Research Of Injury And Molecular Mechanism Of Inflammatory Response Induced By PM_2.5 On Cardiovascular Endothelium

As an important component of air pollutants,PM_2.5 is closely related to the cardiovascular diseases.Now,researches reveals that PM_2.5-induced inflammatory reaction and secondary vascular endothelial injury are important mechanisms of cardiovascular toxicity while most of the attention is focused on the inflammatory effects of PM_2.5.It needs to be further studied about how inflammatory factors are produced,what factors are regulated by inflammatory effects,and how inflammatory factors act on cardiovascular endothelial cells in causing endothelial dysfunction.PM_2.5 is a complex mixture of various chemical components.When evaluating its toxic effects,it is not enough to consider PM_2.5 itself,but also should fully consider the toxic effects of its chemical components.In view of this,this paper firstly studied the relationship between ambient PM_2.5.5 and cardiovascular diseases death in Nanjing residents.Then through the data of population health effects,the repeated measurement study of the fixed group,the cell experiment in vitro and animal experiments in vivo,the molecular mechanism of inflammatory reaction in the cardiovascular endothelial injury induced by PM_2.5 was discussed to find the key genes regulating the cascade reaction,the involved important metal components,sensitive evaluation indexes and potential prevention targets of PM_2.5-induced cardiovascular endothelial inflammatory injury,and provide the theoretical reference for the health protection of the population.Part 1:Time series analysis of the effect of PM_2.5 on cardiovascular disease deathObjective:To explore the daily average concentration of ambient PM_2.5 and the daily cardiovascular disease deaths in Nanjing from 2013 to 2017;to quantitative evaluate the effect of ambient PM_2.5 on cardiovascular disease death in Nanjing.Methods:The daily air pollutants(PM_2.5,SO₂,NO₂,O₃),meteorological data（mean temperature,relative humidity）and cardiovascular diseases deaths were collected and were classified according to International Classification of Diseases,10^th Revision（ICD-10）in Nanjing from January 1,2013 to December 31,2017.The generalized additive model was established by time series analysis.After adjusting the long-term trends,meteorological factors and"days of the week effect",the influence of PM_2.5 on the total deaths of cardiovascular disease,ischemic heart disease,hypertension and arrhythmia for different genders and ages were quantitatively studied in Nanjing in summer,winter and transition season from 2013 to 2017.The excess risk（ER）was calculated for the daily deaths caused by all kinds of diseases when the concentration of PM_2.5 increased by 10μg/m³.Results:（1）From 2013 to 2017,the main air pollutants in Nanjing was PM_2.5.All air pollutants and cardiovascular diseases deaths varied seasonally.Concentration of air pollutants was higher in winter than in summer except O₃.The number of deaths of cardiovascular diseases deaths was higher in winter too.（2）The single pollutant model analysis showed that the effect of PM_2.5 on the total deaths of cardiovascular diseases were statistically significant.At the annual level,with the concentration of PM_2.5 increased by 10μg/m³,the mortality of cardiovascular diseases increased by 0.43%（95%CI:0.08-0.78%）.Among all stratifications,the effect of PM_2.5 on the death risk of cardiovascular disease was the highest in summer,and 45-65 years old group was more sensitive.Among different types of cardiovascular disease,the effect of PM_2.5 on acute myocardial infarction was the most obvious at 7 days lag（Lag7）.When the concentration of PM_2.5 increased by 10μg/m³,the death risk of acute myocardial infarction increased by 1.02%（95%CI:0.40-1.64%）.The effect of PM_2.5 on ischemic heart disease such as atherosclerosis,insufficient coronary blood supply,angina pectoris and hypertension was the most obvious on the day of exposure to PM_2.5 respectively.The death risk of ischemic heart disease and hypertension increased by 1.10%（0.59-1.60%）and 1.11%（0.14-2.09%）when the concentration of PM_2.5 increased by 10μg/m³.