Study On The Effect And Mechanism Of Shenxiong Glucose Injection On Myocardial Injury Based On Myocardial Injury Models In Vivo And In Vitro

Aim:Cardiovascular disease is one of the most common causes of human death.Shenxiong Glucose Injection（SGI）is commonly used in clinical treatment of cardiovascular and cerebrovascular diseases.It has significant curative effects on ischemic stroke,insufficient blood supply of vertebral base artery,myocardial infarction,angina pectoris,heart failure and other diseases.Since its listing,SGI has been widely used in clinic and has remarkable curative effect,but there are few reports on its mechanism of action.Based on the above background,this study used the animal models of myocardial injury in vivo and in vitro to explore the effect and mechanism of SGI on myocardial injury.In order to provide new ideas and methods for the treatment of myocardial ischemia reperfusion injury（MIRI）and arrhythmia,meanwhile,it’s also conducive to the development of new indications of SGI,which has important clinical significance and socio-economic value.Methods:The research is mainly divided into four parts.Experiment 1:To evaluate the efficacy of SGI against MIRI and arrhythmia induced by MIRI through isolated rat heart perfusion model.The changes of relevant electrocardiogram（ECG）indexes at five time points after administration of isolated rat hearts in each group were recorded,and the contents of lactate dehydrogenase（LDH）,creatine kinase（CK）,cardiac troponin I（c Tn-I）in myocardial tissue were measured.The ST segment elevation of ECG and the occurrence of arrhythmia were observed.Experiment 2:Based on network pharmacology and molecular docking,the anti MIRI mechanism of SGI was predicted.Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）,Gene Cards,and Online Mendelian Inheritance in Man（OMIM）databases were used to obtain compounds and disease related targets,and the intersection genes were screened.The"drug-components-disease-targets"network was constructed by Cytoscape software.The protein interaction was analyzed by STRING platform.Gene Ontology（GO）function and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment were analyzed by R software,and the molecular docking between active components and core targets was carried out by Autodock-Vina software.Experiment 3:To explore the protective effect of SGI on ouabain induced arrhythmia in mice.BL-420N biological function experimental system was used to record the lead II ECG of mice,and ouabain was injected into caudal vein to establish the arrhythmia model of mice.The number of ventricular premature contractions,the duration and incidence of ventricular tachycardia,ventricular fibrillation and conduction block within 1 hour after ouabain injection were recorded.The arrhythmia symptoms of mice were scored.After ECG recording,the eyeballs were removed for blood and heart tissue,the content of related enzymes in serum was detected,and the changes of myocardial tissue structure were observed by hematoxylin-eosin staining（H&E staining）.Experiment 4:The effect and mechanism of SGI on zebrafish myocardial injury model induced by aconitine.The changes of heart rate and heart morphology of zebrafish were observed and recorded under the microscope.The pericardial height,ejection fraction,fractional area change,and stroke volume were measured.The apoptosis of cardiomyocytes was detected by acridine orange staining,and the expression of zebrafish related genes was detected by real-time quantitative polymerase chain reaction（RT-qPCR）.Results:Experiment 1:In the isolated rat heart perfusion model,compared with the normal control group,the maximum increase/decrease rate of left ventricular internal pressure(±dp/dt_max)and left ventricular systolic pressure（LVSP）in the model group decreased significantly after administration;Left ventricular end-diastolic pressure（LVEDP）,heart rate,the number of arrhythmias,the incidence of ST segment elevation in ECG,and the contents of LDH,CK,and c Tn-I in myocardial tissue increased significantly.Compared with the model group,the±dp/dt_maxand LVSP in other treatment groups were significantly increased;LVEDP,the number of arrhythmias,and the contents of LDH,CK,and c Tn-I in myocardial tissue decreased significantly.Except the SGI high dose group,the heart rate of other treatment groups decreased compared with the model group.Compared with the model group,the incidence of ST segment elevation in SGI medium and low dose groups decreased,and the incidence of ST segment elevation in SGI high dose group and tanshinone II A group decreased significantly.This shows that SGI has a significant effect on myocardial injury and arrhythmia caused by isolated cardiac perfusion model in rats.Experiment 2:The results show that there are 15 potential targets of Danshensu and Tetramethylpyrazine against MIRI,involving cyclooxygenase pathway,extracellular matrix binding,antioxidant activity,and other processes,and closely related to platelet activation,regulation of lipolysis in adipocytes,and other pathways.Danshensu and Tetramethylpyrazine can form stable conformations with low binding energy with core targets prostaglandin G/H synthase 2（PTGS2）,vascular endothelial growth factor A（VEGFA）,and acetylcholinesterase（ACh E）.Experiment 3:In the ouabain induced mouse arrhythmia