高糖环境通过抑制免疫反应基因1的表达诱导巨噬细胞促炎性M1型极化

doi:10.12122/j.issn.1673-4254.2025.01.01

摘要/Abstract

摘要：

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。

关键词: 巨噬细胞, M1型极化, 炎症因子, 高糖环境, 免疫反应基因1

Abstract:

Objective 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. Conclusion 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.

Key words: macrophages, M1 polarization, inflammatory cytokines, high glucose condition, immune-responsive gene 1

罗维, 王宇航, 刘延松, 王媛媛, 艾磊. 高糖环境通过抑制免疫反应基因1的表达诱导巨噬细胞促炎性M1型极化[J]. 南方医科大学学报, 2025, 45(1): 1-9.

Wei LUO, Yuhang WANG, Yansong LIU, Yuanyuan WANG, Lei AI. High glucose induces pro-inflammatory polarization of macrophages by inhibiting immune-responsive gene 1 expression[J]. Journal of Southern Medical University, 2025, 45(1): 1-9.

图/表 8

图1 高糖环境下巨噬细胞中过表达或沉默IRG1效果的验证

Fig.1 Validation of the efficiency of overexpression or silencing of IRG1 in macrophages under high glucose condition. A: Expression of IRG1 protein detected by Western blotting after transfection with the IRG1-overexpressing plasmid (**P<0.01 vs correspondingNC-OE groups; ##P<0.01 vs corresponding Con groups). B: Expression of IRG1 protein detected by Western blotting after IRG1 silencing (**P<0.01 vs correspondingNC-siRNA groups; ##P<0.01 vs corresponding Con groups). Data are presented as Mean±SD (n=6) if not specified otherwise. HG: High glucose.

图2 IRG1过表达或沉默对高糖条件下巨噬细胞活性的影响

Fig.2 Effects of IRG1 overexpression (A) or silencing (B) on viability of the macrophages cultured in high glucose detected by CCK-8 assay (Mean±SD, n=6).

图3 IRG1过表达或沉默对高糖条件下巨噬细胞形态的影响

Fig.3 Effects of IRG1 overexpression or silencing on morphology of the macrophages cultured in high glucose (phase contrast microscopy, scale bar=50 μm). Arrows indicate M1-like spindle cells and pseudopodia.

图4 IRG1过表达或沉默对高糖条件下巨噬细胞极化标志蛋白表达的影响

Fig.4 Effects of IRG1 overexpression (A) or silencing (B) on expressions of iNOS and Arg-1 in the macrophages cultured in high glucose detected by Western blotting. *P<0.05, **P<0.01 vs corresponding NC-OE group (A) or NC-siRNA group (B); #P<0.05, ##P<0.01 vs corresponding Con group.

图5 IRG1过表达对HG条件下巨噬细胞极化标志蛋白表达的影响

Fig.5 Immunofluorescence assay for assessing the effects of IRG1 overexpression on expressions of iNOS and Arg-1 in the macrophages cultured in high glucose (scale bar=50 μm). **P<0.01 vs corresponding NC-OE group; ##P<0.01 vs corresponding Con group.

图6 IRG1沉默对高糖条件下巨噬细胞极化标志蛋白表达的影响

Fig.6 Immunofluorescence assay for assessing the effects of IRG1 silencing on expressions of iNOS and Arg-1 in the macrophages cultured in high glucose (scale bar=50 μm). **P<0.01 vs corresponding NC-siRNA group; #P<0.05, ##P<0.01 vs corresponding Con group.

图7 IRG1过表达或沉默对高糖条件下巨噬细胞炎症因子表达的影响

Fig.7 Western blotting for assessing the effects of IRG1 overexpression (A) or silencing (B) on expressions of IL-1β and IL-10 in the macrophages cultured in high glucose. *P<0.05, **P<0.01 vs corresponding NC-OE group (A) or NC-siRNA group (B); #P<0.05, ##P<0.01 vs corresponding Con group.

图8 IRG1过表达或沉默对高糖条件下巨噬细胞炎症因子分泌的影响

Fig.8 ELISA for detecting secretions of IL-1β and IL-10 in the supernatant of macrophages with IRG1 overexpression (A) or silencing (B) cultured in high glucose. *P<0.05, **P<0.01 vs corresponding NC-OE group (A) or NC-siRNA group (B); ##P<0.01 vs corresponding Con group.

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