Toxicity And Molecular Mechanism Of PM2.5 Components On Beas-2B Cells

With the development of industry,atmospheric particulate pollution has become more and more serious,causing great harm to human health.PM2.5 refers to airborne particulate matter with an aerodynamic diameter of 2.5?m,which mainly comes from the process of coal combustion,vehicle exhaust,and industrial exhaust emissions,and is the main reason for the formation of hazy weather.At present,the mechanism of PM2.5 toxicity mainly includes the following possible mechanisms.PM2.5 firstly interacts with lung epithelial cells and releases various cytokines after stimulation.PM2.5 can further interact with cells to release active oxygen free radicals,etc.Tissue cells.The early stage of airway inflammation begins with the inflammatory response of bronchial epithelial cells.The change of inflammatory factors is one of the main causes of PM2.5-induced inflammation.In order to promote the research on the relationship between atmospheric pollution and human health in China,it is necessary and urgent to carry out research on the toxicity mechanism of PM2.5.In this study,Beas-2B cells were used to study the toxic effects and mechanisms of PM2.5 on human bronchial epithelial cells.MTT assay was used to detect the inhibition of cell growth by PM2.5 at different concentrations;PM2.5 was observed under a microscope.Effect on cell morphology;NO and ROS oxidative stress levels in cells were detected by NO and ROS fluorescent probe experiments;PM2concentrations were detected by Hoechst 33258 and flow cytometry.5 Effect on the induction of apoptosis.In order to further study the damage effects and mechanisms of PM2.5 and its main polycyclic aromatic hydrocarbons fluoranthene,anthraquinone and phenanthrene on human bronchial epithelial cells,the main components of PM2.5,fluoranthene,anthraquinone and phenanthrene were selected as the research subjects,and the MTT ratio was used.The inhibitory effect of fluoranthene,anthracene,and phenanthrene on cell growth was detected by colorimetry.The effect of mRNA on the expression of cytokines was detected by Real-Time PCR.Western blot was used to detect the expression of NF-?B,p38 MAPK and Nrf2 and activation of signal pathway after treatment of the cells,and the expressions of p53,Bcl2,Bax and caspase1,3,12 were detected.Finally,in order to elucidate the mechanism of the transport of fluoranthene and pyrene,the main polycyclic aromatic hydrocarbons of PM2.5 invivo,BSA was used as a protein model to study the effect of fluoranthene,anthraquinone and BSA on the aromatic residue of BSA protein by UV spectroscopy.Changes in the environment and changes in the secondary structure of BSA molecules;Fluorescence spectroscopy was used to study the interaction between fluoranthene,fluorene,and BSA,and the molecular conformation.The distance between fluoranthene,fluorene,and BSA was calculated by the formula to determine whether it was embedded in the interior of the BSA molecule;Synchronous fluorescence spectroscopy measures changes in the maximum emission wavelength,studies the corresponding changes in the environment of amino acid residues after detection of the interaction between fluoranthene and hydrazine and BSA,and describes the structure of tyrosine and tryptophan residues using three-dimensional fluorescence spectroscopy.Changes and changes in the structure of the polypeptide chain to determine the fluoranthene,hydrazine binding sites within the BSA molecule.The results showed that PM2.5 can significantly inhibit cell proliferation and there is a dose-dependent relationship.Under the microscope,the cell morphology after treatment with different doses of PM2.5 changed from shuttle to circular,and the cells appeared shrinkage and the number of cells decreased significantly with increasing concentrations.With the increase of PM2.5 concentration,the intracellular NO and ROS fluorescence intensity increased significantly,indicating that PM2.5increased the degree of oxidative stress.Hoechst33258 and flow cytometry experimental results show that PM2.5 can effectively induce the apoptosis of Beas-2B cells,and there is a certain concentration dependence.Real Time PCR showed that PM2.5,fluoranthene,anthraquinone and phenanthrene could increase the expression of inflammation-related factors and interleukin after treatment,indicating that PM2.5,fluoranthene,anthraquinone,and phenanthrene could all induce inflammation.Western blot results showed that PM2.5 can activate p38 MAPK,NF-?B pathway and inhibit Nrf2 signaling pathway.The study also found that the results of fluoranthene,anthraquinone,and phenanthrene were the same as those of PM2.5.Only the detection of some apoptosis-related proteins could induce the expression of inflammation-related factors,activate the p38 MAPK,NF-?B pathway,and inhibit the Nrf2 signaling pathway.Spectroscopic studies have shown that the hydrophobic groups of tryptophan and tyrosine residues inside the molecule after the addition of fluoranthene and fluorene are exposed while the BSA peptide chain is extended,the UV spectrum shows a significant color enhancement effect and changes the secondarystructure of the BSA molecule.In the investigation of the fluorescence quenching mechanism,the binding properties of fluoranthene and fluorene to BSA were calculated by the Stern-Volmer equation.All of them were statically quenched,and both the binding sites and the equilibrium constants decreased with the increase of temperature,which also met the static quenching mechanism.According to Forster's theory of non-radiative energy transfer,the binding distance r and the energy transfer efficiency E between tryptophan and fluoranthene interacted with BSA were calculated,and the binding distance between them and BSA tryptophan was calculated.,indicating that there is a high possibility of energy transfer between them.The ?G,?H,and ?S of FLA-BSA and PYR-BSA were calculated by thermodynamic formula under different temperature conditions,and it was judged that the interaction forces were hydrophobic.The effect of FLA and PYR on the tyrosine residue domain and the tryptophan residue domain of the luminescent fluorophore in BSA was determined by synchronous fluorescence spectroscopy.Finally,three-dimensional fluorescence spectroscopy was used to confirm that the binding site was in the hydrophobic cavity of BSA,resulting in a change in the polarity of the hydrophobic microenvironment and eventually causing a change in BSA conformation.In summary,PM2.5 and its major PAH components can also cause cytotoxicity,and can induce inflammation,activate the p38 MAPK,NF-?B pathway,inhibit the Nrf2 signaling pathway,and PM2.5 components.The in vivo transport mechanism is achieved by combining with BSA.This study provides a basis for the theoretical and experimental basis of toxic inflammatory mechanisms for the study of the relationship between environmental pollution and atmospheric pollution in China.