激活转录因子3对巨噬细胞M2型极化的影响及其机制

Effect of activating transcription factor 3 on M2 type polarization of macrophages and its mechanism

目的研究激活转录因子3(ATF3)对巨噬细胞M2型极化的影响及其机制。方法取对数生长期RAW264.7细胞,接种至6孔板中,将细胞分为对照组、M1组和M2组;对照组细胞未做任何处理,M1组细胞用干扰素-γ(10μg·L^(-1))和脂多糖(10μg·L^(-1))诱导6 h,M2组细胞用白细胞介素(IL)-4(15μg·L^(-1))诱导48 h。采用荧光实时定量聚合酶链反应法检测对照组、M1组、M2组RAW264.7细胞中ATF3 mRNA表达水平,采用Western blot法检测3组RAW264.7细胞中ATF3、信号传导及转录激活蛋白(STAT3)及磷酸化STAT3(p-STAT3)蛋白的相对表达量。另取对数生长期RAW264.7细胞分为对照组、空白转染组、M2对照组、M2 ATF3转染组;对照组细胞未做任何处理,空白转染组细胞转染空白小干扰RNA(siRNA),M2组细胞用IL-4(15μg·L^(-1))诱导48 h,M2 ATF3转染组细胞用IL-4(15μg·L^(-1))诱导48 h,然后转染ATF3 siRNA序列。采用荧光实时定量聚合酶链反应检测对照组、空白转染对照组、M2组、M2 ATF3转染组RAW264.7细胞中ATF3、IL-10及转化生长因子-β(TGF-β)mRNA相对表达量,采用Western blot检测4组RAW264.7细胞中ATF3、精氨酸酶-1(Arg-1)、STAT3及p-STAT3蛋白的相对表达量。结果M2组RAW264.7细胞中ATF3 mRNA及ATF3、p-STAT3蛋白相对表达量均显著高于对照组(P<0.05);M1组与对照组RAW264.7细胞中ATF3 mRNA及ATF3、p-STAT3蛋白相对表达量比较差异无统计学意义(P>0.05);M1组、M2组RAW264.7细胞中STAT3蛋白相对表达量与对照组比较差异无统计学意义(P>0.05)。M2对照组RAW264.7细胞中ATF3 mRNA及蛋白相对表达量均显著高于对照组和空白转染组(P<0.05);M2 ATF3转染组RAW264.7细胞中ATF3 mRNA及蛋白相对表达量均显著低于M2对照组(P<0.05);其余各组RAW264.7细胞中ATF3 mRNA及蛋白相对表达量比较差异均无统计学意义(P>0.05)。M2对照组RAW264.7细胞中Arg-1、p-STAT3蛋白相对表达量均显著高于对照组(P<0.05);M2 ATF3转染组RAW264.7细胞中Arg-1、p-STAT3蛋白相对表达量均显著低于M2对照组(P<0.05)。其余各组RAW264.7细胞中Arg-1、STAT3及p-STAT3蛋白相对表达量比较差异均无统计学意义(P>0.05)。M2对照组RAW264.7细胞中IL-10、TGF-βmRNA相对表达量均显著高于对照组(P<0.05);M2 ATF3转染组RAW264.7细胞中IL-10、TGF-βmRNA相对表达量均显著低于M2对照组(P<0.05)。其余各组RAW264.7细胞中IL-10、TGF-βmRNA相对表达量比较差异均无统计学意义(P>0.05)。结论ATF3可能通过STAT3信号通路调节巨噬细胞向M2型极化。

Objective To investigate the effect of activating transcription factor 3(ATF3)on macrophage M2 type polarization and its mechanism.Methods RAW264.7 cells in logarithmic growth stage were inoculated into 6-well plates,and the cells were divided into control group,M1 group and M2 group.The cells in the control group were not treated,the cells in the M1 group were treated with interferon-γ(10μg·L^(-1))and lipopolysaccharide(10μg·L^(-1))for 6 h,and the cells in the M2 group were treated with interleukin(IL)-4(15μg·L^(-1))for 48 h.The relatire expression of ATF3 mRNA in RAW264.7 cells was detected by fluorescence real-time quantitative polymerase chain reaction,and the relatire expressions of ATF3,signal transducer and activator of transcription(STAT3)and phospho-STAT3(p-STAT3)protein in the RAW264.7 cells in the three groups were detected by Western blot.In addition,the RAW264.7 cells in logarithmic growth stage were taken and divided into control group,blank transfection group,M2 control group and M2 ATF3 transfection group.The cells in the control group were not treated,the cells in the blank transfection group were transfected with blank small interfering RNA(siRNA),the cells in the M2 control group were induced with IL-4(15μg·L^(-1))for 48 h,and the cells in the M2 ATF3 group were induced with IL-4(15μg·L^(-1))for 48 h,and then transfected with ATF3 siRNA sequence.The relatire expressions of ATF3,IL-10 and transforming growth factor-β(TGF-β)mRNA in RAW264.7 cells in control group,blank transfection group,M2 control group and M2 ATF3 transfection group were detected by fluorescence real-time quantitative polymerase chain reaction.The relatire expressions of ATF3,arginase-1(Arg-1),STAT3 and p-STAT3 protein in RAW264.7 cells in the above four groups were detected by Western blot.Results The relative expressions of ATF3 mRNA and ATF3,p-STAT3 protein in RAW264.7 cells in the M2 group were significantly higher than those in the control group(P<0.05);there was no significant difference in the relative expressions of ATF3 mRNA and ATF3,p-STAT3 protein in RAW264.7 cells between the M1 group and the control group(P>0.05);there was no significant difference in the relative expression of STAT3 protein in RAW264.7 cells between M1 group,M2 group and control group(P>0.05).The relative expressions of ATF3 mRNA and protein in RAW264.7 cells in M2 control group were significantly higher than those in the control group and blank transfection group(P<0.05);the relative expressions of ATF3 mRNA and protein in RAW264.7 cells in the M2 ATF3 transfection group were significantly lower than those in the M2 control group(P<0.05);there was no significant difference in the relative expressions of ATF3 mRNA and protein in RAW264.7 cells among the other groups(P>0.05).The relative expressions of Arg-1,p-STAT3 protein in RAW264.7 cells in the M2 control group were significantly higher than those in the control group(P<0.05);the relative expressions of Arg-1,p-STAT3 protein in RAW264.7 cells in the M2 ATF3 transfection group were significantly lower than those in the M2 control group(P<0.05).There was no significant difference in the relative expressions of Arg-1,STAT3 and p-STAT3 protein in RAW264.7 cells among the other groups(P>0.05).The relative expressions of IL-10,TGF-βmRNA in RAW264.7 cells in the M2 control group were significantly higher than those in the control group(P<0.05);the relative expressions of IL-10,TGF-βmRNA in RAW264.7 cells in M2 ATF3 transfection group were significantly lower than those in the M2 control group(P<0.05).There was no significant difference in the relative expressions of IL-10,TGF-βmRNA in RAW264.7 cells among the groups(P>0.05).Conclusion ATF3 may regulate the macrophage polarization to M2 through STAT3 signaling pathway.