The Effects Of Endoplasmic Reticulum Stress On PM2.5-induced Apoptosis In A549 Cells Based On Lung Tissue Chip

Fine particulate matter?PM2.5?with aerodynamic diameter less than 2.5?m is the main component of air pollution.Because PM2.5 has a large specific surface area,some toxic and harmful substances including heavy metals/metalloids,micro-organisms and organics in the air are easily adsorbed by PM2.5.Additionally,the small size of PM2.5 can penetrate deeply into human respiratory system.Therefore,exposure to PM2.5 environment poses a great health risk to human beings.However,how the PM2.5 affects the human health and what the molecular mechanism of respiratory system cells injured by PM2.5 are still not illuminated.In this study,polydimethylsiloxane?PDMS?has been utilized to prepare the lung tissue chip.Human alveolar epithelial A549 cells and human umbilical vein endothelial cells?HUVEC?have been co-cultured in the two chambers of this chip.Polycarbonate membrane is a wall between two chambers with different types of cells.Based on this design,a lung tissue chip is established to mimic the key structural features and functions of the alveolar-capillary barrier in vivo.This lung tissue chip is applied to analyze the toxicity of PM2.5 on pulmonary cells.We have studied the effects of PM2.5 induced apoptosis in A549 cells and in lung tissue chip structure,and also discussed the role of endoplasmic reticulum stress and oxidative damage during the apoptosis process caused by PM2.5,which is providing a scientific proof for revealing how PM2.5 injured on lung tissue cells.Objective:1.To establish lung tissue chip model to investigate the toxic effects of PM2.5 on lung tissue cells.2.To explore the role of endoplasmic reticulum stress pathway in apoptosis of A549 cells induced by PM2.5.Methods:1.PM2.5 collection and composition analysisThe medium-flow air particle sampler was used for sampling.The mass concentration of PM2.5 was determined by weighing method;the heavy metals and metalloids were determined by inductively coupled plasma mass spectrometry;the polycyclic aromatic hydrocarbons?PAHs?were determined by gas chromatography-mass spectrometry;and the water-soluble ions were determined by ion chromatography.2.The effect of PM2.5 on apoptosis of A549 cells in isolate culture mode?1?Experimental design:a control group and a series of concentrations?10,20,50,100?g/mL?PM2.5 groups were set up,and exposure time was 24hours.?2?Cell viability was determined by cell counting kit-8?CCK-8?assay.?3?Annexin V-FITC and PI double staining were used to detect cell apoptosis rate by flow cytometry.?4?Detection of oxidative damage and other indicators were detected by biochemical kits such as reactive oxygen species?ROS?,superoxide dismutase?SOD?,glutathione peroxidase?GSH-Px?,and malondialdehyde?MDA?.?5?Western-blot method was used to analyze the expression of endoplasmic reticulum stress-related proteins,such as BIP,PERK,eIF2?,p-eIF2?,CHOP and Caspase-3.3.The effect of PM2.5 on apoptosis of A549 cells in lung tissue chip mode?1?Lung tissue chip was fabricated using PDMS material and polycarbonate film.?2?The function of the model was verified by means of liquid exchange,cell morphology observation,apoptosis rate,ROS level,inflammatory factors?IL-1?,IL-1?,IL-6 and IFN-??.?3?N-Acetyl-L-cysteine?NAC?was used for intervention.This experimental section was designed three groups:control group,PM2.5 group,PM2.5+NAC group.After PM2.5 exposure for 24 h,ROS,cell apoptosis rate and endoplasmic reticulum stress-related proteins'expression were used to assay the role of endoplasmic reticulum stress in A549 cells apoptosis induced by PM2.5.Results:1.The components analysis of PM2.5During the study period,the mass concentration range of PM2.5 was264-487?g/m³,and the average mass concentration was 413.83?g/m³.The content of Al was the highest among the 12 metals and metalloids,followed by Pb,Mn and Cr;16 kinds of PAHs were detected,among which Indeno[1,2,3-cd]pyrene?IP?content was the highest,followed by Benz[a]anthracene?BaA?,Benzo[b]fluorathene?BbF?and Acenaphthylene?Acy?.Among the four inorganic water-soluble ions,the highest content was SO₄^2-,followed by NO₃^-and NH₄⁺,and the