| Background and objective:Because of the high morbidity,high mortality and high hospitalization costs,acute myocardial infarction(AMI)urgently needs our attention and should be provided more complete treatment strategies.Reperfusion is currently the standard treatment of acute myocardial infarction,which can restore myocardial infarction blood flow,to rescue damaged myocardial cells.Reperfusion after myocardial ischemia also can reduce myocardial infarct size,improve long-term myocardial function,and reduce mortality.However,myocardial ischemia and reperfusion is a"double-edged sword",which can aggravate the damage of cardiac function,accelerate the death of myocardial cells and cause various malignant arrhythmias,which is called myocardial ischemia/reperfusion(I/R)injury.Therefore,early treatment of myocardial I/R injury is crucial to improve the prognosis of patients with acute myocardial infarctionThe development of myocardial I/R injury involves multiple mechanisms.The damage of endothelial cells(ECS)and the destruction of microvascular barriers have been proved to be the key process of myocardial I/R injury.Myocardial I/R injury can up-regulate the expression of surface adhesion molecules and recruit neutrophils,disrupt endothelial barrier function and increase microvascular endothelium permeability,thereby aggravating inflammatory infiltration and no-reflow phenomena caused by I/R injury.Therefore,maintaining the integrity of endothelial cells makes it a potential target for the treatment of myocardial I/R injury.Heat shock protein A12B(HSPA12B)is a newly discovered member of the heat shock protein family and specifically expressed in vascular endothelial cells.It may be involved in endothelium-related pathophysiological processes and may reduce the heart remodeling in the period of chronic ischemia of irreversible reperfusion of myocardium.However,myocardial I/R injury is different from permanent myocardial ischemia and has its own unique pathophysiological process.The previous study of our research group have found that HSPA12B has a protective effect on myocardial I/R injury,but its effect on the integrity of endothelial cells induced by acute myocardial I/R injury and its mechanism is still unclear.This subject further studied the effect of HSPA12B on the integrity of endothelial cells induced by myocardial I/R injury on the results of previous experiments,to investigate whether HSPA12B protects myocardial I/R by maintaining the integrity of endothelial cells during I/R injury.Methods:1.The experimental animal model:Transgenic mice(HSPA12B Transgenic,Tg)with human hspa12b gene created in our previous research,aged 8 to 10 weeks were used for the experiment,and the wild type(WT)male mice were used as control.We research group cooperated with the Nanjing University Model Animal Research Institute.The experimental animals used were kept in the SPF animal room of the Animal Model Institute of Nanjing University.2.I/R model:After anesthetized mice with 1.5%-2%isoflurane inhalation,opened its chest and exposed the heart,and then ligated the left anterior descending branch of the coronary artery.After 45 minutes,the ligature was released and the reperfusion began.The sham group did not occlude the left anterior descending coronary artery,and the remaining surgical procedures were the same as the I/R group.3.Myocardial microvascular blood flow during reperfusion:When reperfusion began after myocardial ischemia,25 μ1 FITC-labeled tomato lectin was directly injected into the mice from the caudal vein.After 30 minutes of micro vascular blood flow reconstruction,isolated the heart and made 4μm transverse sections at the level of cardiac papillary muscles,then observed by fluorescence microscopy.4.Evan’s blue dye extravasation:When reperfusion began after myocardial ischemia,Evans dye was injected into mice at the body weight of 3.2 μg/g from the caudal vein.After 30 minutes of myocardial reperfusion,flushed the coronary network from the left ventricle with 4%paraformaldehyde until the perfusion fluid from the right ventricle cleared;isolated the heart and photographed.Ventricular tissue was concussed and dissolved for 24 h at 60 ℃ with formamide;centrifuged,and read supernatant(610 nm)to quantitatively quantify the amount of Evans’ blue dye leakage.Evans ’ blue dye leakage was expressed as absorbance per gram of myocardial tissue.5.Ultrastructure observation of endothelial cells:The heart tissue was cut into 1mm2 small pieces,fixed,dehydrated,embedded,and solidified,and cut into ultra-thin sections of 60-70nm using an ultramicrotome,and then collected with 200 mesh copper mesh.double stained with Uranyl acetate and lead citrate and observed under a transmission electron microscope.6.The expression of tight junction protein ZO-1:The mRNA expression level of ZO-1 in myocardial tissue was detected by real-time quantitative PCR,while the expression of ZO-1 in myocardial endothelial cells was observed by ZO-1 and CD31 double immunofluorescence staining.7.Neutrophil infiltration:Immunofluorescence staining for neutrophil infiltration in the myocardial tissue.8.Expression level of adhesion molecules:Real-time quantitative PCR was used to detect mRNA expression levels of myocardial adhesion molecules ICAM-1 and VCAM-1.9.Statistical Analysis:Each group of measurement data was processed using GraphPad Prism 6.0.All data were expressed as mean ± standard deviation(X±SD).The comparison between the two groups of data was performed using the t-test.The comparison between the multiple groups of data was performed using one-way analysis of variance(ANOVA).P<0.05 indicates statistically significant difference between data.Results:1.Overexpression of HSPA12B reduced the no-reflow phenomenon of myocardial microvascular blood flow during myocardial ischemia-reperfusion.2.Overexpression of HSPA12B reduced the myocardial microvascular high permeability induced by myocardial I/R injury.3.Overexpression HSPA12B increased the mRNA expression of anti-permeability molecules after myocardial I/R injury and decreased the mRNA expression of promoting permeability molecules4.Overexpression of HSPA12B protected the integrity of microvascular endothelial cells structure and function after myocardial I/R injury.5.Overexpression of HSPA12B maintained the expression of ZO-1 in endothelial cells after myocardial I/R injury.6.Overexpression of HSPA12B reduced myocardial neutrophil infiltration after I/R injury.7.HSPA12B reduced myocardial neutrophil infiltration after I/R injury by modulating VCAM-1 expression.Conclusions:HSPA12B can reduce the infiltration of neutrophils induced by myocardial ischemia-reperfusion injury and maintain the integrity of myocardial microvascular endothelium,thus protecting myocardial damage caused by ischemia-reperfusion... |
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