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-SiO2 and 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.