Abstract:
Background
Macrophages are essential components of the natural immune system. They play a significant role in resisting foreign bodies in the respiratory tract and maintaining the homeostasis of the internal environment of lung tissue.
Objective
To investigate the mechanism of macrophage pyroptosis induced by silica dust with different particle sizes.
Methods
The modified murine macrophage cell line, RAW-ASC cells, was cultured and divided into a blank control group, a lipopolysaccharide (LPS) group (1 μg·mL−1 LPS), a nano-SiO2 group (1 μg·mL−1 LPS+100 μg·mL−1 nano-SiO2), a micro-SiO2 group (1 μg·mL−1 LPS+750 μg·mL−1 micro-SiO2), and a positive control group 1 μg·mL−1 LPS+3 mmol·L−1 adenosine triphosphate (ATP). Apart from the blank control group, cells in other groups were pretreated with LPS for 6 h, and then exposed to SiO2 or ATP for 4 h. According to the molecular target NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and reactive oxygen species (ROS), we applied MCC950 (NLRP3 inhibitor) and N-acetyl cysteine (NAC, ROS scavenger) to macrophages. CCK-8 assay was used to detect cell viability; 5-ethynyl-2'-deoxyuridine (EdU) staining was used to detect cell proliferation; lactate dehydrogenase (LDH) assay kit was used to detect LDH in supernatant; calcein AM/PI fluorescent double-staining was applied to evaluate cell rupture; 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescent probe was used to measure the content of ROS; Western blotting was used to measure the expressions of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), Caspase-1, gasdermin D (GSDMD), and interleukin-1β (IL-1β).
Results
Compared with the blank group, 100 μg·mL-1nano-SiO2and 750 μg·mL-1micro-SiO2 dust exposure reduced the cell viability to 40% and 68% (P<0.05), and the cell proliferation rate to 30% and 33% (P<0.01), respectively; they also induced cell lysis and ROS release, upregulated NLRP3, ASC, Caspase-1, GSDMD, and IL-1β at protein level (P<0.05), and induced macrophage pyroptosis. After intervening with MCC950 (10 μmol·L-1) and NAC (10 mmol·L-1), the expressions of NLRP3, ASC, Caspase-1, and IL-1β decreased (P<0.05), and, specifically, NAC effectively reduced ROS levels (P<0.05).
Conclusion
Both nano- and micro-SiO2 dust have cytotoxicity, can upregulate ROS level, activate NLRP3 inflammasome, and promote the release of cytokines, leading to pyroptosis. These results are helpful to reveal the molecular mechanism of macrophage pyroptosis induced by SiO2 dust.
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