The Role Of S100A8/A9 In Fine Particulate Matter-aggravated Myocardial Ischemia And The Intervention Mechanism Of Shengmaiyin

1.Background and objectiveThe rapid development of energy-intensive industries in China in the past 30 years has brought serious environmental pollution,and the impacts on public health represented by fine particulate matter(PM)in the air has become a very important public health problem.PM enters the human blood circulation system,damages normal blood circulation,and causes cardiovascular diseases such as hypertension and coronary heart disease.The morbidity and the mortality of myocardial infarction patients were significantly increased after exposing to PM.However,the toxicological mechanism of PM exacerbating myocardial ischemia is still unclear,and the related drug intervention is still in its infancy.Our previous studies have shown that Shengmaiyin has a good effect on the intervention of PM-aggravated myocardial ischemia.But its mechanism of action needs to be further clarified.In this study,the biological pathways and molecular targets of myocardial ischemia with PM exposure in the previous experimental sample were deeply explored by the combination of transcriptomics and proteomics.We selected S100A8/A9 molecule and speculated that it plays an important role in PM-aggravated myocardial ischemia based on the bioinformatics analysis combined with toxicological studies.We further explored the expression changes of S100A8/A9 level in myocardial ischemia animals after PM exposure,and verified the correlation between S100A8/A9 level and myocardial injury,and explored whether the pharmacological effect of Shengmaiyin in the treatment of myocardial ischemia with PM exposure was related to the expression of S100A8/A9 molecule.2.Methods2.1.Analysis of characteristics of myocardial ischemia model aggravated by fine particulate matter exposure by omics mining2.1.1.Omics analysis on the characteristics of myocardial ischemia model aggravated by fine particulate exposureWe used the myocardial tissue samples of myocardial ischemia(MI)rats with PM exposure established by tracheal instillation of fine PM suspension combined with ligation of the left anterior descending coronary artery.We extracted total mRNA and total protein for transcriptome sequencing and quantitative proteomics detection.The transcriptomics and proteomics data were compared and co-analyzed to obtain differentially expressed mRNAs and proteins.The toxicological mechanism of PM exposure aggravating myocardial ischemia was further explored to screen the key molecules of PM toxicological effect.2.1.2.Preliminary validation of key targets for myocardial ischemia aggravated by fine particle exposureThe total mRNA was extracted from the myocardial tissue of MI mouse with PM exposure using the same method above.Based on the results of omics analysis,we used RT-qPCR to select and verify the key toxicological molecules.2.2.Effect of S100A8/A9 on myocardial ischemia aggravated by fine particulate matterC57/BL6N mice were randomly divided into Sham group,PM group,MI group,and MI+PM group.After adaptive feeding,PM exposure was performed twice in the second week,and myocardial ischemia model was established in the third week.After 48 h of MI modeling,the following tests were performed:(1)TTC staining was used to evaluate the successful establishment of myocardial ischemia model;(2)HE staining to observe the pathological conditions in mice;(3)The content of serum S100A8/9 was detected by ELISA;(3)Immunofluorescence was used to analysis the expression and the co-localization of S100A9 and Ly6g protein;(4)RT-qPCR was used to detect the mRNA expression levels of proteins S100A8,S100A9,TLR4,Cxcl2,Cxcl3,Ccl2,GCSF,IL-1β and IL-6 in mouse myocardial tissues;(5)Western-blot and Simple Western were used to detect the expression levels of HIF-1α,RAGE,TLR4,NF-κB p65 and pNF-κB p65proteins.2.3.Intervention effect of Shengmaiyin on myocardial ischemia injury aggravated by fine particulate matter and the expression of S100A8/A92.3.1.Interventional effect of Shengmaiyin on the expression of S100A8/A9 in PM-aggravated MI in miceC57/BL6N mice were randomly divided into Sham group,MI+PM group,SM group(Shemngmaiyin,7.8 mL/kg),PAQ group(Paquinimod,10 mg/kg)and PAQ+SM group.After administration:(1)TTC staining was used to evaluate the