Protective Effect Of Astragaloside Ⅳ On Myocardial Injury Induced By Overexpression Of MicroRNA-1

ObjectiveTo study the protective effect of Astragaloside Ⅳ（As-Ⅳ）on MicroRNA-1overexpression-induced cardiomyocyte damage,and to further explore the possible molecular mechanism of the protective effect of ASⅣ.MethodsSD rats were selected as the research object and randomly divided into 4groups by random number method:normal control group（Control,C group）,miR-1 negative virus control group（miR-1 mimics NC,NC group）,miR-1 over expression group（miR-1 mimics,miR-1 group）,ASⅣ+miR-1 over expression group（ASⅣ group）.ASⅣ group was given oral administration of AS Ⅳ（80mg/Kg/d）for 1 week;miR-1 group and AS Ⅳ group were given a one-time injection of miR-1 lentivirus（1×10⁷/mL total 10mL,ventricular wall injection）,NC group was given a one-time injection of miR-1 negative virus（1×10⁷/mL total 10mL,ventricular wall injection）;after 2 weeks,Real-time PCR showed that miR-1 overexpression was regarded as a successful model.After successful modeling,ASⅣ group was given ASⅣ by oral gavage for 2 weeks;group C,NC group and miR-1 group were given the same amount of normal saline by gavage.The structure and function of the rat’s heart were examined by echocardiography;rat serum was collected and used to measure the activity of myocardial enzymes;left ventricular tissue was taken and hematoxylin-eosin（HE）staining was used to observe the morphology and arrangement of myocardial cells.Similarly,H9C2 cardiomyocytes were divided into C group,NC group,miR-1 group and ASⅣ group.ASⅣ group was given ASⅣ（10mM）protection for 4h first.Cardiomyocytes of miR-1 group and ASⅣ group were infected with miR-1 lentivirus,and the NC group was transfected with miR-1 negative virus;72 hours later,the expression of miR-1 in H9C2 cardiomyocytes was detected by Real-time PCR;cell viability was determined by MTT method Screening the optimal concentration of AS Ⅳ;Nanjing Jiancheng Ca²⁺kit to detect changes in Ca²⁺concentration in cardiomyocytes;Transmission electron microscopy to observe autophagosomes in cardiomyocytes;Real-time PCR method to detect Ca²⁺/calmodulin-dependent protein kinase（Ca MKⅡ）,ryanodine receptor type 2（Ry R2）,p62 and autophagy-related protein（LC3）Ⅱ/（40）mRNA;Western blot method to detect the expression of Ca MKⅡ,Ry R2,p62 and LC3Ⅱ/（40）protein.ResultsIn animal experiments,compared with the C group,the NC group had no significant difference in the above experimental results.The miR-1 expression in the miR-1 group was significantly increased,and the left ventricular ejection fraction（EF）and left ventricular short axis shortening rate（FS）was significantly reduced,the activities of creatine kinase（CK）,myokinase isoenzyme（CK-MB）and cardiac troponin I（CTn I）were significantly increased,and the abnormal arrangement of myocardial cells was disordered.Compared with the miR-1 group,the ASⅣ group the expression of miR-1 was significantly reduced,the EF and FS values of rats were significantly increased,the activities of CK,CK-MB and CTn I were significantly reduced,and the abnormal arrangement of cardiomyocytes was significantly reduced.In the cell experiment,compared with the C group,there was no significant difference in the results of the appeal experiment in the NC group.Compared with group C,the expression of miR-1 in the miR-1 group was significantly increased,the intracellular Ca²⁺concentration was significantly increased,the number of autophagosomes was significantly increased,and the expression of CamkⅡmRNA and protein and LC3Ⅱ/（40）mRNA and protein were significantly increased.Ry R2 mRNA and protein,and p62 mRNA and protein expression were significantly reduced;compared with miR-1 group,miR-1 expression was significantly reduced in ASⅣ group,intracellular Ca²⁺concentration was significantly reduced,the number of autophagosomes was significantly reduced,and CamkⅡmRNA And LC3Ⅱ/（40）protein expressions were significantly reduced,and Ry R2 mRNA and p62 protein expressions were both significantly increased.ConclusionsOverexpression of miR-1 can obviously cause the abnormal expression of CamkⅡand Ry R2 and autophagy-related proteins in cardiomyocytes,causing imbalance of calcium homeostasis and excessive autophagy,and ultimately leading to myocardial injury and cardiac dysfunction.ASⅣ may inhibit the expression of miR-1 in cardiomyocytes,participate in the regulation of calcium ion concentration in cardiomyocytes,inhibit excessive autophagy,and then play a protective effect on the myocardium.