| Particulate matter (PM) pollution is a serious air pollution, and it is one of the important indicators of evaluation of atmospheric pollution. Therefore, the WHO recommended PM2.5 (particulate matter 2.5, with an aerodynamic diameter?2.5 ?m of the concentration of particulate matter) as a monitor of air quality index. As diameter is small, PM2.5 has a larger specific surface area, and keep longer time at the atmosphere. Therefore, more PM2.5 enters the body with breath and the harm of PM2.5 to human respiratory system is more and more concerned. This research firstly detected the effects of PM2.5 exposure on transcriptomic expression profile of HBE cells. In addition, in vitro and in vivo studies on the toxic effects of PM2.5 exposure were taken to explore the toxic effect of PM2.5 exposure, and provide theoretical basis for further research.1. Effects of PM2.5 exposure on transcriptomic expression profile of HBE cellsTo study the effect of PM2.5 exposure on HBE, firstly, transcriptomic expression profile of HBE cells was detected. Agilent mRNA single-channel expression profiling microarray were used to detect the effects of PM2.5 on mRNA expression profiling of HBE cells after exposed to 100 ?g/ml PM2.5 for 24 h. To analyze the data of expression profile microarray, first of all, we screened differentially expressed genes (DEGs) after PM2.5 treatment. Then, the Gene Ontology (GO) analysis was conducted by Visualization and Integrated Discovery (DAVID) 6.7. The results of present study indicated that there were 96 DEGs, including 75 up-regulated genes and 21 down-regulated genes compared to the control group. Five down-regulated genes (ATP5E, NDUFB1, NDUFB2, NDUFC1, and UQCR10) were founded from top 10 of GO Terms in BP, which related to mitochondria, by using mRNA microarray analysis. Therefore, PM2.5 exposure could induce changes in the mRNA expression profile of HBE cells, and the DEGs were related to oxidative phosphorylation. The results showed exposure of PM2.5 may cause the deterioration of mitochondrial factions.2. The toxic effects of PM2.5 on HBE cellsTo study the toxic effects of PM2.5 on HBE cells in vitro, we took the following experiments:(1) We used qRT-PCR to verify the results of microarray analysis. The trends of down-regulation of ATP5E, NDUFB1, NDUFB2, NDUFCl and UQCR10 genes in qRT-PCR assay were similar to those in the results of mRNA microarray analysis. Very good agreement was observed between the microarray data and qRT-PCR data.(2) Western Blot was used to measure the expressions of NDUFB1 and NDUFB2 in protein level. Western blot results showed that compared with the control group, the expression levels of NUDFB1 in the PM2.5 treated groups were lower. Meanwhile, there was no difference between the expression levels of NUDFB2.(3) Cell Counting Kit 8 (CCK-8) assay was used to evaluate the cell viability of HBE cells exposed to different concentrations (12.5,25,50,100,250,500,1000 ?g/ml) of PM2.5. The results showed that after 24 h,48 h and 72 h of incubation, the cell viability of cells exposed to different concentrations of PM2.5 all decreased compared to the control(P<0.05).(4) Cell Cycle and apoptotic behavior of HBE cells exposed to different concentrations (100,500 ?g/ml) of PM2.5 were examined by flow cytometric analysis. The result of cell cycle assay showed that, the proportion of cells in the G1 phase of the PM2.5 100 ?g/ml group was 52.38%, the PM2.5 500 ?g/ml group was 45.98% and the control group was 64.31%. As the concentrations gone up, there was a tendency to increase in the S phase cells (control group: 14.13%, PM2.5 100 ?g/ml:34.83%, PM2.5 500 ?g/ml group:40.22%). In the G2 phase, a tendency to decrease with the exposure concentration raised (control group:9.84%, PM2.5 100 ?g/ml group:12.79%, PM2.5 500 ?g/ml group:13.81%). The result of apoptosis assay showed that, total cellular apoptosis rate in the PM2.5 100 ?g/ml group (17.43%) and PM2.5 500 ?g/ml group (26.72%) was higher than the control group (0.08%).