model,compared with the normal control group,the model group mice showed obvious arrhythmia symptoms:ventricular premature contraction,ventricular tachycardia,ventricular fibrillation,and conduction block.After ouabain injection,heart rate decreased and RR interval increased in all groups.The heart rate of the model group was significantly lower than that of other treatment groups,and the RR interval was significantly higher than that of other treatment groups.Compared with the normal control group,the level of serum aspartate aminotransferase（AST）in the model group increased significantly.Compared with the model group,the level of serum AST in other treatment groups decreased significantly.Compared with the model group,the incidence and duration of ventricular tachycardia,ventricular fibrillation,and arrhythmia score in the normal control group,SGI administration groups,and lidocaine positive control group were significantly reduced.At the same time,high-dose SGI would not induce arrhythmia symptoms in normal mice.The results of H&E staining showed that compared with the normal control group,pathological damage phenomena such as cell arrangement disorder and lymphocyte infiltration appeared in the myocardial tissue of the model group,while the myocardial tissue damage of other treatment groups was improved to varying degrees.Experiment 4:Compared with the control group,zebrafish in aconitine treated model group showed obvious symptoms of myocardial injury,such as pericardial and yolk sac edema,changes in atrial and ventricular positions,etc;heart rate and pericardial height increased significantly;ejection fraction,ventricular surface area change rate,and stroke volume decreased significantly.Compared with the model group,SGI drug treatment group can significantly reduce the edema of pericardium and yolk sac of zebrafish caused by aconitine;significantly antagonized the increased heart rate and pericardial height of zebrafish caused by aconitine;significantly inhibited the decrease of ejection fraction and ventricular surface area change rate of zebrafish caused by aconitine;restored the decrease of stroke volume caused by aconitine in zebrafish to a certain extent,but the difference was not statistically significant.Acridine orange staining showed that compared with the blank control group,there was obvious dense green fluorescence in the heart of zebrafish in the model group,indicating that aconitine can promote cardiomyocyte apoptosis and cause myocardial injury.The green fluorescence of SGI drug treatment group decreased obviously,indicating that SGI can alleviate the apoptosis of zebrafish cardiomyocytes caused by aconitine.The results of RT-qPCR showed that the expression of zinc finger transcription factor GATA binding protein 4（GATA4）,atrial natriuretic peptide（ANP）,brain natriuretic peptide（BNP）,cysteine aspartic acid protease 3（Caspase-3）,and Caspase-8 m RNA in the model group increased significantly compared with the normal control group.Compared with the model group,SGI drug treatment group can significantly reduce the expression of GATA4,ANP,BNP,Caspase-3,and Caspase-8m RNA.Compared with the normal control group,the m RNA expression of specificity protein 1（Sp1）,Guanylyl cyclase-A（GC-A）,cyclic guanosinc monophosphate（c GMP）-dependent protein kinase 1b（Prkg1b）in the model group decreased significantly.Compared with the model group,SGI drug treatment group could significantly increase the expression of Sp1,GC-A,and Prkg1b m RNA.Compared with the normal control group,the expression of Prkg1a m RNA in the model group decreased to some extent,and compared with the model group,the expression of Prkg1a m RNA in SGI drug treatment group increased slightly,but there was no significant difference.Conclusions:Three animal models of isolated rat heart perfusion,ouabain induced arrhythmia in mice,and zebrafish myocardial injury induced by aconitine were successfully established.According to the experimental results,SGI has a protective effect on myocardial injury and arrhythmia caused by isolated rat heart perfusion model,and also has a significant inhibitory effect on ouabain induced arrhythmia in mice.The realization of the effect of SGI may be related to improving myocardial systolic and diastolic function,reducing the release of LDH,CK,and c Tn-I,inhibiting ST segment elevation,changing heart rate,and slowing down myocardial tissue injury.The mechanism of SGI against MIRI was predicted by network pharmacology and molecular docking.It is found that SGI may treat MIRI through biological processes such as cyclooxygenase pathway,and involves molecular functions such as extracellular matrix binding and antioxidant activity,which is closely related to signal pathways such as platelet activation and the regulation of adipocyte lipolysis.SGI can also antagonize the myocardial injury of zebrafish induced by aconitine,alleviate the enlargement of pericardium and yolk sac,and improve the abnormalities of cardiac function indexes such as heart rate,stroke volume,ventricular surface area change rate,pericardial height,and ejection fraction.Acridine orange staining and RT-qPCR results suggest that the mechanism of SGI antagonizing zebrafish myocardial injury caused by aconitine may be related to SGI inhibiting myocardial hypertrophy and cardiomyocyte apoptosis.