Mechanisms Of Reactive Oxygen-mediated Endoplasmic Reticulum Stress In The Autophagy Of Cardiomyocytes Caused By Particulate Matter 2.5 (PM_2.5)

Air pollution has been proved to be a risk factor for several system-related diseases,including respiratory,cardiovascular,and circulatory and has been a public health issue of global concern.Air pollutants are a complex mixture of mixed gases and particulate matter originated from all aspects of life while particulate matter has a greater impact on health among all the ingredients.Particulate matter with particle size less than 10μm(PM10,PM_2.5,PM0.1)can enter the body through the respiratory route,which leads to severe damages on human health.Among the three types of particles,PM_2.5 is of great concern due to its small particle size,large specific surface area,long residence time in the air and the easy deposition in the alveoli.Numerous epidemiological studies have shown that exposure to atmospheric particulate matter has a strong correlation with a variety of cardiovascular diseases（CVD）,and many people die prematurely every year due to air pollution,which has raised a heavy disease burden worldwide.Therefore,it is particularly important to investigate the toxicity mechanisms of atmospheric particulate matter in depth.A number of studies have already indicated that oxidative stress,inflammatory response,and endothelial dysfunction are the toxic mechanisms causing CVD.In recent years,autophagy and endoplasmic reticulum stress（ERS）have also been increasingly regarded as one of the toxic mechanisms of atmospheric particulate matter.Since the toxicological studies in this area are still not deep and abundant,it is necessary to carry out further exploration.On the basis of previous studies,BALB/c mice and human cardiomyocytes were selected as the main research objects in this study.Considering the comparability of the results,the urban atmospheric particulate reference material PM SRM1648 a was used to simulate and study the toxic mechanism of PM_2.5 on cardiac injury,to explore the role of oxidative stress,ERS and autophagy in cardiac and cardiomyocyte injury,and to clarify the relationship between the three mechanisms to provide more possibilities for the pathogenesis of CVD.The specific research content includes the following three parts:1.Toxic effect and mechanism of atmospheric particulate matter（PM SRM1648a）on myocardial tissue of mice.According to the up-and-down procedure（UDP）revised by the organization for economic co operation and development（OECD）,the acute toxicity test was conducted on BALB/c mice by oropharyngeal inhalation,and half lethal dose LD50 of PM SRM1648 a was obtained.The mice were divided into 4 groups for 28 days of subacute toxicity test.HE staining,Masson staining and TEM observation were used to measure the cardiac injury.Lactate dehydrogenase（LDH）kit was used to detect the changes of cardiac enzymes,and the level of cardiac oxidative damage was assessed by the changes of oxidative stress related indexes of 3’-nitrotyrosine（3’-NT）,malondialdehyde（MDA）,superoxide dismutase（SOD）and glutathione（GSH/GSSG）.Western Blot was used to measure the expression of cardiac ERS marker proteins BIP/GRP78,CHOP and autophagy marker proteins LC3-II/I and P62 in mice heart.The results showed that the exposure dose in the acute toxicity test had reached the upper limit of 2000mg/kg in the UDP middle limit test,and the mice did not die.The results of 28 days subacute toxicity test showed that the myocardial fibers of mice in the high dose group（9.17mg/kg）were edema and the gap was widened,and TEM also observed the rupture and distortion of muscle fibers and the expansion of sarcoplasmic reticulum（a special kind of endoplasmic reticulum）in cardiac tissue,which indicated that the exposure of PM SRM1648 a would cause pathological damage to the heart of mice.Compared with the control group,myocardial enzyme LDH was increased in low,medium and high dose groups.The expression of protein nitrification product 3’-NT,lipid oxidation product MDA and antioxidant enzyme SOD increased in the experimental group,while the GSH/GSSG ratio decreased,indicating that exposure to particulate matter caused oxidative stress in the heart.The results of protein detection showed that the expression of cardiac ERS signature proteins BIP/GRP78 and CHOP and autophagy signature proteins LC3-II/I and p62 were increased in experimental mice,which confirmed the cardiac ERS and autophagy induced by exposure to PM SRM1648 a.2.Mechanism of toxic effects of atmospheric particulate matter（PM SRM1648a）on human myocardial cells.The survival rate of AC16 cardiomyocytes exposed to PM SRM1648 