| BackgroundAtherosclerosis refers to the hardening of arteries and the formation of plaques,leading to the formation of endovascular embolism,resulting in ischemic necrosis of the corresponding organs and greater harm to the body.With the improvement of Chinese people’s living standard and the change of life style,the risk factors of atherosclerosis show a diversified trend,and the incidence of vascular disease increases significantly,which has become the main threat to Chinese people’s health.Therefore,it is urgent to develop new effective methods to prevent and treat atherosclerosis.Inflammation in atherosclerosis has played a very important role in the occurrence and development,the existing research shows that many factors mediated and regulating the inflammatory response,inhibit or promote the mononuclear cell infiltrates in subcutaneous generate macrophages,inhibit or aggravate vascular smooth muscle cell proliferation and migration,thus inhibiting or accelerate the initiation and progression of atherosclerosis.Macrophages play an important role in the chronic inflammatory response to atherosclerosis.Macrophages are the main components of atherosclerotic plaques,which can promote the production of inflammatory factors and contribute to the instability of plaques.Protein tyrosine kinase(PTK)is a group of enzyme proteins that can catalyze the phosphorylation of tyrosine residues of substrate proteins and is involved in many signal transduction pathways.Spleen tyrosine kinase(Syk)is a non-receptor protein tyrosine kinase,which is expressed in a variety of cells,mainly blood cells,platelets,other vascular endothelial cells,vascular smooth muscle cells,liver cells,etc.Recently,it has been found that Syk,as a key mediator,participates in and regulates the occurrence and development of inflammatory responses.Syk promotes the occurrence and development of inflammatory response by activating the downstream signaling pathway.Recent studies have shown that there is an important connection between Syk and vascular endothelial function.Syk is of great significance in the pathogenesis of cardiovascular diseases and the inflammatory response process,especially in the pathogenesis of atherosclerosis,but the specific mechanism is not very clear.Our study researched the Syk in the molecular control mechanism in the inflammation process of atherosclerosis,and its important role in the development of atherosclerotic inflammatory reaction,such as proliferation,migration,apoptosis,and reveal the possible regulation rule.Syk is likely to be the potential target of atherosclerosis,drug research of the Syk will be one of the new directions for the development of atherosclerosis drug treatment.Obj ectives1.To investigate the expression of Syk and inflammatory cytokines in macrophages,and to observe the expression of Syk and inflammatory cytokines in tumor necrosis factor-alpha(TN F-a)induced macrophages.2.To observe the effect of Syk inhibition on the expression of Syk and inflammatory cytokines in TNF-a induced macrophages.3.To investigate the effect of TNF-a induction on the proliferation,migration and apoptosis of macrophages.To investigate the effect of Syk inhibition on TNF-a induced macrophage proliferation,migration and apoptosis.4.To investigate the effect of Syk on TNF-α induced macrophages.Methods1.Macrophage culture and interventionRAW264.7 macrophages were used for subculture.In the time-effect experiment,10ng/ml TNF-a was used to stimulate macrophages for 0,2,4,8,12 and 24h.In order to ensure that macrophages would not degrade or inactivate due to prolonged stimulation,the medium was changed every 8 hours.In the dose-response experiment,TNF-a at the concentrations of 0,5,10,20,40 and 80ng/ml were used to stimulate macrophages for 12 hours,respectively,and the cells were collected.Different concentrations of Syk inhibitor Piceatannol(0、1、10、20μmol/l)were applied to collect cells.2.Western blotMacrophages of each group were collected and proteins were extracted.The expression of Syk protein in time-effect experiment and dose-effect experiment were detected respectively.The expression of Syk protein was detected after different concentrations of Syk inhibitor were used.Test the protein expression of interleukin 6(IL-6),interleukin 12(IL-12),matrix metalloproteinases 9(MMP-9),monocyte chemotactic protein 1(MCP-1)and related signaling pathways phosphatidyl inositol 3 kinase(PI3K),silk/threonine kinase(AKT),nuclear factor kappa B predominate kinase(IKK),nuclear factor kappa B(NF-κB)before and after the application of Syk inhibitor.3.RT-PCRMacrophages of each group were collected and RNA was extracted.The expression of Syk mRNA in time-effect experiment and dose-effect experiment were detected respectively.The expression of Syk mRNA was detected with different concentrations of Syk inhibitor.4.Detection of macrophage proliferationCKK-8 assay was used to detect the proliferation of macrophages before and after TNF-a stimulation and inhibition of Syk,5.Detection of macrophage apoptosisThe apoptosis of macrophages before and after TNF-a stimulation and inhibition of Syk was detected with the apoptosis detection kit.6.Detection of macrophage migrationThe migration of macrophages before and after TNF-a stimulation and inhibition of Syk was detected by Transwell chamber method.7.Statistical analysisEach set of results came from three separate experiments.All data were represented by mean ± standard deviation(SD).Anova was used for comparison between different groups,and then Dunnett multiple comparison test was applied.SPSS 17.0 software was used for statistical analysis of all the data,and P<0.05 showed significant statistical difference.Results1.Stimulation of TNF-a on macrophagesTNF-a promoted the expression of Syk compared with the normal group.In the time-effect experiment,lOng/ml TNF-a promoted protein and mRNA expression