High glucose induces pro-inflammatory polarization of macrophages by inhibiting immune-responsive gene 1 expression

doi:10.12122/j.issn.1673-4254.2025.01.01

Abstract

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

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.

Figures/Tables 8

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.

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).

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.

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.

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.

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.

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.

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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