高糖通过miR-125b对小鼠RAW264.7巨噬细胞极化的调控作用

Regulatory effect of high glucose on polarization of RAW264.7 macrophages via miR-125b in mice

目的:以小鼠单核巨噬细胞系RAW264.7为研究对象,体外探讨高糖微环境对小鼠巨噬细胞极化的影响,并阐明miR-125b的调控作用。方法:RAW264.7细胞分为正常对照(NG)组、正糖抑制(NG+miR-125b inhibitor)组、高糖刺激(HG)组和高糖抑制(HG+miR-125b inhibitor)组,进行相应的干预处理。各组细胞体外培养72 h后留取细胞及上清,采用实时荧光定量PCR(RT-qPCR)法检测各组细胞中M1/M2极化相关因子mRNA和miR-125b表达水平,酶联免疫吸附测定(ELISA)法检测各组细胞上清中M1/M2极化相关细胞因子水平,流式细胞术检测M1/M2极化表面标记物CD86(M1标记)和CD206(M2标记)阳性表达率。通过miR-125b inhibitor沉默细胞中miR-125b的表达,进一步采用RT-qPCR和Western blotting法检测各组细胞中M1/M2极化相关因子干扰素调节因子4(IRF4)、干扰素调节因子5(IRF5)和过氧化物酶体增殖物激活受体(PPAR)mRNA及蛋白水平表达。结果:高糖处理72 h后,与NG组比较,HG组细胞中白细胞介素10(IL-10)mRNA表达水平和细胞上清中IL-10水平明显降低(P<0.01),白细胞介素1β(IL-1β)和Toll样受体4(TLR4)mRNA表达水平明显升高(P<0.01),细胞上清中IL-1β、IL-12和IL-6水平明显升高(P<0.01)。与NG组比较,HG组细胞中CD86阳性表达率明显升高(P<0.05),CD206阳性表达率明显降低(P<0.05),miR-125b表达水平明显升高(P<0.01)。采用miR-125b inhibitor沉默miR-125b表达后,分别与NG和HG组比较,NG+miR-125b inhibitor组和HG+miR-125b inhibitor组细胞中miR-125b表达水平均明显降低(P<0.01),细胞中IL-10 mRNA表达水平和细胞上清中IL-10水平升高(P<0.01),细胞中IL-1β和TLR4 mRNA表达水平及细胞上清中IL-12、IL-6和IL-1β水平均明显降低(P<0.05或P<0.01),细胞中CD86阳性表达率降低(P<0.05或P<0.01),CD206阳性表达率升高(P<0.01)。与NG组比较,HG组细胞中IRF4和PPAR mRNA和蛋白表达水平均明显降低(P<0.01),IRF5 mRNA和蛋白表达水平均明显升高(P<0.01);沉默miR-125b表达后,与NG组比较,NG+miR-125b inhibitor组细胞中IRF4和PPAR mRNA及蛋白表达水平均明显升高(P<0.01),IRF5 mRNA和蛋白表达水平明显降低(P<0.05);与HG组比较,HG+miR-125b inhibitor组细胞中IR4和PPAR mRNA及蛋白水平明显降低(P<0.01),IRF5 mRNA表达水平明显降低(P<0.01)。结论:高糖刺激可以诱导巨噬细胞向促炎M1表型极化,同时miR-125b表达水平明显升高,沉默miR-125b表达可部分逆转高糖刺激诱导的巨噬细胞向促炎M1表型的极化,该过程可能是通过上调IRF4和PPAR及下调IRF5实现的。

Objective:To use the mouse macrophage cell line RAW264.7 as the research object and explore the effect of high glucose microenvironment on the polarization of mouse macrophages in vitro,and to clarify the regulatory effect of miR-125b.Methods:The RAW264.7 cells were divided into normal control(NG)group,normal glucose inhibition(NG+miR-125b inhibitor)group,high glucose stimulation(HG)group and high glucose inhibition(HG+miR-125b inhibitor)group.The cells in each group were cultured in vitro for 72 h,and the cells and supernatant were collected.RT-qPCR method was used to detect the expression levels of M1/M2 polarization-related genes and the expression levels of miR-125b in the cells in various groups.ELISA method was used to detect the levels of M1/M2 polarization-related cytokines in supernatant of the cells in various groups;flow cytometry was used to detect the positive expression rates of M1/M2 polarization surface markers CD86(M1 marked)and CD206(M2 marked).The expression of miR-125b in the cells was silenced through miR-125b inhibitor,and RT-qPCR and Western blotting methods were used to detect the expression levels of key transcription factors interferon regulator 4(IRF4),interferon regulator 5(IRF5),and peroxisome-activated receptor(PPAR)mRNA and proteins associated with M1/M2 polarization in the cells in various groups.Results:After treated with high glucose for 72 h,compared with NG group,the IL-10 mRNA expression level and the level of IL-10 in the cell supernatant in HG group were significantly decreased(P<0.01),while the expression levels of IL-1βand TLR4 mRNA were significantly increased(P<0.01),and the levels of IL-1β,IL-6,and IL-12 in the cell supernatant were increased(P<0.01).Compared with NG group,the positive expression rate of CD86 in HG group was significantly increased(P<0.05),while the positive expression rate of CD206 was significantly decreased(P<0.05);at the same time,the expression level of miR-125b was significantly increased(P<0.01).The expression level of miR-125b was silenced by miR-125b inhibitor,compared with NG and HG groups respectively,the expression levels of miR-125b after transfection in NG+miR-125b inhibitor group and HG+miR-125b inhibitor were decreased significantly(P<0.01),the IL-10 mRNA expression levels in the cells and the levels of IL-10 in the cell supernatant were significantly increased(P<0.01),while the expression levels of IL-1βand TLR4 mRNA and the levels of IL-12,IL-6,and IL-1βin the cell supernatant were decreased(P<0.05 or P<0.01);the positive expression rates of CD86 in the cells were significantly decreased(P<0.05 or P<0.01),and the positive expression rates of CD206 were significantly increased(P<0.05).Compared with NG group,the expression levels of IRF4,PPAR mRNA and proteins in HG group were significantly decreased(P<0.01),while the expression levels of IRF5 mRNA and protein were increased(P<0.01);after silencing the expression of miR-125b,compared with NG group,the mRNA and protein expression levels of IRF4 and PPAR in the cells in NG+miR-125b inhibitor group were increased significantly(P<0.01),while the expression levels of IRF5 mRNA and protein were decreased(P<0.05).Compared with HG group,the expression levels of IR4 and PPAR mRNA and proteins in the cells in HG+miR-125b inhibitor group were decreased(P<0.01),while the expression level of IRF5 miRNA was significantly decreased(P<0.01).Conclusion:High glucose stimulation can induce to the macrophage polarization to the pro-inflammatory M1 phenotype,and the expression level of miR-125b is also significantly increased.Silencing the expression of miR-125b can partially reverse the macrophages induced by high glucose stimulation to the pro-inflammatory type change,and this process may be achieved by up-regulating IRF4,PPAR and down-regulating IRF5.