| BackgroundAcute myocardial infarction(AMI)has become an important risk factor for human health.Percutaneous coronary intervention(PCI)is the most effective strategy to restore blood flow in time and improve clinical outcomes.However,myocardial ischemia/reperfusion(I/R)injury caused by reperfusion can increase the infarct size and eventually lead to myocardial necrosis and remodeling.After I/R,the immune response will be initiated,and then the acute inflammatory response will be activated.Inflammatory cells such as neutrophils and monocytes will infiltrate the damaged myocardium one after another,while activated neutrophils will produce excessive biological functions and damage the myocardial tissue.The exudation of neutrophils depends on the interaction of integrins and adhesion molecules expressed on neutrophils and endothelial cells.At present,a large number of studies have shown that CD11b,a member of the integrin family,is involved in the occurrence and development of various cardiovascular diseases by mediating the adhesion and migration of inflammatory cells,but its mechanism in myocardial I/R injury is still unclear.ObjectiveTo clarify the related role and pathophysiological mechanism of CD11b in regulating the exudation and adhesion of inflammatory cells such as neutrophils and monocytes after I/R injury,and to provide experimental basis for clinical diagnosis and treatment of myocardial I/R injury and remodeling and screening new therapeutic targets.Methods1.Establishment and treatment of myocardial I/R injury modelC57BL6J male mice,aged 8-10 weeks,were randomly divided into 4 groups:sham+anti-IgG group,sham+anti-CD11b neutralizing antibody group,anti-IgG+I/R72 h group and anti-CD11b neutralizing antibody+I/R72 h group,n=10.Three days before preoperative treatment,200μL of CD11b neutralizing antibody was injected into the tail of mice every day.During the operation,the left anterior descending branch of the left coronary artery was ligated to cause myocardial ischemia for 30 min and coronary reperfusion for 6-72 h.I/R model was established.Then cardiac function was evaluated by echocardiography.2.Echocardiography detectionAn ultrasound probe(model M400;frequency 30-MHZ)was used to collect short-axis M-mode ultrasound images and long-axis slice images of the left ventricle around the left chest of the mice.Then use Vevo 1100 software to measure the inner diameter of the left ventricle and the thickness of the anterior and posterior walls of the left ventricle when the mouse heart is in the end-diastole and end-systole respectively.At least 5 inner diameters of the left ventricle should be measured for each mouse and the average value should be obtained.Finally,ejection fraction(EF%)and fractional shortening(FS%)were obtained.3.Histopathological examinationMyocardium was stained pathologically with triphenyltetrazolium chloride(TTC)and Evans blue to observe the ischemic area after ligation;myocardial tissue H&E staining was used to observe the infiltration of myocardial inflammatory cells;myocardial tissue MASSON staining was used to observe the degree of myocardial fibrosis;FAP-αstaining was used to observe the activation of cardiac fibroblasts;α-SMA staining was used to observe the transdifferentiation of cardiac fibroblasts;TUNEL staining was used to observe the degree of myocardial cell apoptosis.4.Flow cytometryFlow cytometry was used to detect the percentage of CD45~+cells and CD11b~+Ly6G~+neutrophils in the blood of mice.The inflammatory cells in the myocardial infarction area tissue of the mice were further detected by myocardial tissue flow cytometry,and the percentages of CD45~+cells and CD11b~+Ly6G~+F4/80~-neutrophils in the myocardial infarction area tissue of the mice were detected.5.Technical analysis of molecular biologyWestern Blot was used to detect the protein expression levels of CD11b,p-P65,P65,Bax,Bcl-2,Casepase-3,FAP-α,α-SMA,Collenge-III,TGF-β1,Smad2,Smad3,p-Smad2 and p-Smad3 in myocardial tissue,and to observe myocardial inflammation,fibrosis and apoptosis.The mRNA levels of integrin receptors and adhesion molecules in myocardial infarction area after myocardial I/R were detected by gene expression microarray.The mRNA levels of ICAM-1 and VCAM-1 in myocardial infarction area were detected by real-time fluorescence quantitative PCR.6.Statistical analysisSPSS22.0 software was used for statistical analysis of the experimental data,and the mean±standard deviation was used to describe.We used the t-test to compare the means of two independent samples with normal distribution,and the Mann-Whitney test for the mean of samples with non-normal distribution.And when P<0.05,the data difference is statistically significant.Result1.Ischemia/reperfusion(I/R)induced increased expression levels of CD11b(Itgam)and adhesion molecules(ICAM-1 and VCAM-1)in damaged myocardial tissue,and increased infiltration of CD11b~+neutrophils.2.Using CD11b neutralizing antibody to reduce the percentage of granulocytes in the circulating blood of mice induced by I/R and inhibit the adhesion of neutrophils to endothelial cells.3.The application of CD11b neutralizing antibody reduces the infiltration of neutrophils in the ischemic site and myocardial inflammatory response.4.Use CD11b neutralizing antibody to alleviate I/R-induced cardiac dysfunction,reduce myocardial infarct size and myocardial cell apoptosis.5.Apply CD11b neutralizing antibody to inhibit I/R-induced myocardial fibroblast activation and fibrosis,and improve myocardial remodeling.ConclusionThis study found that I/R treatment can significantly increase the levels of CD11b and ligand adhesion molecules(ICAM-1 and VCAM-1)and the proportion of CD11b~+cells in myocardial damaged tissues.The application of CD11b neutralizing antibody can significantly inhibit I/R-induced neutrophil endothelial cell adhesion and infiltration in myocardial tissue,and reduce myocardial inflammatory response,myocardial cell apoptosis and fibrosis. |
PDF Full Text Request