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-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.
ABSTRACT
OBJECTIVE: To detect the expression of differential expression genes(DEGs) on microarray chips of macrophages exposed to nano-silicon dioxide(SiO_2) dust, and to screen the leading signaling pathway of nano-SiO_2 dust exposure-related diseases. METHODS: The gene chip GSE13005 of RAW264.7 macrophage intervened by nano-SiO_2 dust was obtained from the public gene chip database developed by the National Center for Biotechnology Information. The macrophages in the control group were cultured in complete medium without adding SiO_2 dust, whereas the macrophages in the exposure group were treated with SiO_2 dust with the final concentrations of 5, 20, and 50 mg/L. The gene expression data of macrophages was analyzed by RMA Express 1.2.0 software and R language 3.5.1. The Kyoto Encyclopedia of Genes and Genomes(KEGG) was used to screen DEGs and perform gene ontology(GO) enrichment analysis on related genes and signaling pathways. RESULTS: A total of 67 DEGs of macrophages were screened after SiO_2 dust treatment, of which 48 genes were up-regulated and 19 genes were down-regulated. GO enrichment analysis results showed that the main functional items of participating DEGs were reaction of amine, regulation of viral genome replication,negative regulation of amino acid transport, ovulation, bronchodilator response, chemokine activity, negative regulation of muscle cell differentiation, response to lack of amino acid, positive regulation of glomerular mesangial cell proliferation, and positive regulation of vascular smooth muscle cell proliferation. KEGG signaling pathway analysis results suggested that DEGs could function through 7 signaling pathways including nuclear transcription factor-κB(NF-κB) signaling pathway, p53 signaling pathway, glioma, melanoma, toll-like receptor signaling pathway, renal cell carcinoma and salmonella infection. Further functional enrichment revealed that NF-κB signaling pathway changed most significantly after macrophages were exposed to nano-SiO_2 dust. CONCLUSION: Exposure to nano-SiO_2 could induce the abnormal expression of 67 genes in macrophages. The genes that participated in macrophage activation process induced by nano-SiO_2 dust exposure are related to NF kappa B signaling pathway.