Study On The Pathological Changes Of Hyperacute Myocardial Infarction And The Effectiveness Of Salvianolate Injection

Acute myocardial infarction(AMI)remains one of the most common causes of death globally.The hyperacute phase(reversible phase)of myocardial infarction refers to 30 min after myocardial infarction.Generally considering,this short duration is a problematic diagnosis with reversible myocardial injury,so the related pathophysiological mechanism is seldom studied.However,the hyperacute phase of myocardial infarction can easily cause ventricular fibrillation,which increases the risk of sudden death in patients with myocardial infarction and causes severe adverse consequences.With the development of proteomic technology,these changes can be qualitative and quantitative,which means the pathological changes,diagnosis and drug intervention of the beginning of myocardial infarction could be deeply researched.This may benefit to understand the pathological and regulation mechanism of myocardial infarction.The discovery of related biomarkers will be helpful for clinical prevention and diagnosis;The recognition of regulatory effect helps to discover drug targets and to explore the mechanisms of tradition Chinese medicine.Aims:To discuss the pathological changes in the time course of hyperacute myocardial infarction in vivo and in the protein level.Then,to explore the intervention effect and mechanism of salvianolate injection in this period,based on the key protein involved in the course of myocardial infarction,HSPB6.Method:Chapter 1:The hyperacute myocardial infarction model was established by ligation of left anterior descending(LAD)branch.SD rats were randomly divided into sham group,1 min group,5 min group,10 min group,15 min group,20 min group,30 min group and 60 min group.The elevation change of the ST segment was recorded by an electrocardiogram;and the chocardiography was used to detect cardiac function.After the animals were sacrificed according to time after operation,their blood was collected from abdominal aorta.The platelet aggregation rate,hemorheology index and coagulation(PT,APTT,TT,FIB)were detected by biochemical method.The heart tissues were collected for HE staining and ultrastructure observation by transmission electron microscopy.The serum was separated to determine the concentration of the cell factors by using ELISA kits.The cell factors were including myocardial damage factors:creatine kinase isoenzyme(CK-MB)and lactate dehydrogenase(LDH);the endothelial damage factors:reactive oxygen species(ROS),endothelin-1(ET-1),endothelial nitric oxide synthase(eNOS);the platelet aggregation factors:prostacycline I2(PGI2),thromboxane A2(TXA2);and the myocardial stress factors:cardiac natriuretic peptide(ANP),5-hydroxytryptamine(5-HT).The energy metabolism member,ATP,was detected by chemiluminescence kit.Chapter 2:The model was established as above.The left ventricular tissue of 2 mm below the ligation site was collected and processed by FASP method.After proceeding cleavage,quantification,reduction,alkylation,and digestion,the label-free proteomic analysis was performed using a Q-Exactive plus mass spectrometey.Proteome Discoverer software and Maxquant software were used for qualitative and quantitative analysis of protein information.Then,the of differential expression proteins was analyzed by bioinformatics methods to investigate their classification,function,cell localization,biological pathway,biological function and interaction network.The ggplot tool of R package,OriginGraphPad Prism 6.0.1 and BioLadder website were used to draw the analysis diagrams.The results of proteomics were verified by animal experiments.The model establishment was the same as the first chapter.The oxygenation level of peripheral artery and venous blood was detected by using a blood gas analyzer;The hypoxia level of myocardial tissue was investigated by using Hypoxyprobe-1 hypoxia probe;And the myocardial microvessels was counted by using immunohistochemical(IHC)method.Meanwhile,the contents of hemoglobin subunit(HBB)and band 3 protein(Slc4a1,BAND3)in myocardial tissue were detected by Western Blot.The content and distribution of heat shock protein B6(HSPB6)and p-HSPB6 in myocardial tissue were determined by IHC and Western Blot.Chapter 3:Based on the results of proteomics and bibliometrics,salvianolate injection was identified as a potential intervention drug in hyperacute myocardial infarction.The binding potential of HSPB6 and the chemical components of salvianolate injection was predicted by molecular docking analysis.Then,the surface plasmon resonance(SPR)assay was introduced to verified the molecular affinity between magnesium salvia acetate and HSPB6.Animal experiments were used to verify the above hypotheses.SD rats were randomly divided into sham group,model group,diltiazem group,salvianolate injection 42 and 21 mg/kg groups.The animals were anesthetized and given corresponding drugs by caudal intravenous injection.Then the operation was performed 15 minutes after administration.Twenty minutes after the operation,the cardiac function of the animals was measured by echocardiography,Then the myocardial blood perfusion was measured immediately by using myocardial contrast