ABSTRACT
OBJECTIVE: To explore the molecular mechanism of curcumin in inhibiting the nucleotide-binding oligomerization domain like receptor family pyrin domain-containing(NLRP3) inflammatory bodies induced by silica(SiO_2) in mouse alveolar macrophages(AM). METHODS: AMs were isolated from the bronchoalveolar lavage fluid of specific pathogen free C57 BL/6 mice and divided into 6 groups. Among them, the AM of the control group received no stimulation; the AM in the SiO_2 stimulation group was stimulated with SiO_2 suspension at the final mass concentration of 50 mg/L; the AM in nuclear factor(NF-κB)inhibition group was pretreated with 5-(4-fluorophenyl)-2-urea-thiophene-3-formamide with a final concentration of 200 nmoL/L for 1 hour, the AM in the low-, medium-and high-dose curcumin groups were pretreated with curcumin with the final concentrations of 20, 40 and 50 μmol/L for 1 hour, respectively, and then stimulated with SiO_(2 )suspension with a final concentration of 50 mg/L. Samples were collected after 6 hours of incubation. The mRNA expression of NLRP3 inflammasome related genes such as NLRP3, Caspase-1 and interleukin(IL)-1β was detected by real-time fluorescence quantitative polymerase chain reaction. The secretion level of maturation IL-1β(mIL-1β) and IL-18 in AM was detected by enzyme-linked immunosorbent assay. The protein expression and secretion level of cleaved Caspase-1, precursor-IL-1β(pro-IL-1β) and mIL-1β were analyzed by Western blotting. RESULTS: The mRNA relative expression of NLRP3, Caspase-1 and IL-1β, and the secretion levels of mIL-1β and IL-18, and the protein relative expression of Caspase-1, pro-IL-1β and mIL-1β, as well as the secretion levels of cleaved Caspase-1 and mIL-1β increased in the SiO_2 stimulated group compared with the control group(P<0.05). Except for the relative expression and the secretion level of cleaved Caspase-1, the other 8 indexes in the NF-κB inhibition group were lower than that in the SiO_2 stimulation group(P<0.05). Except for the relative expression of cleaved Caspase-1 and mIL-1β proteins in the low-dose curcumin group, the relative expression of all the above 10 indexes was lower in the three curcumin treated groups than that in the SiO_2 stimulation group(P<0.05). In addition, all the above indexes decreased with the increase of curcumin intervention dose(P<0.05). The mRNA relative expression of NLRP3 and IL-1β, and the protein relative expression of pro-IL-1β increased in the medium-dose curcumin group(P<0.05), the secretion levels of mIL-1β and IL-18, as well as the protein relative expression and secretion levels of cleaved Caspase-1 and mIL-1β decreased(P<0.05), compared with the NF-κB inhibition group. CONCLUSION: Curcumin can inhibit SiO_2-induced AM NLRP3 inflammasome activation in a dose-response relationship. This process may be related to the inhibition of NF-κB signaling pathway by curcumin and the down-regulating NLRP3 inflammasome-related genes at the transcriptional level. The important mechanism may be that curcumin directly blocks the activation, assembly, and downstream shearing of NLRP3 in inflammasomes.
ABSTRACT
Objective@#To observe the repairing effect of adipose mesenchymal stem cells (ADSCs) on lung injury induced by silica in rats.@*Methods@#Primary ADSCs-GFP was obtained from rats. ADSCs-GFP was injected into tail vein of silicosis model rats. The expression of green fluorescence in lungs was observed regularly to determine the homing ability of ADSCs. Primary ADSCs of rats were obtained and randomly divided into control group, exposure group, vehicle group and ADSCs group. Silicosis rat model was established by non-exposed tracheal drip method. 24 hours after silica exposure, rats in ADSCs group were injected with ADSCs of 1×106/kg body weight through tail vein, and the pathological changes of lung tissue were observed and evaluated 28 days after intervention. To explore the early intervention mechanism of ADSCs on pulmonary fibrosis in silicosis model rats, apoptosis-related proteins were detected by immunohistochemistry.@*Results@#28 days after exposure to silica, rats in the exposure group showed obvious pulmonary fibrosis. Compared with exposure group and vehicle group, ADSCs group showed less pulmonary inflammation, less silica nodules and less collagen deposition area. Immunohistochemical results showed that the expression of Caspase-3 and cytochrome C protein decreased and Bcl-2 protein increased after ADSCs transplantation.@*Conclusion@#ADSCs infusion has an obvious intervention effect on postponing early silicosis fibrosis in rats exposed to silica, and its mechanism is related to the regulation of apoptotic process.
ABSTRACT
OBJECTIVE: To analyze the difference in gene expression profiles and screen silicosis development related differentially expressed genes(DEGs) and signaling pathways in peripheral blood specimens of rats exposed to silica dust using bioinformatics and gene chip. METHODS: GSE27023 gene expression profiles of peripheral blood samples of rats exposed to silica dust were downloaded from Gene Expression Omnibus database. After screening of the DEGs through paired-sample t-test and fold change method,DEGs related to rats exposed to silica dust was performed by Gene Ontology functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. The protein-protein interaction(PPI) network was constructed with the Search Tool for the Retrieval of Interacting Genes database and visualized using the software Cytoscape. RESULTS: Of the 2 767 DEGs screened,1 363 were up-regulated and1 404 were down-regulated. KEGG pathway enrichment analysis showed that 39 signaling pathways,such as the calcium signaling pathway,neuroactive ligand-receptor interaction,Ras-related protein 1 signaling pathway,were significantly enriched. DEGs enrichment and significance reached the highest level in the calcium signaling pathway. PPI network and module analysis suggested that mitogen-activated protein kinase 14(Mapk14),DNA-directed RNA PolymeraseⅡ,V-erb-a erythroblastic leukemia viral oncogene homolog 4,B-cell lymphoma-2(Bcl-2),receptor interacting serine/threonine kinase 4,PH domain leucine-rich repeat protein phosphatase 2,DNA-directed RNA polymerase Ⅲ,neurogenic locus notch homolog protein 1,histone deacetylase 1,yeast switch in mating type/Sucrose non fermentation related,matrix associated,actin dependent regulator of chromatin,subfamily a,member 4 were top 10 hub-proteins. Mapk14 had the highest node degree in up-regulated DEGs,and Bcl-2 had the highest node degree in down-regulated DEGs. CONCLUSION: We found 39 signaling pathways and 10 DEGs that are related to toxicity caused by exposure to silica dust in rats by analysis of peripheral blood gene chip data. Among them,calcium signaling pathway,Mapk14 and Bcl-2 may play important roles in the development and progress of silicosis in rats.