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Interplay of Oxidative Stress, Inflammation, and Autophagy in RAW 264.7 Murine Macrophage Cell Line Challenged with
Loredana Stanca1, Ovidiu Ionut Geicu1, Andreea Iren Serban1
1Preclinical Sciences Department, Faculty of Veterinary Medicine, University of Agronomical Sciences and Veterinary Medicine Bucharest, 105 Splaiul Independentei, 050097 Bucharest, Romania.
Abstract:
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/SiO2 QDs challenge. Cells were exposed to 5 and 15 μg/mL Si/SiO2 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/SiO2 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.
Insights
Silicon/silicon dioxide quantum dots (Si/SiO2 QDs) induce significant immunotoxicity in RAW 264.7 macrophages, increasing oxidative stress and inflammatory responses. Further research is needed to develop strategies mitigating these adverse effects in biological applications.
Area of Science:
- Nanotechnology
- Cell Biology
- Immunotoxicology
Background:
- Photostable quantum dots (QDs) are useful for imaging but their impact on living cells remains unclear.
- Understanding cellular responses to nanomaterials is crucial for safe application.
Purpose of the Study:
- To investigate the effects of Si/SiO2 QDs on RAW 264.7 murine macrophage cell line.
- To assess cellular viability, oxidative stress, membrane integrity, and inflammatory markers following QD exposure.
Main Methods:
- Exposure of RAW 264.7 cells to varying concentrations and time points of Si/SiO2 QDs.
- Assays for cell viability (MTT, live/dead), oxidative stress (superoxide, MDA), membrane integrity (LDH).
- Analysis of antioxidant enzymes, cytokines (ELISA, magnetic bead assay), and protein expression (Western blot).
Main Results:
- Si/SiO2 QDs demonstrated immunotoxic effects, with an average IC50 of 16.1 μg/mL.
- Exposure led to increased oxidative stress, elevated pro-inflammatory cytokines (TNF-α, IL-6), and enhanced autophagy.
- Inhibition of superoxide dismutase and increased malondialdehyde indicated oxidative damage, while some antioxidant enzymes were upregulated.
Conclusions:
- Si/SiO2 QDs induce significant immunotoxicity in macrophages, impacting cellular functions and inflammatory pathways.
- The findings highlight the need for strategies to mitigate adverse effects of Si/SiO2 QDs in biological systems.
- Further investigation into QD-cell interactions is essential for their safe and effective use in biomedical applications.
