The longer the worse: a combined proteomic and targeted study of the long-term versus short-term effects of silver nanoparticles on macrophages

Despite considerable research effort devoted to the study of the effects of silver nanoparticles on mammalian cells in recent years, data on the potential long terms effects of this nanomaterial remain scarce, and centered on epithelial cells. The aim of this study was to explore the effects of silver nanoparticles on macrophages. To this end, RAW 264.7 murine macrophages were exposed to either 1 µg/ml silver nanoparticles for 20 days, i.e. a chronic exposure scheme, or to 20 µg/ml silver nanoparticles for 24 hours, i.e. an acute exposure scheme. A proteomic study was then conducted to study and compare the cellular responses to both exposure schemes. They proved to be essentially different, and stronger for the chronic exposure scheme. Targeted validation studies showed effects of chronic exposure to silver nanoparticles on detoxifying enzymes such as biliverdin reductase B, which was increased, and on central metabolism enzymes such as triose phosphate isomerase, which activity decreased under chronic exposure to silver nanoparticles. Chronic exposure to silver nanoparticles also induced a decrease of reduced glutathione content, a decreased phagocytic activity and reduced macrophages responses to lipopolysaccharide, as exemplified by nitric oxide and interleukin 6 production. Overall, chronic exposure to silver nanoparticles induced stronger effects than acute exposure on macrophages in the metabolic (glutathione level, mitochondrial potential) and functional (phagocytosis, cytokine production) parameters tested.

尽管近年来针对银纳米颗粒（silver nanoparticles）对哺乳动物细胞的影响已开展了大量研究，但有关该纳米材料潜在长期效应的数据仍较为稀缺，且现有研究多聚焦于上皮细胞。本研究旨在探究银纳米颗粒对巨噬细胞的影响。为此，将RAW 264.7小鼠巨噬细胞分别施以两种暴露方案：以1 µg/ml浓度的银纳米颗粒进行20天慢性暴露，或以20 µg/ml浓度的银纳米颗粒进行24小时急性暴露。随后开展蛋白质组学研究，分析并对比两种暴露方案下的细胞应答反应。结果证实二者的细胞应答存在本质差异，且慢性暴露组的应答更为强烈。靶向验证实验显示，慢性暴露于银纳米颗粒会影响两类酶的功能：一类是解毒酶，如胆绿素还原酶B（biliverdin reductase B），其表达量上调；另一类是中枢代谢酶，如磷酸丙糖异构酶（triose phosphate isomerase），其活性在慢性银纳米颗粒暴露后降低。此外，慢性银纳米颗粒暴露还可诱导还原型谷胱甘肽（reduced glutathione）含量下降、吞噬活性降低，以及巨噬细胞对脂多糖（lipopolysaccharide）的应答减弱，具体体现为一氧化氮（nitric oxide）与白细胞介素6（interleukin 6）的生成量减少。综上，在所检测的代谢（还原型谷胱甘肽水平、线粒体膜电位）与功能（吞噬作用、细胞因子生成）参数中，银纳米颗粒的慢性暴露对巨噬细胞产生的影响均强于急性暴露。