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。
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-1βand 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-1βand 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-1βexpressions and IL-1βsecretion,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-1βand 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.
作者
罗维
王宇航
刘延松
王媛媛
艾磊
LUO Wei;WANG Yuhang;LIU Yansong;WANG Yuanyuan;AI Lei(School of Sport and Health,Nanjing Sport Institute,Nanjing 210014,China;Jiangsu Research Institute of Sports Science,Nanjing 210033,China)
出处
《南方医科大学学报》
北大核心
2025年第1期1-9,共9页
Journal of Southern Medical University
基金
国家自然科学基金项目青年科学基金(32200944)
江苏省高校“青蓝工程”
江苏省体育科学研究所基金项目(BM2023-03)。
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
macrophages
M1 polarization
inflammatory cytokines
high glucose condition
immune-responsive gene 1
作者简介
罗维,博士,副教授,E-mail:wei.luo@nsi.edu.cn;通信作者:艾磊,博士,副研究员,E-mail:ailei_982@163.com。
