Study On Acute Lung Inflammation Induced By Iron Oxide Nanoparticles In Mice

Objective:Iron oxide is the main component of the particulate matter produced by the friction of the traffic track.It mainly exists in the form of nanometers in the urban rail traffic environment and has a vital impact on human respiratory health.In this study,iron oxide nanoparticles（Fe₂O₃ NPs）with a particle size of<50 nm were used for in vitro and in vitro experiments,aiming to explore its role in causing acute lung inflammation and related mechanisms,and provide further experimental and theoretical basis for the risk assessment of particulate matter.Not only for occupational contact susceptible people,but also for the prevention and treatment of respiratory health of the general population has great practical significance.Methods:The vivo experiment adopted BALB/c male mice which were further divided into three groups,namely,control group,low,medium dose group and high dose group.Blood was collected from the apex,serum and alveolar lavage fluid after the tracheal instillation of Fe₂O₃ NPs（0.1ml）for 24 hours.For the purpose of analyzing cytokine levels in alveolar lavage fluid and serum,the experiment adopted enzyme-linked immunosorbent assay（ELISA）.In this experiment,in order to classify and count the cells in the alveolar lavage fluid,a whole blood cell analyzer was used;in order to observe the inflammatory cell infiltration and epithelial mucus secretion,this study used paraffin sections of lung tissue,HE and PAS staining,etc.technology.In order to detect the expression of epithelial cadherin and eosinophil basic protein,immunohistochemistry was used in this experiment.Through in vitro extraction of mouse RAW264.7macrophages,this experiment was divided into four groups:control group,low-dose group,middle-dose group,and high-dose group.After 12 hours,in order to detect the toxicity of macrophages,the study collected the cell supernatant,lactate dehydrogenase（LDH）,and measured the level of tumor necrosis factor-α（TNF-α）by using ELISA method to detect the cytotoxicity c.RNA and protein were extracted from the cells.In order to detect the gene and protein expression of TLR2,TLR4,My D88,TRAF6,NF-κB and TNF-α,RT-q PCR and Western experiments were performed in this experiment.After adding TLR2/TLR4 inhibitor（Ox PAPC）and My D88 inhibitor（ST2825）,the expression of downstream related proteins and the secretion of inflammatory factors were detected.The experimental results adopt SPSS19.0 to conduct statistical analysis.Results:1.According to the experimental results,it can be determined that the number of total cells,neutrophils,eosinophils and lymphocytes in the alveolar lavage fluid（P<0.05）and the exposure concentration show a significant positive correlation,while macrophages There is no obvious correlation between the number of cells and the exposure concentration.2.The levels of interleukin（IL）-5,IL-12,IL-17A,IL-33,TNF-α,thymic stromal lymphopoietin（TSLP）,keratinocyte chemical attractant（KC）,eosinophil chemotactic factor（eotaxin）and monocyte chemotactic protein（MCP）-3 in mouse alveolar lavage fluid increased significantly（P<0.05）.Serum IL-5,KC,eotaxin and MCP-3 increased significantly（P<0.05）,but TNF-αwas not detected.3.Through the analysis of HE staining,the results showed that the amount of inflammatory cell infiltration in the control group was small,while the infiltration of neutrophils,lymphocytes,and eosinophils in the treatment group increased.By analyzing the results of PAS staining,there was no apparent pathological changes detected in the control group,and the increase in exposure concentration resulted in an increase in mucus in epithelial tissue.4.By analyzing the results of immunohistochemistry,there was an apparent decrease detected in the expression of cadherin in the lung tissue of the treatment group,and an obvious increase found in the expression of eosinophil basic protein.5.In vitro experiments showed that with the increase of exposure concentration of Fe₂O₃ NPs,cell mortality increased significantly（P<0.05）,and TNF-αlevels in cell culture supernatant increased significantly（P<0.05）.6.By analyzing the results of in vitro experiments,it was found that cell death rate,cell culture supernatant TNF-αlevel and Fe₂O₃ NPs concentration showed a significant positive correlation.7.There was a great increase in the gene expression of TLR2,TLR4,My D88,TRAF6,NF-κB,and TNF-α（P<0.05）,and the expression of TLR2,TLR4,My D88,TRAF6,and NF-κB（P<0.05）without any TNF-αprotein detected.With the addition of Ox PAPC,the expression of TLR2,TLR4,My D88,TRAF6 and NF-κB showed an apparent decrease trend（P<0.05）,and with the addition of ST2825,the expression of My D88,TRAF6 and NF-κB showed an apparent decrease trend（P<0.05）,The level of TNF-αin cell supernatant was greatly reduced（P<0.05）.Conclusion:1.Fe₂O₃ NPs can destroy the lung epithelial barrier function of mice,resulting in the increase of inflammatory cells and cytokines in alveolar lavage fluid and infiltration of inflammatory cells in lung tissue,which further resulted in the acute lung inflammation and systemic inflammatory reaction in mice.2.Fe₂O₃ NPs activates TLR2/TLR4/My D88 signaling pathway,leading to cytotoxicity and inflammation of RAW264.7 macrophages.