（3）After adjusting other pollutants in the double pollutant model and multi-pollutant model,the effect of PM_2.5 on different kinds of cardiovascular diseases changed in different degrees compared with the single pollutant model,suggesting that there may be interaction between pollutants.Conclusion:PM_2.5 can increase the death risk of ischemic heart disease such as acute myocardial infarction,atherosclerosis,insufficient coronary blood supply,angina pectoris and hypertension in Nanjing residents,especially in summer,45-65 years old group.No correlation between PM_2.5 and arrhythmia death was observed in this study.Part 2:Population repeated measurement study on effects of PM_2.5 and metal components on serum metal and cytokines related to cardiovascular endothelium inflammatory injuryObjective:To investigate the effects and mechanisms of cardiovascular endothelium inflammatory injury of PM_2.5 and metal components from the population level,to find the main metal components that cause cardiovascular endothelium inflammatory injury,and to evaluate the sensitive indicators of PM_2.5 cardiovascular endothelium inflammatory injury in peripheral blood.Methods:During the temporary period of air quality improvement because of air pollution control of Nanjing Youth Olympics,31 health adults were recruited.Then five longitudinal and repeated measurements study was used respectively in pre-Olympic period,intro-Olympic period,post-Olympic period,to explore the concentration fluctuation of PM_2.5,the heavy metals of PM_2.5,the heavy metals in blood serum（Al,Cr,Mn,Hg,Sb,Pb,Cd,Ni,As）,and the level of cytokines of cardiovascular endothelium inflammatory injury in blood serum.To explore the effects and mechanism of adverse cardiovascular events,the linear mixed-effect models was used to examine the associations between the heavy metals of PM_2.5.5 and the heavy metals in blood serum,the heavy metals of blood serum and cytokines of cardiovascular endothelium inflammatory injury（IL-18,IL-10,IL-1β,TNF-α,CRP,MCP-1,P-selectin,ICAM-1,VCAM-1,sCD40L）in blood serum.Results:The restrictive air pollution control policy has allowed the concentration of PM_2.5 in the atmosphere to decrease by 33.82%during Olympic,and the concentration of the metal component of PM_2.5 such as Al,Cr,Mn,Hg,Pb,Cd,Ni,Tl,Se,Sb,As all decreased except Be（75%of the sample Be is lower than the detection limit）.The top 6 metal was Hg,Mn,As,Cd,Pb and Se,and the decreasing range is 37.85%,33.13%,30.77%,29.45%,18.89%and 16.18%,respectively.After Olympic,the concentration of PM_2.5 and the concentration of the above-mentioned metal components increased,and the top 6 metal was Cd,Be,Hg,Pb,As and Ni,and the upwelling amplitude was63.04%,50.00%,30.45%,21.31%,21.20%and 15.40%,respectively.The concentration of heavy metals of blood serum had similar change,decreasing during Olympic period,and then increasing after Olympic period for the removal of air pollution control policies.The concentration of serum nickel in time window（24-48 hours）was significantly correlated with the nickel of PM_2.5,and the concentration of serum cadmium in time window（0-7 days）was associated with the cadmium of PM_2.5.Within the ten cytokines related to inflammation response,vascular adhesion and platelet activation,the concentration of IL-1β,IL-18,sCD40L,VCAM-1 and CRP decreased during Olympic period and then increased after Olympic period.Through the linear mixed-effect models,we found that the relation between the concentration of PM_2.5 and IL-1β,IL-18,sCD40L,ICAM-1,VCAM-1and P-selectin was positive,especially in time window（13-24 hours）and time window（0-24 hours）.Moreover we also found that PM_2.5 was negative correlated with IL-10 in time window（7-12 hours）and time window（0-24 hours）.There was significantly relevance among the concentration of heavy metals and cytokines in blood serum,especially antimony,which had positive relation with MCP-1、TNF-α和IL-18.The relation between arsenic of blood serum and ICAM-1,aluminum and IL-18,and manganese