of Syk in macrophages,reaching the maximum effect at 12 hours.In the dose-effect experiment,with the increase of TNF-a concentration,Syk protein and mRNA levels gradually increased,and the maximum effect was reached at 20ng/ml.TNF-a promoted the expression of IL-6,IL-12,MMP-9 and MCP-1 protein compared with the normal group.2.Inhibition of Syk on macrophagesCompared with the normal group and TNF-a stimulation group,Syk inhibitor could inhibit the expression of Syk protein and mRNA.Syk inhibitor Piceatannol inhibited the expression of IL-6,IL-12,MMP-9 and MCP-1 proteins compared with the normal group and TNF-a stimulation group.3.TNF-a can increase the expression of regulatory proteins in the NF-κB signaling pathway.Inhibition of Syk can inhibit the expression of regulatory proteins in the NF-κB signaling pathway.Compared with the normal group,TNF-a promoted the expression of NF-κB and its upstream regulatory proteins PI3K,AKT and IKK in the NF-κB signaling pathway.Compared with the normal group and TNF-a stimulation group,Syk inhibitor Piceatannol inhibited the expression of NF-κB and its upstream regulatory proteins PI3K,AKT and IKK in the NF-κB signaling pathway.4.TNF-a can increase the proliferation of macrophages,while Syk inhibition can inhibit the proliferation of macrophages.TNF-a increased macrophage proliferation compared with the normal group.Compared with the normal group and TNF-a stimulation group,the Syk inhibitor Piceatannol with different concentrations can significantly inhibit the proliferation of macrophages.5.TNF-a can promote the migration of macrophages,while Syk inhibition can inhibit the migration of macrophages.TNF-a promoted macrophage migration compared with the normal group.Compared with the normal group and TNF-a stimulation group,the Syk inhibitor Piceatannol with different concentrations can significantly inhibit the migration of macrophages.6.TNF-a can inhibit the apoptosis of macrophages,while Syk inhibition can promote the apoptosis of macrophages.TNF-a inhibited the apoptosis of macrophages compared with the normal group.Compared with the normal group and TNF-a stimulation group,Syk inhibitor Piceatannol of different concentrations can significantly promote the apoptosis of macrophages.Conclusion1.TNF-a can promote the expression of Syk and related inflammatory factors in macrophages.Inhibition of Syk can inhibit the expression of Syk and related inflammatory factors in macrophages.2.Inhibition of Syk can inhibit the proliferation and migration of macrophages in inflammatory response and promote the apoptosis of macrophages.3.Inhibition of Syk can inhibit the expression of related inflammatory factors in the inflammatory response and changes in macrophage function,which may be related to the NF-κB signaling pathway.BackgroundCoronary atherosclerotic heart disease(CHD)is a chronic inflammatory disease.Damage of endothelial function,formation of atherosclerotic plaques and infiltration of inflammatory cells are important mechanisms for the occurrence and development of coronary atherosclerosis(AS).CHD includes stable angina pectoris,non-st-segment elevation acute coronary syndrome,and acute st-segment elevation myocardial infarction.Inflammatory response plays an extremely important role in the occurrence and development of coronary atherosclerosis,so how to regulate inflammatory response has become a hot topic in the study of CHD.A number of studies have shown that a variety of factors mediate and regulate the inflammatory response,inhibit or promote monocyte infiltration into the endodermis to generate macrophages,inhibit or intensify the proliferation and migration of vascular smooth muscle cells,thereby inhibiting or accelerating the occurrence and development of atherosclerosis.Monocytes play an important role in the inflammatory response of CHD.Mononuclear cells are the source of a lot of inflammatory cells,mononuclear cells can differentiate into macrophages,it can promote the generation of inflammation factors causing unstable plaques.In addition,mononuclear cells are also the main components of coronary atherosclerotic plaques.Mononuclear cells can promote the transport of cholesterol to lipid nuclei,leading to the decline of plaque stability.After plaque rupture,corresponding vascular stenosis or even occlusion will be caused,resulting in ischemic necrosis of the corresponding organs,which will do great harm to the body.With the improvement of Chinese people’s living standard and the change of life style,the risk factors of CHD show a diversified trend,and the incidence of cardiovascular disease increases significantly,which has become the main threat to Chinese people’s health.Therefore,it is urgent to develop new effective methods for the prevention and treatment of CHD.Spleen tyrosine kinase(Syk)is a non-receptor protein tyrosine kinase,which is expressed in a variety of cells.Recently,it has been found that Syk,as a key mediator,participates in and regulates the occurrence and development of inflammatory responses.Recent studies have shown that there is an important connection between Syk and vascular endothelial function.Syk is of great significance in the pathogenesis of cardiovascular diseases and the inflammatory response process,especially in the pathogenesis of atherosclerosis,but the specific mechanism is not very clear.In summary,Syk plays an important role in the occurrence and development of inflammatory responses,and inflammatory response plays an important role in the occurrence and development of CHD.However,the expression and clinical significance of Syk in patients with CHD are still unclear.Therefore,this study aims to investigate the expression and possible relationship of Syk and related inflammatory factors in patients with different CHD.In order to further prove the important role of Syk in the occurrence and development of CHD,we