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OBJECTIVE: To investigate the role of high mobility group protein 1(HMGB1) in toluene diisocyanate(TDI) induced nucleotide-binding oligomerization domain like receptor family pyrin domain-containing 3(NLRP3) inflammasome activation in human bronchial epithelial cells(HBECs). METHODS: i) The TDI-human serum albumin(HSA) stimulation experiment: the HBECs in logarithmic growth phase were randomly divided into control group, low-, medium-and high-dose groups that were pretreated with TDI-HSA with the final concentration of 0.00, 40.00, 80.00 and 120.00 mg/L for 12 hours. ii) The HMGB1 expression inhibition experiment: the HBECs in logarithmic growth phase were divided into control group, TDI-HSA group, TDI-HAS+negative-siRNA group, and TDI-HAS+HMGB1-siRNA group. The cells in TDI-HAS+negative-siRNA group and TDI-HAS+HMGB1-siRNA group were infected with HBECs with negative-siRNA lentivirus and HMGB1-siRNA lentivirus, respectively. Cells in these two groups and the TDI-HSA group were treated with 120.00 mg/L of TDI-HSA for 12 hours. The cells in the control group were not treated with TDI-HAS. iii) The expression of HMGB1, NLRP3, apoptosis-associated speck-like protein containing CARD(ASC), pro-caspase-1 and caspase-1 p20 proteins in all groups were detected by Western blot. The number of NLRP3 and caspase-1 inflammasome in TDI-HSA stimulation experiment was observed by immunofluorescence method. RESULTS: i) TDI-HSA stimulation experiment: the relative protein expression of HMGB1 and ASC was higher in HBECs of medium-and high-dose groups than that of control group(all P values were <0.01). The relative protein expression of NLRP3 and casepase-1 p20 and the number of NLRP3-caspase-1 inflammasome were higher in HBECs of 3 dose groups than that of control group(all P values were <0.01). The number of NLRP3-caspase-1 inflammasome in HBECs increased obviously in low-, medium-and high-dose groups as compared to the control group(all P values were <0.05). The number of NLRP3-caspase-1 inflammasome in HBECs increased with the increase of TDI-HSA dose(all P values were <0.01). ii) The HMGB1 expression inhibition experiment: the relative protein expression of HMGB1, NLRP3, ASC, pro caspase-1 and caspase-1 p20 in HBECs were higher in the TDI-HSA group and TDI-HSA + negative-siRNA group than those of the control group(all P values were <0.01). The above indexes of HBECs were lower in the TDI-HAS + HMGB1-siRNA group than those in the TDI-HSA group and TDI-HSA + negative-siRNA group(all P values were <0.01).CONCLUSION: TDI treatment in HBECS can induce the increase of HMGB1 protein expression and activate NLPR3 inflammasome. Inhibition of HMGB1 expression can down-regulate the expression of NLPR3 and its related proteins.
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OBJECTIVE: To explore the effect of reactive oxygen species(ROS) and cell cycle in bone marrow cells in benzene-induced aplastic anemia(AA) mouse model. METHODS: Specific pathogens free male CD1 mice were randomly divided into control group and exposure group(n=10, each group). The mice in exposure group were subcutaneously injected with benzene at a dose of 2 mL/kg body weigh diluted 1 ∶1 in corn oil, while the mice in control group were treated with equal volume of corn oil, 3 times a week for a total of 25 times. After exposure, the blood routine and reticulocyte percentage of peripheral blood of mice were examined. The femur histopathology was performed. The levels of benzene and its metabolites hydroquinone, and phenol in blood, liver and bone marrow were tested by solid-phase extraction gas chromatography mass spectrometry. The level of ROS and the changes of cell cycle in bone marrow mononuclear cells(BMMNCs) were determined by flow cytometry. The protein expression of Cyclin D1 and cyclin-dependent kinase 4(CDK4) in BMMNCs was detected by Western blot. RESULTS: Since the 10 th benzene exposure, the body mass of mice in the exposure group was lower than that in the control group at the same time point(P<0.05). After the benzene exposure, all the counts of white blood cell, red blood cell, platelet, and hemoglobin level and reticulocyte percentage in peripheral blood of mice in the exposure group were decreased when compared with the control group(P<0.05). Bone marrow histopathological examination showed that bone marrow hematopoietic cells were decreased and non-hematopoietic cells were increased in the exposure group. In this study, a mouse model of AA induced by benzene was successfully established. The levels of benzene, hydroquinone and phenol in exposure group increased in blood, liver, and bone marrow compared to control group(P<0.05). Furthermore, the level of benzene from high to low were blood, liver and bone marrow, while the levels of hydroquinone and phenol were mainly stored in the blood and bone marrow in exposure group. Compared with the control group, the level of ROS, S phase fraction, and the relative protein expression of Cyclin D1 and CDK4 in BMMNCs increased, while the G1 phase fraction decreased in exposure group(P<0.01). CONCLUSION: The results suggest that benzene and its metabolites induce an increase of ROS level and S phase cell arrest, that play an important role in the pathogenesis and development of benzene-induced AA.