and VCAM-1 were positive too.Conclusion:PM_2.5 can stimulate systemic inflammatory cascade reaction and secrete cytokines related to inflammation,adhesion and platelet activation,with cardiovascular endothelium as the target,causing endothelial dysfunction.PM_2.5 metal components are involved in this process,in which Sb,As,Al and Mn have significant cardiovascular injury effects.The combined of serum inflammatory factor IL-1β,IL-18,IL-10,molecule involved in vascular adhesion ICAM-1,VCAM-1 and platelet activation marker sCD40L,P-selectin can be used as synthetic evaluation index for the cadiovascular toxicity induced by PM_2.5.Part 3 The cytotoxicity of PM_2.5 to EA.hy926 and the analysis of mRNA transcriptional group Objective:To clarify how PM_2.5 stimulates the inflammatory cascade of vascular endothelial cells,how various inflammatory factors interact to cause vascular endothelial cell damage,which mechanisms are regulated by this process,and whether metal ions are involved?Transcriptomics was used to fully understand the potentially complex mechanism of vascular endothelial cell injury induced by PM_2.5,and to provide clues for further molecular mechanism.Methods:EA.hy926 cells treated with PM_2.5 were sequenced by microarray analysis.The dose of PM_2.5 was determined by MTT assay,cell cycle test and apoptosis test.The results were analyzed to know the interaction between genes,the main biological processes and pathways of differential gene enrichment through differentially expression genes screening,GO analysis,KEGG pathway,Gene signal transduction network.Results:mRNA microarray sequencing had two treatment groups:2.5μg/cm² group with no obvious cell death,apoptosis and cell cycle change,cell survival rate nearly 80%,and 10μg/cm² group with obvious apoptosis and S phase arrest.Differentially expression gene screening results showed that107 genes were changed in the 2.5μg/cm² group,of which 75 genes were up-regulated and 32 genes were down-regulated,compared with the control group.In 10μg/cm² group,the expression of 440genes were changed,of which 297 genes were up-regulated and 143 genes were down-regulated.The number of differentially expression genes in high dose group was significantly higher than that in the low dose group.By GO analysis,KEGG pathway,the mechanism of vascular endothelial cells injury induced by PM_2.5 was mainly inflammatory response in 2.5μg/cm² group,multiple inflammatory related pathways were activated,and the burden of ROS began to increase.Metal ions might be involved in these biological processes.In 10μg/cm² group,oxidative stress and inflammatory cascade reaction were further aggravated.In which,adhesion effect,platelet activation and vasoconstriction were prominent.In addition,phagocytosis,abnormal lipid metabolism and fibrosis could not be ignored.In order to test the reliability of sequencing results and provide clues for the mechanism of the next chapter,10 differentially expression genes related to inflammation,adhesion and oxidative stress were selected for RT-qPCR verification.The RT-qPCR results of 8 genes were coherent with the results from microarray,which indicated that the sequencing results were reliable.Conclusion:PM_2.5 activates a variety of biological processes and pathways mediated by membrane receptors related to oxidative stress,inflammatory response,adhesion effects,endocytosis,and metabolic abnormalities,and these processes may involve multiple metal ions.mRNA signal network analysis suggests that the toxicity of PM_2.5 to vascular endothelial cells is regulated and interacted by several genes involved in oxidative stress,inflammation and adhesion.Part 4 The role of NOX1 in the cardiovascular inflammatory response induced by PM_2.5Objective:To know the regulation of NOX1 in vascular endothelial inflammation induced by PM_2.5.Methods:The EA.hy926 cells exposed to PM_2.5 were treated with NOX1 inhibitor ML090.Changes in cytokine involved in inflammatory injury to vascular endothelial cells such as IL-1β,IL-18,IL-10,ICAM-1,VCAM-1,P-seletin and the ROS levels were compared before and after ML090 