studied the relationship between the changes of Syk and related inflammatory factors and the severity and risk factors of CHD by detecting the changes of Syk and related inflammatory factors in patients with different CHD.In this study,on the one hand,we hope to predict the occurrence of acute coronary events through the changes in the levels of Syk and related inflammatory factors;on the other hand,Syk is likely to be a potential target of anti-atherosclerosis,and drug development for Syk will be one of the new directions for the development of drugs for CHDObjectives1.To investigate the independent risk factors of CHD.2.To investigate the changes of expression levels of Syk and inflammatory factors in patients with CHD.3.To explore the correlation between the expression level of Syk and the expression level of inflammatory factors in patients with CHD.4.To explore the correlation between the expression level of Syk in patients with CHD and independent risk factors of CHD.Methods1.Patients grouping and peripheral blood collectionFrom October 2017 to March 2018,226 patients with coronary angiography who were hospitalized in the department of cardiology of Heze municipal hospital were selected,excluding those with lung disease,kidney disease,liver disease,heart failure,mental disease and those who could not participate in the study.In patients with coronary angiography when extracting peripheral blood in patients with 15 ml,the use of heparin anticoagulation,extraction into 4℃ refrigerator for follow-up after extraction of peripheral blood mononuclear cells.According to the results of coronary angiography and clinical diagnosis,the patients were divided into four groups:Control group(Control),stable angina pectoris group(SAP),non-st-elevation acute coronary syndrome group(NSTEACS),and st-elevation myocardial infarction group(STEMI).Patients in CHD group included SAP group,NSTEACS group and STEMI group.All the selected patients signed the informed consent,and this study was approved by the ethics committee of Heze municipal hospital.2.Collection of clinical data of patientsClinical data were collected according to the inpatient data of each patient.3.Separation of monocytesPeripheral blood monocytes were extracted from the received peripheral blood.4.Western blotPeripheral blood monocytes of each patient were collected,protein was extracted,and the expression of Syk,interleukin-6(IL-6),interleukin-12(IL-12),matrix metalloproteinase-9(MMP-9)and monocyte chemotactic protein-1(MCP-1)in peripheral blood monocytes of each patient was detected by Western blot.5.Statistical analysisMean± standard deviation(SD)represents the data of all measurement data,and t test is used for the comparison of measurement data between groups.Counting data were described by sample number and percentage,and the comparison of counting data between groups was conducted by chi-square test.The expression level of Syk between different groups and the expression level of related inflammatory factors were compared by ANOVA,and then Tukey multiple comparative analysis was applied.Pearson correlation analysis was used to study the correlation between the expression level of Syk and the expression level of related inflammatory factors.Multivariate logistic regression was used to analyze the risk factors of patients.Independent risk factors were analyzed by multivariate linear correlation analysis.Pearson correlation analysis was used to study the correlation between Syk expression level and independent risk factors.Each set of results came from three separate experiments.All analyses were conducted using SPSS 17.0,and a value of P<0.05 was considered statistically significant.Results1.Independent risk factors for CHDIndependent risk factors for CHD patients,including:gender,history of smoking,history of drinking,white blood cells(WBC),N-terminal pro brain natriuretic peptide(NT-proBNP),creatine kinase(CK),creatine kinase isoenzyme(CK-MB),fibrinogen(FIB),aspertate aminotransferase(AST),total amount of the target vascular lesions,number of stent placement and left ventricular fractional shortening(LVFS).2.Changes in the expression levels of Syk and inflammatory factors in patients with CHDThe protein expression levels of Syk,IL-6,IL-12,MMP-9 and MCP-1 in patients with STEMI were significantly higher than those of NSTEACS,SAP and Control groups.The protein expression levels of Syk,IL-6,IL-12,MMP-9 and MCP-1 in patients with NSTEACS were significantly higher than those of SAP and Control groups.The protein expression levels of Syk,IL-6,IL-12,MMP-9 and MCP-1 in patients with SAP were significantly higher than those of Control groups.3.Correlation between the expression level of Syk and the expression level of inflammatory factors in patients with CHDThe expression level of Syk in patients with CHD was positively correlated with the expression levels of IL-6,IL-12,MMP-9 and MCP-1.4.Correlation between the expression level of Syk and independent risk factors of CHDThe expression level of Syk in CHD patients was positively correlated with smoking history,drinking history,WBC,NT-proBNP,CK,CK-MB,FIB,AST,number of target vessel lesions and number of stent implantation in independent risk factors of CHD.The expression level of Syk in CHD patients was negatively correlated with the gender and LVFS of CHD independent risk factors.Conclusion1.The expression of Syk and related inflammatory factors were different in different CHD patients.2.The higher expression level of Syk in CHD patients,the higher expression of related inflammatory factors and the stronger inflammatory response.3.The expression level of Syk is correlated with the severity of CHD.The higher expression level of Syk in CHD patients,the higher severity of CHD patients.4.The expression of Syk is associated with independent risk factors of CHD. |
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