successful establishment of myocardial ischemia model;(2)HE staining was used to observe the pathological conditions in mice;(3)The molecular level of serum S100A8/9 was detected by ELISA;(4)RT-qPCR was used to detect the mRNA expression levels of S100A8,S100A9,TLR4,Cxcl2,Cxcl3,Ccl2,G-CSF,IL-1β and IL-6 in mouse myocardial tissues;(5)Western-blot and Simple Western was used to detect the changes in the expression levels of HIF-1α,RAGE,TLR4,NF-κB p65,and p-NF-κB p65 proteins,and the correlation between Shengmaiyin and S100A8/A9 in the treatment of PM-aggravated MI injury.2.3.2.Effect of Shengmaiyin on cell injury and expression of S100A8/A9 in H9c2 cells exposed to glucose and oxygen deprivation combined with PMH9c2 cells were divided into NC group,PM group,LO group,LO+PM group,PAQ group,SM(1 μg/mL)group,SM(2 μg/mL)group and SM(4 μg/mL)group.Cells were simulated by adding PM to the medium for 24 h,and myocardial ischemia was simulated by deprivation of glucose and oxygen for 8 h.The morphological changes of cells were observed.The mRNA expression levels of S100A8 and S100A9 were detected by RT-qPCR,and the protein expressions of RAGE,NF-κB p65 and p-NF-κB p65 were detected by Simple Western,to explore the stress response of myocardial cells to PM exposure combined with ischemia and hypoxia and the intervention effect of Shengmaiyin on the cell’s deprivation of glucose and oxygen.3.Results3.1.Analysis of characteristics of myocardial ischemia model aggravated by fine particulate matter exposure by omics miningBased on the bioinformatics analysis of transcriptomics and proteomics,the biological process of PM exposure model and the molecules enriched in the process were obtained,that is chemokine signaling pathways(Cxcl2,Cxcl3,S100A8,S100A9,Vegfa,C5,Lgals3,Pf4),apoptosis(Bcl211,Vegfa,Bcl2111,S100A8,S100A9,Rnf34,Casp2,Becn1,Apip,Xiap,Ntnl),natriuretic peptide(Npr3,Atpla2,Corin,Tnni3k,Agt,Nppb,Nppa),angiogenesis(C3,Lgals3,C5,Flt1,Vegfa,Pgf,Nppb,Agt,Pf4),complement-coagulation cascade(C9,C7,C5,C4a,C3,Serp2,Serpingl).According to the results of RT-qPCR,it is speculated that S100A8 and S100A9 are key molecules in PM exposure-aggravated myocardial ischemia.3.2.Effect of S100A8/A9 on myocardial ischemia aggravated by fine particulate matterIn the PM-aggravated myocardial ischemia mice:(1)TTC staining showed that the Sham group and PM group were red without obvious ischemic area;the overall color of MI group and MI+PM group was pale,and there were obvious ischemic areas;(2)Lung and heart histopathology:Sham group had complete lung tissue structure and thin alveolar wall.Compared with Sham group,PM and MI+PM group had thicker alveolar wall and disordered structure;Sham group had complete myocardial tissue structure,neat arrangement of myocardial cells,clear muscle striation,and no tissue necrosis and inflammatory tissue infiltration.Compared with Sham group,MI+PM group showed obvious myocardial fiber breakage and cell necrosis,accompanied by obvious bleeding,inflammatory infiltration and connective tissue hyperplasia;compared with MI group,myocardial structural damage and inflammatory infiltration in MI+PM group were more serious.(3)The effect of PM exposure combined with myocardial ischemia on the expression of S100A8/A9:Compared with Sham group,MI+PM group showed a large number of S100A9 positive expression in myocardial tissue,and the S100A9 positive cell rate in myocardial tissue was significantly upregulated(P<0.001),and the average fluorescence intensity was significantly increased(P<0.001).The S100A8 mRNA was significantly up-regulated(P<0.001),and the S100A9 mRNA was significantly up-regulated(P<0.001).Compared with MI group,the positive rate of S100A9 and the average fluorescence intensity were significantly different(P<0.005,P<0.05),S100A8 and S100A9 mRNA levels were significantly different(P<0.05,P<0.001).(4)Effects on the infiltration of Ly6g positive cells in myocardial tissue:Compared with Sham group,MI+PM group had an up-regulate of Ly6g expression in myocardial tissue,and the cell rate of Ly6g positive expression in myocardial tissue was significantly up-regulated(P<0.001),fluorescence intensity increased(P<0.001).Compared with MI group,the positive cell expression rate and fluorescence intensity were significantly different(P<0.001,P<0.001).