Toxic Effect Of PM2.5 On Macrophage RAW264.7 And Its Mechanism

PM_2.5 refers to fine particles with an aerodynamic diameter?2.5?m in ambient air,which can be suspended in air for a long time.Because of its small particle size,large specific surface area,and strong activity,it is easy to attach toxic and harmful substances.It is easy to be inhaled and precipitated into the alveoli.It has a great impact on the lungs and harms human health.Prolonged exposure to PM_2.5 can harm the human respiratory system and cause related diseases.Oxidative stress and apoptosis are one of the important mechanisms of PM_2.5-induced respiratory damage.After exposure to PM_2.5 cells,excess reactive oxygen species?ROS?are produced to cause oxidative damage to the cells,which in turn causes apoptosis,and more toxic effects impair the normal physiological functions of the cells,causing respiratory tract infections and tumors and other diseases.In this experiment,in vitro exposure experiments were used to investigate the toxic effects of atmospheric PM_2.5 on RAW264.7 cells in Hangzhou urban area.The specific process and results are as follows:?1?After soxhlet extraction and GC-MS determination of the content of organic components in PM_2.5,the use of mass spectrometry to detect the content of metal elements in PM_2.5 to determine the main components of particulate matter,the atmospheric PM_2.5 in Hangzhou City The main organic components are n-alkanes,PAHs and PAEs with the highest n-alkanes content.The metal elements mainly studied Al,Cr,Mn,Fe,Ni,Cu,Zn,As,Cd,Pb,with the most Zn content.It can be inferred from the experimental results that the source of PM2.5 in Hangzhou is mainly the burning of fossil fuels and the volatilization of biomass.?2?Inverted microscope,fluorescence microscope and transmission electron microscope were used to observe the cell surface,organelle damage and ROS distribution.It was found that the morphology of the exposed cells changed significantly,the cells were broken,the cell membrane edge was not obvious,and there were vacuoles in the organelles.The ROS distribution increases significantly with increasing exposure concentration.From this it can be concluded that PM_2.5 can cause changes in cell morphology and affect oxidative damage in cells.?3?The viability of the cells was measured by MTT assay.Apoptosis rate,cycle change,and mitochondrial membrane potential were measured by flow cytometry.Then the amount of NO leakage in the cells was measured and the immune-related proteins TNF-?,IL were measured by ELISA.-1,IL-6 production changes.The survival rate of cells was significantly decreased by MTT assay.When the maximum exposure concentration?800?g/mL?was reached,the cell viability was only 47%.The results of flow cytometry showed that apoptosis increased.When the exposure concentration?400?g/mL?was reached,the early apoptosis rate was 14.7%and the late apoptosis rate was 5.2%.However,the cell cycle and mitochondrial membrane potential were not observed.Significant changes occurred,the production of immune-related factors TNF-?,IL-1,and IL-6 increased,and the amount of NO leakage increased,indicating that PM_2.5 produced a greater toxic effect on cells.?4?The expression of apoptosis-related genes Bax,Bcl-2,P53 and oxidative stress-related genes Sod1,Sod2,Gpx1,Gpx2,INOs and Cat were detected by fluorescence quantitative PCR at the molecular level to determine the apoptosis of the cells.Pathways and antioxidant pathways.The expression of apoptosis gene P53 and Bax were up-regulated and Bcl-2 expression was down-regulated.The expression of oxidative stress-related genes also showed corresponding changes,and the expression was up-regulated.The results showed that the cytotoxicity of PM_2.5 on the cells had a significant effect on the molecular level,and it had a significant effect on the apoptotic pathway of P53,and the antioxidant activity was reduced.?5?In order to further search for more metabolic pathways causing cytotoxicity of PM_2.5,we used transcriptomics technology to use a large data platform to enrich pathways of altered metabolic pathways.The results showed that 20 differentially expressed genes were up-regulated.Involves PI3K-Akt signaling pathway,NOD-like receptor signaling pathway,Jak-STAT signaling pathway,TNF signaling pathway,glutathione metabolism pathway,DNA-sensing signaling pathway,mineral absorption pathway,vasoconstriction system,and oxidative stress Nitrogen metabolism pathways,NF-kappa B signaling pathways and other metabolic pathways,indicating that cells in a number of metabolic pathways affect the physiological activities of cells.In summary,increased production of ROS,reduced cell viability,increased expression of apoptosis and oxidative stress-related genes,and increased release of inflammatory factors,suggest that PM_2.5.5 has a greater cytotoxic effect on cells,and involves Synergistic effects of multiple metabolic pathways such as NOD-like receptor signaling pathway and Jak-STAT signaling pathway.Through this study,the mechanism of PM_2.5-induced cytotoxicity can be found,providing a new prevention and control strategy for the pathogenic mechanism of PM_2.5.