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OBJECTIVE: To investigate the effect of toluene diisocyanate(TDI) on the activation of autophagy and expression of inflammatory cytokines interleukin(IL)-4 and IL-6 in normal human bronchial epithelial cells(16 HBE). METHODS: i) We prepared TDI-human serum albumin(HSA) and determined the mass concentration of TDI in TDI-HSA. ii) The cells were treated with TDI-HSA and HSA at concentrations of 0.00-400.00 mg/L for 12 hours. CCK-8 assay was used to determinate the cell viability, and TDI-HSA and HSA doses were selected for subsequent experiments. iii) The cells were treated with TDI-HSA and HSA at doses of 0.00-120.00 mg/L for 12 hours, and the levels of reactive oxygen species(ROS) in the cells were detected by flow cytometry. The levels of IL-4 and IL-6 in the cell supernatant were measured by enzyme-linked immunosorbent assay. iv) The cells were treated with TDI-HSA at doses of 0.00-120.00 mg/L for 12 hours, and the autophagy activity was observed under transmission electron microscope. Western blot was utilized to detect the expression of Beclin1, microtubule-associated protein 1 light chain(LC3β) and P62. RESULTS: i) The mass concentrations of TDI in 40.00, 80.00 and 120.00 mg/L TDI-HSA groups were 0.44, 0.89 and 1.33 mg/L respectively. ii) The results of CCK-8 showed that TDI-HSA and HSA at doses below 120.00 mg/L did not affect cell viability, and 0.00-120.00 mg/L was selected as the TDI-HSA and HSA treatment doses for subsequent experiments. iii) The level of ROS in cells and the levels of IL-4 and IL-6 in the supernatant of 16 HBE cells in the TDI-HSA group at 40.00, 80.00, and 120.00 mg/L were higher than that in HSA group at the same dose(P<0.01). The level of ROS in cells and the levels of IL-4 and IL-6 in the supernatant of 16 HBE cells increased with the increase of TDI-HSA doses(P<0.01). iv) Transmission electron microscopy showed that the number of autophagic lysosomes in 16 HBE cells increased significantly, and the number of mitochondrial vacuoles increased in 40.00, 80.00, 120.00 mg/L TDI-HSA group compared with 0.00 mg/L group. With the increase of TDI-HSA dose, the relative expression of Beclin1 protein and LC3β-Ⅱ/Ⅰ ratio in 16 HBE cell supernatant increased(P<0.05), and the relative expression of P62 protein decreased(P<0.05). CONCLUSION: TDI-HSA induces increased expression of ROS and inflammatory factors and induces autophagy activation in 16 HBE cells. Autophagy may be an important factor for the development of airway inflammation in TDI-induced occupational asthma.
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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.
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Objective@#To explore the changes in the autophagy marker microtubule-associated protein 1 light chain 3 (LC3) and yeast autophagy-related gene 6 (Beclin1) in rat lungs exposed to free silica (SiO2) dust for different periods.@*Methods@#A total of 72 male specific pathogen-free Wistar rats were randomly divided into solvent control group and SiO2 model group. The SiO2 model group received one-time non-exposed intratracheal instillation of suspension of SiO2 particles to establish a model of silicosis. The solvent control group received an equal amount of saline. Six rats each were sacrificed at 1, 7, 14, 21, 28, and 60 days after model establishment. The pathological changes and fibrosis of rat lungs at different time points were evaluated by H&E staining and Masson staining, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of transforming growth factor-β (TGF-β) , interleukin-1 (IL-1) , and tumor necrosis factor-α (TNF-α) in lung tissue homogenate. Western blot was used to determine the relative expression levels of LC3 and Beclin1 in the lung tissue.@*Results@#The results of H&E staining showed that the model group had continuous inflammation in the lung tissue from day 1 to day 60, and the inflammatory scores were significantly higher in the model group than in the control group (P<0.05) . The results of Masson staining showed that rats in the model group had a small amount of collagen fibers in the lung tissue on day 14 and a large amount of collagen fibers on day 60. The fibrosis score was significantly higher in the model group than in the control group (P<0.05) . No collagen fibrosis was observed in the lung tissue in the control group. The results of ELISA showed that the model group had significantly higher levels of IL-1, TNF-α, and TGF-β in lung tissue homogenate than the control group at each time point after exposure (P<0.05) . The results of Western blot showed that the model group had decreased expression of Beclin1 protein in the lung tissue on days 7, 14, 21, 28, and 60, which was significantly higher than that of the control group (P<0.05) . The model group also had a decreased ratio of LC3II/LC3I on days 1, 7, 14, 21, 28, and 60, which were significantly higher than that of the control group (P<0.05) .@*Conclusion@#In the rat model of silicosis induced by free SiO2 dust, the expression levels of autophagy-related proteins, LC3 and Beclin1, are correlated with different stages of silicosis. In the early stage of silicosis, the lung tissue has inflammation, substantially increased ratio of LC3II/LC3I and expression of Beclin1, and active autophagy. With the progression of silicosis, the ratio of LC3II/LC3I and expression level of Beclin1 gradually decrease and autophagy becomes weak.