(5)Colocalization of S100A9 and Ly6g in myocardial tissue:the Pearson Correlation Coefficient and Mander’s Overlap Coefficient of S100A9 and Ly6g in myocardial tissue of each group were ranged from 0.6 to 0.8.(6)The changes of the upstream and downstream related molecules of S100A8/A9 involved pathways:Compared with the Sham group,RAGE protein levels decreased(P<0.005);the expression levels of TLR4 at mRNA and protein levels in MI+PM group had no significant change;NF-κB p65 phosphorylation was also increased(P<0.05).(7)The expression levels of chemokines and inflammatory factors:compared with the Sham group,the levels of G-CSF,Ccl2,Cxcl2 and Cxcl3 in MI+PM group were increased(P<0.05,P<0.005,P<0.05,P<0.001).IL-1β and IL-6 levels were significantly increased(P<0.001,P<0.001);compared with the MI group,IL-6 and Ccl2 were further up-regulated(P<0.001,P<0.005),and Cxcl2 expression was down-regulated(P<0.001).3.3.Intervention effect of Shengmaiyin on myocardial ischemia injury aggravated by fine particulate matterAfter Shengmaiyin administration,PM exposure combined with myocardial ischemia mice model:(1)TTC staining results showed that the ischemic area of SM group,PAQ group and PAQ+SM group were improved in varying degrees.(2)Lung and heart tissue histopathology:Compared with MI+PM group,the pathological structure of lung tissue in SM group was improved and the alveolar wall was thin;the pathological structure of SM myocardial tissue was improved,the number of necrotic cells was reduced,and the inflammatory infiltration was alleviated.(3)Effect of Shengmaiyin on the expression of S100A8/A9:Compared with MI+PM group,the fluorescence intensity of S100A9 in myocardial tissue was decreased,but the positive rate of S100A9 cells in myocardial tissue was not significantly decreased,S100A8 mRNA expression was significantly decreased(P<0.001),and S100A9 mRNA expression was decreased(P<0.01).(4)Compared with MI+PM group,the positive expression rate and the fluorescence intensity of Ly6g decreased in myocardial tissue of SM group(P<0.001,P<0.005)(5)The changes of the upstream and downstream related molecules of S100A8/A9 involved pathways:Compared with MI+PM group,SM group has no significant changes in RAGE protein levels;the expression of TLR4 mRNA in myocardial tissue was up-regulated(P<0.05),and no significant change in protein expression;the expression level of HIF-1α had a trend of adjustment but no statistical significance.NF-κB p65 phosphorylation was not significantly decreased.(6)The expression of chemokines and inflammatory factors:compared with the MI+PM group,the IL-1β and IL-6 levels in myocardial tissue of SM group were significantly decreased(P<0.001,P<0.001);G-CSF level did not change significantly,while the levels of Ccl2,Cxcl2 and Cxcl3 increased(P<0.005,P<0.001,P<0.001).(7)In H9c2 cells,compared with NC group,S100A8 and S100A9 mRNA levels were downregulated in PM group(P<0.05,P<0.05).In LO+PM group,S100A8 mRNA showed an upward trend,S100A9 mRNA expression was up-regulated(P<0.005),and RAGE protein levels and NF-κB p65 phosphorylation levels were increased(P<0.001,P<0.001).Compared with LO+PM group,the expression of S100A8 was decreased in SM(2 μg/mL)group(P<0.05),and the expression of S100A9 was decreased in SM(1μg/mL)group,SM(2 μg/mL)group and SM(4 μg/mL)group(P<0.01,P<0.05,P<0.001).RAGE level did not change significantly;the phosphorylation of NF-κB p65 in SM(1μg/mL)group and SM(4μg/mL)group decreased(P<0.005,P<0.01).4.Conclusion1)In the mice model of PM exposure combined with myocardial ischemia,the expression levels of S100A8 and S100A9 in ischemic myocardial tissue were significantly increased,and neutrophil infiltration was obvious.The expression levels of inflammatory factors IL-6 and chemokine Ccl2 in myocardial tissue were further increased.The expression of S100A9 in myocardial cell PM exposure combined with glucose-oxygen deprivation model was significantly up-regulated.S100A8/A9 is one of the key molecules of PM exposure aggravating myocardial ischemia injury.2)Neutrophils are one of the main sources of increased S100A8/A9 expression in ischemic myocardium under PM exposure,and myocardial cells may be partially involved in this pathological process.3)The regulatory effect of Shengmaiyin on the expression of S100A8 and S100A9 is one of the ways to alleviate the myocardial ischemia injury aggravated by PM exposure.