2/3(Smad-2/3)、磷酸化Smad家族蛋白2/3(p-Smad-2/3)蛋白的表达水平;ELISA法检测巨噬细胞培养上清炎症因子水平TNF-α、白介素12(IL-12)、IL-10含量变化。结果IRF5干扰载体(IRF5 siRNA)显著抑制Ang II诱导的巨噬细胞中ROS的水平(P<0.05)和MDA含量(P<0.01);IRF5 siRNA明显抑制Ang II促进巨噬细胞M1亚型的极化,IRF5 siRNA转染后可显著抑制Ang II诱导的巨噬细胞iNOS和TNF-α的表达,与此同时,IRF5表达的减少降低了MyD88/TGF-β1/Smads信号通路的活性;此外,IRF5 siRNA显著减弱Ang II组巨噬细胞培养上清中TNF-α和IL-12的分泌(均P<0.01),而增加了IL-10含量(P<0.001)。结论IRF5通过TLR-MyD88/TGF-β1/Smads信号通路调控巨噬细胞极化和炎症反应的诱发。

To investigate the regulatory effects of transcription factor interferon regulatory factor 5(IRF5)on RAW264.7 macrophage polarization,mouse macrophages(RAW264.7)were introduced and divided into four groups:normal control group,angiotensin II(Ang II)treatment group,IRF5 siRNA transfection group,and Ang II+IRF5 siRNA transfection group.The levels of reactive oxygen species(ROS)in macrophages were determined by2’,7’-dichlorofluorescein diacetate(DCHFDA)method;the expressions of nitric oxide synthase(iNOS)and IRF5 were detected by immunofluorescence staining;thecontent of malondialdehyde(MDA)in macrophageswas detected by reagent test kit.QRT-PCR andWestern blot were adopted to detect the mRNAexpression levels of iNOS,tumor necrosis factorα(TNF-α)and IRF5 in macrophages,as well as the proteinexpression levels of IRF5,myeloid differentiation factor88(MyD88),transforming growth factor(TGF-β1),Smad family protein 2/3(Smad-2/3),and phosphorylated Smad family protein 2/3(p-Smad-2/3).ELISA kit was used todetect the levels of inflammatory factors,such as TNF-α,interleukin 12(IL-12)and IL-10,in the cell culturesupernatant.Data showed that IRF5 interference vector(IRF5 siRNA)significantly inhibited the level of ROS(P<0.05),MDA content(P<0.01)and M1 polarization in macrophages of Ang II-induced group;IRF5 siRNAtransfection could significantly inhibit the expression of iNOS and TNF-αin macrophages induced by Ang II.At thesame time,the reduction of IRF5 expression downregulated the activity of MyD88/TGF-β1/Smads signalingpathway.In addition,IRF5 siRNA significantly attenuated the secretion of TNF-αand IL-12 in the macrophageculture supernatant(both P<0.01)in Ang II group,while upregulated the content of IL-10 was increased(P<0.001).Taken together,IRF5 regulates the polarization and inflammatory response of macrophages via TLR-MyD88/TGF-β1/Smads signaling pathway.