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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.
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OBJECTIVE: To explore the changes of retinoic acid related orphan receptor-γt( ROR-γt),interleukin( IL)-17 A and forkhead / winged helix protein 3( Foxp3) mRNA expression and promoter methylation in the process of asthma induced by toluene-diisocyanate( TDI). METHODS: Specific pathogens free grade healthy male BALB / c mice were randomly assigned into asthma group and control group with 18 animals in each group. In the asthma group,the mice were sensitized with 0. 30% TDI( mass-volume concentration) dropped on the dorsum of both ears( 20 μL / ear) on day 1 and day 8. On day 15,the mice were challenged with 20 μL 0. 01% TDI( mass-volume concentration) by the trachea. The control group mice were sensitized and challenged by the same procedures with the same amount of solvent( acetone / olive oil). The mice were challenged 24 hours,the pathological changes of trachea and lung tissues were observed. The bronchoalveolar lavage fluid( BALF) from each group was collected,and the inflammatory cells in BALF were counted and classified. IL-4and Interferon-γ( IFN-γ) levels in BALF supernatant were measured by enzyme-linked immunosorbent assay. ROR-γt,IL-17 A and Foxp3 mRNA expression in the lung tissue were measured by real-time fluorescent quantitative polymease chain reaction. The degree of ROR-γt,IL-17 A and Foxp3 promoter methylation in lung issue were detected by Mass Array system.RESULTS: The asthmatic group demonstrated the symptoms of acute asthma,such as breathing deeply and fastly,bowing the back,lifting the forelimbs,et al. But the control group had no such symptoms in mice. Hematoxylin-Eosin staining showed obvious inflammatory lesions in the trachea and lung tissue of asthmatic mice. Compared with the control group,the white blood cell count,the neutrophil and eosinophil percentages in BALF,the IL-4,IFN-γ levels in BALF supernatant in asthma group were all significantly increased( P < 0. 01),meanwhile the lymphocyte and monocyte percentages in BALF were reduced( P < 0. 01); ROR-γt mRNA expression was significantly increased( P < 0. 01),and the degree of promoter methylation from sites 3,4,5,6,8,11 and 12 was significantly reduced( P < 0. 05); IL-17 A mRNA expression was significantly increased( P < 0. 01),and the degree of promoter methylation from sites 6 and 7 was significantly reduced( P < 0. 01); Foxp3 mRNA expression was significantly reduced( P < 0. 01),and the degree of promoter methylation from sites 1 and 10 was significantly increased( P < 0. 01). CONCLUSION: Th17 / Treg cell immune imbalance occurs in asthma induced by TDI. ROR-γt,IL-17 A and Foxp3 gene promoter methylation abnormalities may be involved in Th17 / Treg cell immune imbalance.
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<p><b>OBJECTIVE</b>To investigate the effects of malathion on the testicular spermatogenic function of male rats and its working mechanism.</p><p><b>METHODS</b>Forty specific pathogen-free male Wistar rats were randomly and equally divided into four groups: three exposure groups and a control group. Malathion was administered orally to male rats in the exposure groups at 33.75, 54, and 108 mg/kg (1/32 LD₅₀, 1/20 LD₅₀, and 1/10 LD₅₀) for 60 days. Rats in the control group received an equal volume of water. The body weights of rats were measured after exposure. The organ weights and coefficients of the testes and epididymes were determined as soon as rats were sacrificed. The sperm motility, counts, and malformation rates were measured in the left epididymis. Histopathological changes, cell apoptosis, and the expression levels of Bcl-2/Bax in the testes of rats were observed using HE staining, terminal deoxynucleotidyl transferase-mediated dUPT-biotin nick end labeling, and immunohistochemistry SABC method.</p><p><b>RESULTS</b>The body weights and the testis weights in the exposure groups were significantly lower than those in the control group (P < 0.01). The exposure groups had significantly lower sperm motility and significantly higher sperm malformation rates than the control group (P < 0.01). The sperm counts were significantly lower in the exposure groups than in the control group (P<0.01). The sperm counts and motility were negatively correlated with exposure dose (r = -0.81, P < 0.01; r = -0.51, P < 0.01), while the sperm malformation rate was positively correlated with exposure dose (r = 0.85, P 0.01). The exposure groups had significantly higher spermatogenic cell apoptosis rates than the control group (P<0.01). The expression level of Bax was significantly higher in the exposure groups than in the control group (P<0.01), while the expression level of Bcl-2 was significantly lower in the exposure groups than in the control group (P < 0.01). Histopathological examination of the testes showed degenerative changes in the seminiferous tubules at various doses along with the increase in malathion exposure dose.</p><p><b>CONCLUSION</b>Malathion affects the testicular spermatogenic function of male rats and its working mechanism may involve cell apoptosis induced by down-regulation of Bcl-2 and up-regulation of Bax.</p>