intervention.EA.hy926 cells was exposed to aluminum powder with an average particle size of 1μm.The effect of metal Al on the expression of NOX1 in EA.hy926 cells was detected to speculate whether the metal components of PM_2.5 were involved in NOX1-mediated oxidative stress and vascular inflammatory injury.Finally,the mice were inhaled dynamically with PM_2.5,and the changes in the concentration of pro-inflammatory factors in serum and the expression of NOX1 and ICAM-1 in the vascular endothelium of heart,lung and kidney were observed in animals.Results:After exposure to PM_2.5,the expression level of NOX1 and ROS level were significantly increased in the lower dose group of 2.5μg/cm² which had not been significantly changed in cytokines（IL-18,IL-10,VCAM-1,ICAM-1 and P-seletin）associated with vascular endothelial injury.The overexpression of NOX1 induced by PM_2.5 was decreased by 54.38%by ML090 intervention.At the same time,the levels of ROS,IL-1β,IL-18,and ICAM-1 were significantly lower than in PM_2.5 group,but the level of IL-10 was higher than that in PM_2.5 group.C57BL/6 mice were inhaled with 0.4mg/m³PM_2.5 for 3 and 7 days and the serum levels of IL-18 and IL-10 in the mice were significantly higher than in control group.After 14 days of exposure,the results of immunohistochemistry showed that PM_2.5 significantly increased the levels of NOX1 and ICAM-1 protein in alveolar wall vascular endothelial cells and glomerular vascular endothelial cells,while the ICAM-1 protein levels in myocardial interstitial vascular endothelial cells were significantly increased,but the NOX1 protein levels in myocardial interstitial vascular endothelial cells were not significantly increased compared with the control group.In addition,in this study,EA.hy926 cells were exposed to Al,which was high metal composition ratio of PM_2.5,to observe the changes of NOX1 expression.It was found that Al significantly upregulated the expression of NOX1.Conclusion:NOX1 is involved in the accumulation of ROS and vascular inflammatory response induced by PM_2.5.NOX1 may be the regulatory gene of vascular endothelial injury induced by PM_2.5.Down-regulating NOX1 can reduce ROS and relieve the dysfunction of vascular endothelial cells induced by PM_2.5.The up-regulation of NOX1 expression by metal Al suggests that metal components of PM_2.5 may be involved in the regulation of ROS production and vascular inflammatory response by NOX1.In summary,this study further confirmed that PM_2.5-induced vascular endothelial inflammatory injury is an important mechanism of acute heart attack,atherosclerosis,coronary heart disease and other heart diseases,and the process is the result of the participation and interaction of multiple genes such as oxidative stress,inflammatory reaction,adhesion,multiple biological functions and pathways.The cascade reaction of PM_2.5 inflammation activates a variety of vasoactive substances,among which serum inflammatory factor IL-1β,IL-18,IL-10,molecule involved in vascular adhesion ICAM-1,VCAM-1 and platelet activation marker sCD40L,P-selectin can be used as synthetic evaluation index for the cadiovascular toxicity induced by PM_2.5.ROS can not be ignored in the process of PM_2.5-induced cadiovascular endothelial inflammatory reaction activation and cascade reaction enhancement.NOX1 can influence the level of IL-1β,IL-18,IL-10,ICAM-1 through ROS to regulate vascular endothelial injury caused by PM_2.5,which result from PM_2.5 acting directly on cadiovascular endothelial cells,or systemic response produced by PM_2.5 entering the body through the respiratory tract and affecting ROS and vascular inflammatory factors in multiple organs such as lung and kidney.Metal components of PM_2.5 play an important role in the process of cadiovascular endothelial inflammatory injury which involved in many biological processes and pathways related to oxidative stress,inflammatory response,and adhesion reaction.In the cardiovascular endothelial inflammatory injury caused by metal components of PM_2.5,the toxic effects of Sb,As,Al and Mn should be paid close attention to.