(5) JC-1 assay was used to evaluate the mitochondrial membrane potential of HBE cells exposed to different concentrations (100,500 ?g/ml) of PM2.5. The result showed that mitochondrial membrane potential in HBE cells exposed to PM2.5 decreased compared with that in the control group.(6) ATP contents were measured in HBE cells exposed to different concentrations(100, 500 ?g/ml) of PM2.5. The result showed that compared with control group, the contents of ATP in PM2.5 treated groups significantly decreased(P<0.05)(7) ROS and MDA assays were used to evaluate the oxidative stress of HBE cells exposed to different concentrations(100,500 ?g/ml) of PM2.5. ROS and MDA results showed that the contents of ROS and MDA in HBE cells exposed to PM2.5 increased compared with that in the control group (P<0.05)(8) Mitochondrial respiratory chain complex I results showed that the complex I vitality in HBE cells exposed to the PM2.5 groups decreased compared with that in the control group (P<0.05)As a result, PM2.5 exposure could decrease gene expression levels of mitochondria, ATP produces and mitochondrial respiratory chain complex I activity. Meanwhile, PM2.5 exposure could increase the produces of ROS and MDA. PM2.5 exposure could disorder oxidative phosphorylation process, and oxidative stress response, cause mitochondrial, damage. The results showed exposure of PM2.5 has cytotoxicity on HBE cells, and the target spot is mitochondria.3. Pulmonary toxicity of PM2.5 caused by intratracheal instillation in miceWith a mice model by intratracheal instillation of PM2.5, we explored the toxic effects of PM2.5 on the lungs of mice in vivo.Experimental animals were C57 mice, dividing into 3 treated groups, one control group, and each one including 6 animals. PM2.5 was administered to mice for one time by intratracheal instillation at the dose of 2 mg (50 mg/ml,40 ?L), and with the same volume of PBS for the control group. The mice were sacrificed 7,14 or 28 days after the treatment.(1) GC-TOF-MS analysis was performed to measure serum metabolome. The result showed that amino acid metabolism sugar metabolism and lipid metabolism disordered in mice of 7-day and 14-day groups, comparing with those in the mice of control group.(2) Lung tissue was preserved in 4% PFA for H&E, PAS, Masson, IHC and TUNEL staining. The result showed that athological changes of lung tissue, including hemorrhage, alveolar wall thickening, inflammatory cells infiltration and lung parenchyma, atypical hyperplasia and so on could be obviously observed in 7-day,14-day and 28-day groups, Apoptotic cells in the lung tissue of the PM2.5 treated mice were more than those in the control group (P<0.05).(3) Total mRNA of lung tissue was extracted for qRT-PCR to detect the expression levels of 4 mitochondrial genes (Atp5e, Ndufb2, Ndufcl, and Uqcr10). The expression level of Atp5e in the lung tissue of the PM2.5 treated mice in 14-day (0.5798 ± 0.1974) group was significantly decreased. In addition, there was no significant difference among the expression levels of Ndufcl in the lung tissues of the control group and the PM2.5 treated groups. The expression levels of Ndufb2 in the lung tissue of the PM2.5 treated mice in 7-day (0.7524 ± 0.1364) and 14-day (0.6893 ± 0.2892) groups were significantly lower than that in the control group. The expression level of Uqcr10 in the lung tissue of the PM2.5 treated mice in 14-day (0.4325 ± 0.1970) and 28-day (0.5260 ± 0.3690) groups were significantly lower than that in the control group (P<0.05)As a result, PM2.5 exposure by intratracheal instillation on mice could decrease mitochondrial gene expression levels of lung tissues, and lead to pathological changesin lung tissues. In addition, the metabolism of PM2.5 treated mice disordered. The results showed exposure of PM2.5 has toxic effects on mice, and the target organ is lung. |
PDF Full Text Request