Study On The Mechanism Of PM_2.5 And Vesicular Stomatitis Virus On Programmed Death Of Human Lung Adenocarcinoma Cells And Mice

Background:Fine particulate matter(PM_2.5)refers to particulate matter with an aerodynamic diameter of≤2.5μm,which will be suspended in the air for a long time and enter the human lungs through the respiratory tract,causing toxic effects on the lungs and other organs.PM_2.5,as the main air pollutant,has always been the focus of environmental pollution research.In recent years,it has been discovered that PM_2.5can carry various pathogenic microorganisms,and it can be used as an intermediary for the transmission of pathogenic microorganisms.VSV（Vesicular Stomatitis Virus）is a kind of negative-strand RNA virus,which has the characteristics of simple structure and high replication ability,and has been widely used in the model studies of RNA viruses.However,the understanding of the ways in which PM_2.5 and viruses invade the body is not fully understood,and it is not clear whether programmed cell death can be induced to have an impact on virus replication.Therefore,in this study,the effect of PM_2.5 on VSV replication in A549 cells and the mechanism of action were preliminary explored,and further verified by mice in vivo experiments,so as to provide a basis for in-depth study of the interaction between PM_2.5 exposure and viral infection,and to provide clues for revealing the impact of PM_2.5 exposure on patients with RNA virus pulmonary infection.Methods:（1）vitro cell experiment:A549 cells were infected with VSV（MOI=1）and exposed to different concentrations of PM_2.5 for 12 h and 24 h.At the same time,Control group and VSV（MOI=1）alone infection group were set up.The cell proliferation-toxicity experiment was carried out by the CCK-8 method and the cell cycle was observed by flow cytometry.Real-time quantitative polymerase chain reaction（RT-q PCR）,inverted fluorescence microscope and Western-Blot experiment were used to detect VSV m RNA expression,VSV-GFP virus with green fluorescent markers and VSV-G protein expression.Western-Blot experiment was used to observe the expression of apoptosis,autophagy and pyrolysis proteins,such as apoptosis-related apoptosis proteins（β-actin,pro-caspase-3,pro-caspase-9,Bcl-2,Bax,PARP）,autophagy proteins（LC3,Beclin-1 and ATG-5）.Real-time quantitative polymerase chain reaction（RT-q PCR）was used to observe the expression of apoptosis,autophagy and pyrolysis m RNA,such as caspase-3,caspase-9,Bax,Bcl-x,autophagy-related factor 5（Atg5）,Autophagy related genes Beclin-1,caspase-1,GDSMD.The autophagy inhibitor（Bafilomycin A1）and 3-MA were pretreated for 2h,and the expression of VSV m RNA in the VSV group and PM_2.5+VSV treatment was observed.Enzyme-linked immunosorbent assay（ELISA）and RT-q PCR were used to detect the expression of IL-1βand IL-6 in the cells.（2）Vivo experiment:32 C57BL/6J mice aged 6-8 weeks were randomly divided into Control group,PM_2.5 group,VSV group and PM_2.5+VSV group by random number table method,with 8 mice in each group（half male and half female）.The model of PM_2.5exposure combined with pulmonary VSV infection was established.Mice in Control group were treated with PBS as blank.Observe the changes in body weight,food intake and water consumption within 10 days of the four groups of mice;observe the wet weight of the lungs,liver and spleen of the four groups of mice.Lung tissues were taken for HE staining and was used to observe the m RNA expression of apoptotic autophagy pyrolysis by RT-q PCR experimentResults:（1）With the increase of PM_2.5 concentration and exposure time,the survival rate of A549 cells infected by VSV decreased significantly.PM_2.5 with a final concentration of 200μg/m L and a stimulation time of 24 h had stronger inhibitory effects on the survival rate of A549 cells than other concentrations,the difference was statistically significant（P<0.05）,and the cell cycle arrest was in S phase.（2）PM_2.5can promote the replication of VSV in A549 cells.With the increase of PM_2.5concentration,through RT-q PCR and fluorescence inverted microscope experiments,it can be observed that compared with the VSV group alone,the amount of virus replication in A549 with PM_2.5+VSV increased.（3）PM_2.5and VSV can increase the inflammatory factors IL-1βand IL-6 of A549 cells.（4）PM_2.5 and VSV can jointly up-regulate the apoptosis level of A549 cells.It can be seen from the detection results of apoptosis m RNA and protein levels that compared with the PM_2.5 alone group and the VSV alone group,the apoptosis level of the PM_2.5+VSV group was significantly up-regulated.（5）PM_2.5 and VSV can jointly up-regulate the autophagy level of A549cells.As can be seen from the results of autophagy m RNA and protein levels that the autophagy level of the PM_2.5+VSV group was significantly increased compared with the PM_2.5 alone group and the VSV alone group.（6）PM_2.5 and VSV together up-regulate the level of pyrolysis in A549 cells.From the results of pyrolysis m RNA,it can be seen that compared with the PM_2.5 alone group and the VSV alone group,the level of pyrolysis in the PM_2.5+VSV group was significantly increased.（7）Autophagy inhibitors（Bafilomycin A1）and 3-MA can inhibit the occurrence of autophagy,thereby inhibiting the replication of VSV virus in A549 cells.（8）PM_2.5combined with VSV infection can jointly up-regulate the level of lung apoptosis in mice.RNA was extracted from lung tissues of mice in each group,and RT-q PCR was used to detect the expression of apoptosis genes in the lung of mice in different treatment groups.The results showed that the expression levels of apoptosis-related factors Bcl-2 and Bax in the PM_2.5+VSV group were significantly higher than those in the other groups,and the difference was statistically significant（P<0.05）.It is consistent with the results of in vitro study.（9）The co-stimulation of PM_2.5 and VSV in mice can up-regulate the autophagy level in the lungs of mice.The results showed that the expressions of autophagy-related factors Atg5 and Beclin-1 in the PM_2.5+VSV group were significantly higher than those in the other groups,and the difference was statistically significant（P<0.05）.It is consistent with the results of in vitro studies.（10）PM_2.5 combined with exposure to VSV-infected mice can up-regulate the level of lung pyrosis in mice.RT-q PCR experiment was used to detect the expression of pyroptosis genes and inflammatory factors in the lungs of mice in different treatment groups.The results showed that the expression levels of inflammatory factors IL-1β,IL-6 and Caspase-1 in the PM_2.5+VSV group were significantly higher than those in the other groups,and the differences were statistically significant（P<0.05）.Compared with the VSV group alone,the protein expression level of GSDMD in the PM_2.5+VSV group was lower.It is consistent with the results of in vitro studies in this study.Conclusion:In vitro experiments,A549 cells were co-infected with PM_2.5 exposure and VSV,and results showed that,with the increase of PM_2.5 concentration,the survival rate of A549 cells co-infected with VSV decreased and S-phase cell cycle arrest occurred and the proliferation of VSV in cells was promoted.Further results showed that PM_2.5 led to VSV replication in cells through apoptosis,autophagy and pyrotosis.In vivo studies,it was found that VSV infection and simultaneous exposure to PM_2.5 can increase the amount of drinking water and diet in mice for 10 days,and up-regulate the levels of apoptosis,autophagy and pyrostosis to promote VSV replication in mice,which is consistent with in vivo experiments.