a at different concentrations at different times was measured by CCK-8 assay,which was used to determine the exposure time and the dose in subsequent experiments.DCFH-DA fluorescent probe was used to detect the intracellular ROS level after 24 h of exposure to different doses.Autophagy and endoplasmic reticulum damage were observed by TEM.q RT-PCR was used to detect the activation of three signal pathways IRE1,PERK and ATF6 of UPR when ERS occurred in cells at different doses,and preliminarily determine the specific signal pathways.The m RFPGFP-LC3 autophagy double-labeled lentivirus was used to transfect the cells and intracellular autophagy flow was observed under confocal microscope.The expression changes of ERS signature proteins BIP/GRP78,CHOP,PERK-e IF2α-ATF4 signaling pathway and autophagy proteins LC3-II/I and p62 were further verified at the protein level.The results showed that the survival rate of AC16 cardiomyocytes exposed to PM SRM1648 a for 24 h decreased significantly compared with that 6h and 12 h,and the lower doses of 5μg/m2,10μg/m2 and 20μg /m2 produced certain toxicity to the cardiomyocytes,which were used as subsequent experimental conditions.The results of fluorescence probe showed that intracellular ROS levels increased with increasing dose and the exposure of particulate matter caused cellular oxidative stress.According to the results of q RT-PCR,three UPR signaling pathways IRE1,PERK,and ATF6 were all activated.The protein results also showed that the expression of ERS proteins BIP/GRP78,CHOP,and PERK-e IF2α-ATF4 signaling pathway of protein were elevated,thus this signaling pathway was selected for further subsequent experimental investigation.TEM results showed that PM SRM1648 a caused the production of a large number of autophagosomes and autolysosomes in cardiomyocytes,morphological changes of endoplasmic reticulum were observed,and the expression of autophagic proteins LC3-II/I and P62 were up-regulated,which was consistent with the results of subacute experimental proteins in animals,suggesting that not only autophagy occurs in animals and cells,the autophagic flow may also be blocked.The results of autophagy double label lentivirus showed that both red and green fluorescence were enhanced in the experimental group,which further indicated that the autophagy flux was blocked.3.Mechanism of reactive oxygen species and endoplasmic reticulum stress in autophagy.AC16 cardiomyocytes were pretreated with the antioxidant n-acetyl-l-cysteine（NAC）for 24 h,and the expression of ERS protein BIP/GRP78,CHOP,and PERK-e IF2α-ATF4 signaling pathway proteins were detected by Western Blot to evaluate the role of oxidative stress in ERS,followed by the determination of the role of oxidative stress in autophagy according to the changes of dual fluorescence in autophagy flow and the expression of autophagic proteins LC3-II/I and P62.The PERK inhibitor GSK2606414 was used to inhibit the activation of this pathway in cardiomyocytes,and the expression levels of autophagic dual fluorescence and autophagic proteins LC3-II/I and p62 in cardiomyocytes were detected to evaluate whether the PERK-e IF2α-ATF4 signaling pathway has a regulatory effect on autophagy.The results showed that the expression of ERS protein BIP/GRP78,CHOP,and PERK-e IF2α-ATF4 signaling pathways were down-regulated after NAC treatment,and the dual fluorescence of autophagy flow was significantly decreased,while the expression of autophagy proteins LC3-II/I and p62 decreased,suggesting that ROS may be the upstream factor regulating ERS and autophagy.After inhibiting the activation of this pathway with PERK inhibitor（GSK2606414）,the autophagic stream double fluorescence was somewhat attenuated and the expression of autophagy protein LC3-II/I and p62 was also decreased,which,to some extent,indicates that the activation of PERK-e IF2α-ATF4 signaling pathway promotes autophagy and inhibition of this pathway has some alleviating effect on autophagy,but it was not completely alleviated.In summary,the results of in vivo experimental studies indicate that exposure to PM SRM1648 a can cause pathological damage to the heart.In addition,combined with the results of in vitro cell experiments,exposure to particulate matter can induce oxidative stress,ERS and autophagy in heart and human cardiomyocytes,and ROS production may be the upstream factor leading to ERS and autophagy.ERS activates UPR signaling pathway,which leads to autophagy,while PERK-e IF2α-ATF4 signaling pathway plays a certain role among them.The results of this study provide a valuable reference for the study of the mechanism of cardiovascular system damage caused by PM_2.5.