Stanca Loredana, Geicu Ovidiu Ionut, Serban Andreea Iren, Dinischiotu Anca
Preclinical Sciences Department, Faculty of Veterinary Medicine, University of Agronomical Sciences and Veterinary Medicine Bucharest, 105 Splaiul Independentei, 050097 Bucharest, Romania.
Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, 91-95 Splaiul Independentei, 050095 Bucharest, Romania.
Materials (Basel). 2023 Jul 19;16(14):5083. doi: 10.3390/ma16145083.
Quantum dots (QDs) with photostable fluorescence are recommended for imaging applications; however, their effect on living cells is incompletely understood. We aimed to elucidate the RAW 264.7 murine macrophage cell line's response to the Si/SiO QDs challenge. Cells were exposed to 5 and 15 μg/mL Si/SiO QDs for 6 h, 12 h, and 24 h. Cell metabolic activity and viability were assessed by MTT, live/dead, and dye-exclusion assays. Oxidative stress and membrane integrity were assessed by anion superoxide, malondialdehyde, and lactate dehydrogenase activity evaluations. Antioxidative enzyme activities were analyzed by kinetic spectrophotometric methods. Cytokines were analyzed with an antibody-based magnetic bead assay, PGE2 was assessed by ELISA, and Nrf-2, Bcl-2, Beclin 1, and the HSPs were analyzed by western blot. Autophagy levels were highlighted by fluorescence microscopy. The average IC50 dose for 6, 12, and 24 h was 16.1 ± 0.7 μg/mL. Although glutathione S-transferase and catalase were still upregulated after 24 h, superoxide dismutase was inhibited, which together allowed the gradual increase of malondialdehyde, anion superoxide, nitric oxide, and the loss of membrane integrity. G-CSF, IL-6, TNF-α, MIP-1β, MCP-1, Nrf-2, PGE2, and RANTES levels, as well as autophagy processes, were increased at all time intervals, as opposed to caspase 1 activity, COX-2, HSP60, and HSP70, which were only upregulated at the 6-h exposure interval. These results underscore that Si/SiO QDs possess significant immunotoxic effects on the RAW 264.7 macrophage cell line and stress the importance of developing effective strategies to mitigate their adverse impact.
具有光稳定荧光的量子点（QDs）被推荐用于成像应用；然而，它们对活细胞的影响尚未完全了解。我们旨在阐明RAW 264.7小鼠巨噬细胞系对Si/SiO量子点攻击的反应。将细胞暴露于5和15μg/mL的Si/SiO量子点中6小时、12小时和24小时。通过MTT、活/死和染料排除试验评估细胞代谢活性和活力。通过阴离子超氧化物、丙二醛和乳酸脱氢酶活性评估来评估氧化应激和膜完整性。通过动力学分光光度法分析抗氧化酶活性。用基于抗体的磁珠分析法分析细胞因子，通过ELISA评估PGE2，并通过蛋白质免疫印迹法分析Nrf-2、Bcl-2、Beclin 1和热休克蛋白。通过荧光显微镜突出显示自噬水平。6小时、12小时和24小时的平均半数抑制浓度（IC50）剂量为16.1±0.7μg/mL。尽管24小时后谷胱甘肽S-转移酶和过氧化氢酶仍上调，但超氧化物歧化酶受到抑制，这共同导致丙二醛、阴离子超氧化物、一氧化氮逐渐增加以及膜完整性丧失。在所有时间间隔内，粒细胞集落刺激因子（G-CSF）、白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、巨噬细胞炎性蛋白-1β（MIP-1β）、单核细胞趋化蛋白-1（MCP-1）、Nrf-2、PGE2和调节激活正常T细胞表达和分泌因子（RANTES）水平以及自噬过程均增加，而半胱天冬酶1活性、环氧化酶-2（COX-2）、热休克蛋白60（HSP60）和热休克蛋白70（HSP70）仅在暴露6小时时上调。这些结果强调Si/SiO量子点对RAW 264.7巨噬细胞系具有显著的免疫毒性作用，并强调制定有效策略减轻其不利影响的重要性。