content of Cl^-was lowest.2.The effect of PM2.5 on apoptosis of A549 cells in isolate culture mode?1?With the increasing of the PM2.5 concentration,the proliferation rate of A549 cells gradually decreased?P<0.05?,which indicated that PM2.5inhibited the survival of A549 cells and there was a dose-dependent relationship between PM2.5 concentration and proliferation of cells.?2?The ROS level increased gradually with the increasing of the concentration of PM2.5?P<0.05?;the activity of SOD and GSH-Px decreased gradually with the increasing of the concentration of PM2.5?P<0.05?;the content of MDA increased with the increasing of the concentration of PM2.5?P<0.05?,which indicated that PM2.5 could induce oxidative damage in A549cells.?3?The apoptosis rate of A549 cells increased gradually with the increasing of the concentration of PM2.5?P<0.05?,which indicated that PM2.5 could induce apoptosis in A549 cells.?4?The expression levels of BIP,PERK,p-eIF2?and Caspase-3 in the cells increased with the increasing of PM2.5 concentration?P<0.05?,and the ratio of p-eIF2?protein to eIF2?protein increased?P<0.05?;the expression level of eIF2?protein in A549 cells decreased when PM2.5 concentration was100?g/mL?P<0.05?;the expression level of CHOP protein in A549 cells increased with PM2.5 concentration increasing?P<0.05?.The results of protein expression indicated that PM2.5 caused endoplasmic reticulum stress in A549 cells.PERK-eIF2?pathway was activated,and the expression of CHOP and Caspase-3 apoptotic proteins increased the apoptosis of A549 cells.3.The effect of PM2.5 on apoptosis of A549 cells in lung tissue chip mode?1?The apoptotic rate of A549 cells in lung tissue chip mode was higher than that in isolate culture mode,which indicated that the lung tissue chip model was successfully established to study the toxicity of PM2.5 on A549cells.The reason for the increase of apoptosis were at least partly caused by ROS,IL-1?and IL-6.?2?After NAC intervention,the ROS level in the PM group was significantly higher than that in the control group?P<0.05?.The ROS levels in the NAC group and the PM+NAC group were not significantly different from those in the control group?P>0.05?.The ROS level in the PM+NAC group was significantly lower than that in the PM group?P<0.05?,which indicated that NAC can reduce ROS levels.?3?After NAC intervention,the apoptotic rate of PM group was significantly higher than that of the control group?P<0.05?.The apoptosis rate of PM+NAC group was significantly lower than that of PM group?P<0.05?,which indicated that NAC intervention was going to inhibit apoptosis of A549cells.?4?After NAC intervention,the expression levels of BIP,PERK,p-eIF2?and Caspase-3 in PM group were significantly higher than those in control group?P<0.05?.The expression of BIP protein in PM+NAC group was significantly lower than that in PM group?P<0.05?;the expression of eIF2?protein in PM group was lower than that in control group?P<0.05?,and the expression of eIF2?protein in PM+NAC group was higher than that in PM group?P<0.05?;The ratio of eIF2?protein to eIF2?protein of PM group and PM+NAC group was higher than that of the control group?P<0.05?.The ratio of p-eIF2?protein to eIF2?protein in PM+NAC group was significantly lower than that in PM group?P<0.05?.The expression of CHOP protein in PM group and PM+NAC group was higher than that of the control group?P<0.05?.The expression of CHOP protein in PM+NAC group was significantly lower than that in PM group?P<0.05?.All the results indicated that oxidative stress was an important factor for activing endoplasmic reticulum stress in cells,and PM2.5 was able to cause apoptosis through endoplasmic reticulum stress pathway.Conclusions:1.A co-culture model of lung tissue chip A549 cells and HUVEC cells was successfully established.2.PM2.5 induced apoptosis of A549 cells,which was more obvious in lung tissue chip mode than in isolate culture mode,which may be related to the interaction among inflammatory factors secreted between cells.3.PM2.5 can induce apoptosis of A549 cells,which was more obvious in lung tissue chip mode than in isolate culture mode,which may be related to endoplasmic reticulum stress pathway induced by oxidative damage.