echocardiography(MCE)before the animals were sacrificed.After blood collecting,the serum was separated,and the levels of endothelial nitric oxide synthase(eNOS)and prostacycline 12(PGI2)were detected by ELISA.Myocardial microvessels were counted by IHC staining of heart tissue.The content and distribution of HSPB6 and p-HSPB6 in myocardial tissue were determined by IHC and Western Blot.The contents of Bax,p-Akt,Bcl-2,Caspase 3,PLN,p-PLN and Ywhaz in myocardial tissue were determined by Western Blot.Results:Chapter 1:During hyperacute myocardial infarction,cardiac function has been damaged.This damage is manifested as poor cardiac function,increased levels of CK-MB and LDH,mitochondrial edema and structure destruction in myocardial cells,and decreased level of ATP in myocardial tissue.The result indicated that the most severe damage occurred on 15 minutes after myocardial infarction.However,due to the compensatory action,the heart function is slightly improved for a short time,but it is still below normal level.This compensatory effect may be associated with increased levels of cytokines that play a role in vasodilating,reduced blood viscosity,and prolonged clotting time.Chapter 2:Proteomics suggests that 15 min may be the turning point of compensatory regulation failure in hyperacute myocardial infarction.The pathogenesis of hyperacute myocardial infarction may involve oxygen transport,organic acid binding,and serine-type endogenous peptidase inhibitor activity.Besides,a series of proteins may be involved in compensatory regulation,such as:oxygen metabolism pathway proteins:Hbb-b2,Hba1,Hbb-b1,Cal,Car2,Slc4a1;Heat shock protein:HSPB6;key node proteins in PPI:Statl,Apoa1,Murc and Tpm2.Animal experiments verified that oxygen metabolism pathway and HSPB6 protein were involved in the compensatory regulation of hyperacute myocardial infarction.The results showed that the O2 content of cervical venous blood decreased.At the same time,the tissue oxygen consumption increased from 10 to 20 min,which imply the extra oxygen consumption compensated by body.During this period,at 15 min,the tHb concentration decreased in carotid blood.This may mean that part of Hb was distributed to the internal blood circulation in heart and supply O2.Meanwhile,the result shows that the number of microvessl increased,and the myocardial HBB and Slc4a1 levels increased.These may suggest that capillaries was dilated and the number of red blood cells in the anoxic area increased in this time.In the hypoxia probe experiment,the heart began to form a well-defined anoxic zone at 30 min,indicating that the compensatory regulation of the body ended and irreversible anoxic injury began to occur.During compensatory regulation,the concentration of HSPB6 and p-HSPB6 increased.It proves that HSPB6 and p-HSPB6 could actively respond and participate in the compensatory effect of myocardial protection.Chapter 3:Salvianolate injection has the potential to effect on the hyperacute stage of myocardial infarction.The molecular docking result displays that the major compound of salvianolate injection,magnesium lithospermate B,has the lowest binding energy with HSPB6,which was-14.532 kcal·mol-1.SPR assay shows that the KD between magnesium lithospermate B and HSPB6 was 13.9 μmol·L-1.The results of animal experiments demonstrated that salvianolate injection had a certain ameliorative effect on hyperacute myocardial infarction injury in rats.It may increase the level of PGI2,expand the opening of the left ventricle intimal capillaries by compensatory regulation,improve blood perfusion,and thus improve left ventricular function and protect myocardium.At the same time,low dose of salvianolate injection could better promote the content of HSPB6 and p-HSPB6 protein in myocardial infarction in the hyperacute stage.This further increases p-PLN levels to regulate myocardial shrinkage and protect cardiac function.Conclusions:Both significant myocardial injury and transient compensatory regulation are present in the hyperacute stage of myocardial infarction.The myocardial injury was manifested as decreased cardiac function,increased levels of CK-MB and LDH,decreased levels of ATP in myocardial tissue,mitochondrial edema and structure destruction,and the myocardial tissue formed an anoxic zone with clear boundaries in the end.In the compensatory regulation,the level of vasodilator factors increased firstly,then telangiectasia happened,and the red blood cells growing distribute in cardiac anoxic area to supply the tissue oxygen consumption.Besides,the blood viscosity decreased,and the clotting time prolonged.What is more,the content of HSPB6 and p-HSPB6 increased.They actively respond to and participate in the compensatory regulation of myocardial infarction in the hyperacute phase.Salvianolate injection has a certain ameliorative effect on myocardial injury in hyperacute myocardial infarction rats.It could mediate the increase of HSPB6 and p-HSPB6 protein content.On the one hand,It could improve the compensatory regulation in opening the intimal capillaries in left ventricular,and increase the level of PGI2,finally improve the blood perfusion.On the other hand,it regulates myocardial shrinkage and improves left ventricular function.