Objectives: To investigate the effect of high glucose on macrophage polarization and the role of immune-responsive gene 1 (IRG1) in mediating its effect.
Methods: RAW264.7 cells were transfected with IRG1-overexpressing plasmid or IRG1 siRNA via electroporation and cultured in either normal or high glucose for 72 h to observe the changes in cell viability and morphology using CCK-8 assay and phase contrast microscopy. The protein levels of IRG1, iNOS, Arg-1, IL-1β and IL-10 in the treated cells were detected with Western blotting, and the fluorescence intensities of iNOS and Arg-1 were detected using immunofluorescence assay. The protein levels of IL-1β and IL-10 in the culture medium were determined with ELISA.
Results: High glucose exposure significantly reduced IRG1 and Arg-1 expressions, increased iNOS and IL-1β expressions and IL-1β secretion, and decreased IL-10 level in RAW264.7 cells. Transfection with the IRG1-overexpressing plasmid provided the cells with obvious resistance to high glucose-induced changes in iNOS, Arg-1, IL-1β and IL-10, whereas IRG1 knockdown further enhanced the effects of high glucose exposure on Arg-1 expression and the expression and secretion of IL-10.
Conclusions: High glucose promotes M1 polarization of the macrophages possibly through a mechanism to inhibit the expression of IRG1 protein, thus leading to chronic inflammatory response.
目的: 研究高糖环境对巨噬细胞极化的影响及其潜在分子机制。方法: 将离体培养的RAW264.7细胞随机分为过表达对照组（NC-OE组）、免疫反应基因1过表达组（IRG1 OE组）、沉默对照组（NC-siRNA组）和IRG1沉默组（IRG1 siRNA组），电穿孔进行质粒转染后再组内随机分为对照组（Con组）和高糖组（HG组），60 mmol/L葡萄糖干预72 h后收集细胞进行检测。CCK-8检测细胞活性，相差显微镜观察细胞形态，Western blotting检测细胞中IRG1、iNOS、Arg-1、IL-1β和IL-10蛋白表达，免疫荧光染色检测细胞中iNOS和Arg-1蛋白荧光水平，ELISA法检测细胞培养基中IL-1β和IL-10蛋白水平。结果: 与对应Con组相比，HG组IRG1表达均下降（P<0.01），同时出现较多梭形和多突形细胞且两极可见伸展的伪足，iNOS表达升高（P<0.01）、Arg-1表达下降（P<0.05），IL-1β表达和分泌升高（P<0.05）、IL-10分泌下降（P<0.01）。转染IRG1过表达质粒后，与对应NC-OE组相比，IRG1 OE组IRG1水平均升高（P<0.01）；高糖环境下，HG-IRG1 OE组梭形和多突形细胞明显减少、iNOS表达下降（P<0.01）、Arg-1表达升高（P<0.01）、IL-1β表达和分泌下降（P<0.01）、IL-10表达和分泌升高（P<0.05）。IRG1沉默后，与对应NC-siRNA组相比，IRG1 siRNA组IRG1水平降低（P<0.01）；高糖环境下，HG-IRG1 siRNA组多突形细胞和伪足进一步增加、Arg-1表达降低（P<0.01）、IL-10表达和分泌减少（P<0.05）。结论: 高糖环境诱导巨噬细胞促炎性M1型极化进而诱发慢性炎症反应，其作用机制可能与抑制巨噬细胞中IRG1蛋白表达有关。.
Keywords: M1 polarization; high glucose condition; immune-responsive gene 1; inflammatory cytokines; macrophages.