Study On Immune Organ Injury Induced By Fine Particles From Subway Platform In Mice

Objective:Urban subway system was a relatively closed environment,with large crowds and limited natural ventilation,and high-speed trains produce large amounts of fine particulate matter(PM_2.5)that threaten human health.In the present study,subway PM_2.5was used to conduct experiments on mice,aiming to explore the role in causing immune organ damage and related mechanisms,providing experimental and theoretical basis for the risk assessment of subway PM_2.5,and also providing important clues for exploring markers of subacute immune system damage.It is of great practical significance to prompt relevant departments to improve the air quality of subway platforms and passengers to strengthen their awareness of self-protection.Methods:The content of chemical elements and polycyclic aromatic hydrocarbons in subway PM_2.5and atmospheric PM_2.5were analyzed.In the dose-effect relationship experiment,BALB/c male mice were divided into control group,30μg/mouse,100μg/mouse,and 300μg/mouse of subway PM_2.5group.The mice were intratracheally administrated subway PM_2.5suspensions of various concentrations for 4 times at one-week interval.One day after the last intratracheal administration,blood was collected from the heart and serum was separated.Subway PM_2.5was observed by transmission electron microscopy in spleen and thymus of mice.The levels of cytokines were analyzed in serum by enzyme linked immunosorbent assay.The pathological changes were observed in spleen and thymus by HE staining.The infiltration degree of neutrophil and macrophage were detected by immunohistochemistry.T cells and their subsets were detected by flow cytometry.Tissue RNA and protein were extracted,and RT-qPCR and Western were performed.The mRNA and protein expression of toll-like receptor（TLR）-2,TLR4,myeloid differentiation factor 88（MyD88）,nuclear factor（NF）-κB,tumor necrosis factor（TNF）-α,interleukin（IL）-6,IL-1βand monocyte chemotactic protein（MCP）-1 were detected.Wild type（WT）C57BL/6 mice and Rag1^-/-（C57BL/6 background）mice were used to explore the role of T cells,B cells and ILC2 in inflammatory damage of immune organs.SPSS 25.0 was used for statistical analysis of the data.Results:1.Subway PM_2.5resulted in increased levels of TNF-αand IL-6 in serum of mice（P<0.05）.2.The subway PM_2.5were observed in phagocytic bodies of macrophages of spleen and thymus by transmission electron microscopy.3.The weight change of mice in subway PM_2.5groups was significantly lower than that in control group（P<0.01）;Spleen coefficient of mice had no significant change,and thymus coefficient of mice in subway PM_2.5group was significantly decreased（P<0.001）.4.HE staining results showed that the tissue structure of the control group was normal,while different degrees of bleeding,fibrosis,macrophage and lymphocyte infiltration were observed in the tissues of the subway PM_2.5groups.5.Immunohistochemical results showed that the infiltration degree of neutrophils and macrophages in subway PM_2.5groups were significantly increased（P<0.05）.6.Flow cytometry showed that the levels of CD3⁺,CD4⁺and CD4⁺/CD8⁺in subway PM_2.5were decreased in the spleen of mice,while the level of CD8⁺cells was increased（P<0.05）.The levels of CD3⁺,CD4⁺and CD8⁺cells in subway PM_2.5groups were decreased in thymus of mice,while the level of CD4⁺CD8⁺cells was increased（P<0.05）.7.The mRNA and protein expressions of TLR2,TLR4,MyD88,NF-κB,TNF-α,IL-6,IL-1βand MCP-1 in subway PM_2.5groups were increased in a dose-dependent manner in immune organs of mice（P<0.05）.8.Compared with WT mice,the contents of TNF-αand IL-6 in subway PM_2.5groups were significantly decreased in serum of Rag1^-/-mice（P<0.05）.9.After PM_2.5and IL-33treatment,the thymus coefficient of Rag1^-/-mice was significantly lower than that of WT mice（P<0.001）.10.Pathological changes and inflammatory cell infiltration in the subway PM_2.5groups were significantly reduced in immune organs of Rag1^-/-mice compared with WT mice.11.The mRNA and protein expression levels of TNF-α,IL-6,IL-1βand MCP-1 in subway PM_2.5and IL-33 groups were significantly decreased in the immune organs of Rag1^-/-mice compared with those of WT mice（P<0.05）.Conclusion:1.Subway PM_2.5could enter thymus and spleen,induced pathological changes in tissue structure,inflammatory cell infiltration and T lymphocyte subgroup toxicity,and increased secretion of inflammatory cytokines in tissue and serum,leading to inflammation of thymus and spleen and systemic inflammatory response of mice.2.The TLR2/TLR4-MyD88 signaling pathway was involved in the inflammation induced by subway PM_2.5.3.The damage of thymus and spleen in mice caused by subway PM_2.5depends on T cells and B cells,but not ILC2.