The Role Of ROS-pyroptosis In The Damage Of Air-blood Barrier Induced By PM_2.5

Objective:In recent years,air pollution has led to an increase in the global burden of disease,and PM_2.5has attracted increasing attention due to its small particle size and significant threat to human health.Numerous studies have shown that PM_2.5is associated with various diseases,such as acute lung injury,lung cancer,atherosclerosis,and neurodegenerative diseases.As PM_2.5is first inhaled through the respiratory tract and causes damage to the lungs,and the air-blood barrier is an important structural and functional barrier of lung tissue,this study mainly explores the damage and potential mechanisms of PM_2.5to the air-blood barrier through in vitro experiments.Methods:1.Collection and preparation of PM_2.5particlesPM_2.5particles were collected in Dalian during autumn and winter seasons using a four-stage cascade impactor,and the collected particles were suspended in physiological saline solution to prepare a 40mg/ml PM_2.5suspension for the following experiments.2.The system in vitro has obtained and experimental groupingHUVEC cells were seeded on the back of Transwell culture plates,and after overnight attachment,A549 cells were seeded in the small chamber of the Transwell.After several days of co-culture,an in vitro co-culture system was obtained.The constructed in vitro system was divided into 5 groups:（1）control group:in vitro co-culture model treated with physiological saline solution for 24 hours;（2）PM_2.5group:in vitro co-culture system treated with PM_2.5for 24 hours;（3）PM_2.5+NAC group:cells were pre-treated with NAC for 2 hours before PM_2.5treatment,followed by 24-hour PM2.5 treatment;（4）PM_2.5+DSF group:cells were pre-treated with disulfiram for 2hours before PM_2.5treatment,followed by 24-hour PM_2.5treatment;（5）PM_2.5+PBS group:cells were pre-treated with PBS for 2 hours before PM_2.5treatment,followed by24-hour PM_2.5treatment.3.Detection of relevant indicators（1）Cell survival rate and oxidative stress detectionThe CCK-8 assay kit was used to detect the effect of PM_2.5on the survival rate of A549 and HUVEC cells.The superoxide dismutase（SOD）assay kit was used to detect the SOD activity of cells and the malondialdehyde（MDA）assay kit was used to detect the MDA content of cells in the in vitro co-culture system.（2）Detection of tight junctions in vitroThe gene expression levels of ZO-1,Occludin,and Claudin-5 were detected by real-time fluorescence quantitative PCR,and the expression of TJ proteins was detected by Western blot.The expression of ZO-1 protein was detected by immunofluorescence in the in vitro co-culture system.（3）Detection of pyroptosis-related indicatorsThe gene expression levels of NLRP3,Caspase-1,GSDMD,IL-1β,and IL-18 in the in vitro co-culture system were detected by qRT-PCR.The protein expression levels of NLRP3,Caspase-1,GSDMD,IL-1β,and IL-18 in the in vitro co-culture system were detected by Western blot,and the expression level of GSDMD protein in the in vitro co-culture system was detected by immunofluorescence staining.The release of LDH and SYTOX Green fluorescence staining were used to evaluate the degree of cell membrane damage in the co-culture system.Results:1.Detection of damage to the in vitro co-culture system after exposure to PM_2.5qRT-PCR and Western blot results showed that compared with monolayer cell culture,the expression levels of ZO-1,Occludin,and Claudin-5 in the co-culture system increased,indicating that the co-culture system can obtain a tighter cell connection and better simulate the air-blood barrier in vitro.CCK-8 results showed that the toxicity of PM_2.5to A549 and HUVEC cells was concentration dependent.qRT-PCR and Western blot results showed that PM_2.5 treatment caused a decrease in the expression levels of tight junction proteins in the in vitro co-culture system.The ZO-1 results of immunofluorescence showed that the tight junction would break and discontinuous after PM_2.5exposure.2.Detection of oxidative stressResults of SOD and MDA assay kits showed that compared with the control group,PM_2.5stimulation can induce a decrease in SOD activity and an increase in MDA content in the in vitro co-culture system.These results indicate that exposure to PM_2.5can enhance the oxidative stress response.3.Detection of classical pyroptosis pathwayqRT-PCR results showed that after PM_2.5stimulation,the m RNA expression of NLRP3,Caspase-1,GSDMD,IL-18 and IL-1βincreased in the in vitro co-culture system.Western blot results suggested that the protein expression of NLRP3,Caspase-1,GSDMD,IL-18,and IL-1βalso increased in the in vitro co-culture system after exposure to PM_2.5.Immunofluorescence results showed that the expression level of GSDMD increased significantly in the co-culture system after PM_2.5treatment,indicating that exposure to PM_2.5can induce cell pyroptosis in the in vitro co-culture system.LDH assay results showed that the release of LDH from the in vitro co-culture system cells increased significantly after PM_2.5stimulation,and the increase in the number of SYTOX Green-positive cells stained with propidium iodide indicates that exposure to PM_2.5can cause damage to the cell membrane in the in vitro system.4.Detection of changes in relevant indicators of co-culture system in vitro after N-acetylcysteine（NAC）treatmentObservations made after pre-treatment with NAC showed a decrease in oxidative stress levels in the PM_2.5+NAC group compared to the PM_2.5+PBS group in the in vitro co-culture system.qRT-PCR and Western blot results indicated that NAC pre-treatment alleviated PM_2.5-induced damage to tight junctions,and immunofluorescence experiments for ZO-1 showed that NAC could reduce the tight junction damage induced by PM_2.5.The qRT-PCR results showed that NAC treatment reduced the gene expression of NLRP3,Caspase-1,GSDMD,IL-18,and IL-1βinduced by PM_2.5,and Western blot results showed a similar trend in protein expression for NLRP3,Caspase-1,GSDMD,IL-18,and IL-1β,with expression in the PM_2.5+NAC group lower than in the PM_2.5+PBS group.Immunofluorescence experiments showed a decrease in GSDMD expression in the PM_2.5+NAC group compared to the PM_2.5+PBS group,and a reduction in LDH release and the number of SYTOX Green-positive cells was also observed in the PM_2.5+NAC group.5.Detection of changes in relevant indicators of the co-culture system in vitro after treatment with disulfiram（DSF）After pre-treatment with DSF,there was a significant decrease in the high expression of NLRP3,Caspase-1,GSDMD,IL-1β,and IL-18 genes and proteins induced by PM_2.5,as well as a reduction in LDH release and the number of SYTOX Green-positive cells.Immunofluorescence results showed a significant decrease in GSDMD expression in the PM_2.5+DSF group compared to the PM_2.5+PBS group.qRT-PCR and Western blot results showed that DSF pre-treatment reduced PM_2.5-induced damage to tight junction proteins,and ZO-1 immunofluorescence experiments also showed an enhancement in the continuity of tight junctions between cells in the PM_2.5+DSF group compared to the PM_2.5+PBS group.Conclusion:Exposure to PM_2.5can cause damage to the air-blood barrier,and its potential mechanism may be that PM_2.5increases oxidative stress,leading to the activation of NLRP3 inflammasome and GSDMD,ultimately resulting in pyroptosis.The oxidative stress-pyroptosis pathway may play an important role in the damage of air-blood barrier caused by PM_2.5exposure.And disulfiram may be a potential viable drug for the treatment of PM_2.5-related lung injury.