| Background and Objective:Background:The protection of ischemic preconditioning(IPC)on ischemic myocardium is one of the research hotspots in the prevention and treatment of ischemic cardiomyopathy in recent years.In recent years,a large number of studies have confirmed that IPC can protect ischemic myocardium.IPC enhances myocardial resistance to subsequent ischemic damage through transient and reversible myocardial ischemic attacks.As a kind of stress,exercise of a certain intensity can also cause ischemia and hypoxia of myocardial cells.Repeated short exercise of a certain intensity can induce temporary and repeated ischemia of the myocardium,thus producing IPC and protecting the ischemic myocardium,which is exercise preconditioning(EP).Studies have shown that EP can activate multiple signaling pathways in vivo through a variety of different ways to regulate the expression of apoptotic related genes,thereby reducing the apoptosis of cardiomyocytes and protecting ischemic myocardium.Our previous study found that EP can increase the expression of the anti-apoptotic gene Bcl-2,reduce the expression of the pro-apoptotic gene Bax,and reduce the apoptosis of cardiomyocytes.In addition,some studies have found that the number of mitochondrial autophagy in cardiomyocytes increases after EP,while the number of apoptotic cells significantly decreases.EP may exert the self-protection effect of cardiomyocytes by inducing mitochondrial autophagy.The specific mechanism by which EP alleviates the apoptosis of cardiomyocytes has not been fully clarified yet.EP may stimulate the body to produce many signals and activate multiple signal transduction pathways in vivo,such as phosphatidlinositol-3-kinase(PI3K)/protein kinase B(AKT)(PI3K-AKT),AMPK-mTOR-UKL1,etc.Among them,PI3K-Akt signaling pathway is one of the most important signal transduction pathways in cells.However,so far,which signal pathway is the specific one through which EP regulates the expression of apoptotic related genes,is there any correlation between mitochondrial autophagy and apoptosis,and what is the specific relationship between the two?It is not yet fully understood.Objective:To Explore the role of PI3K-Akt signaling pathway in exercise preconditioning in inducing mitochondrial autophagy and reducing cardiomyocyte apoptosis.Methods:1.The experimental group:Select 40 healthy SD rats and randomly divide them into exercise preconditioning+acute myocardial ischemia group with 20 rats(group A),PI3K-Akt inhibitor+exercise preconditioning + acute myocardial ischemia group with 20 rats(group B);2.Model establishment:The exercise preadaptation model was established in group A and group B by intermittent swimming training for 4 weeks with A weight load of 3%on the tail of rats(the metal thread was wrapped around the tail of the rats).After the last exercise,rats in group B were immediately given PI3K-Akt inhibitor LY294002(0.3 mg/kg)via rat tail vein,and rats in group A were simultaneously injected with the same amount of normal saline.30min later,the acute myocardial ischemia model of rats in group A and group B was established by intraperitoneal injection of isoproterenol(3mg/kg body weight).3.Heart sampling:After completing the above steps,the rats were killed immediately to take heart tissue and divided into several parts.Part was used for TUNEL assay to detect myocardial cell apoptosis and immunohistochemical assay.Some of them were made into sections,and the ultrastructure of mitochondria was observed by electron microscopy and the autophagosomes were counted.The remaining tissues were placed in EP tubes and stored in a refrigerator at-80℃ for detection of the protein expression of Akt,p-Akt,LC3-Ⅱ and Beciln-1.4.Apoptosis of cardiomyocytes:The apoptosis of cardiomyocytes was detected by TDT-mediated dUTP notched end labeling(TUNEL)method,and the apoptosis index(AI)was calculated,AI(%)=number of apoptotic cells/total number of cardiomyocytes×100%;5.Mitochondrial autophagy detection:light microscopy was used to observe the changes of myocardial cell microstructure;The ultrastructural changes of cardiomyocytes and the formation of autophagosomes were observed by transmission electron microscopy and the number of autophagosomes was counted.6.Related protein detection:The protein expressions of Akt,p-Akt,LC3-Ⅱ and Beciln-1 were determined by Western blot.7.Comparison of results:The apoptosis index(AI),the number of autophagosomes,and the expression of Akt,p-Akt,LC3-Ⅱ,and Beciln-1 protein were compared between A and B groups of rats,respectively.Results:1.Survival rate of rats in the two groups:After the experiment,18 rats in group A and 15 rats in group B survived.2.The protein expression levels of Akt,P-Akt,LC3-Ⅱ and Beciln-1 in the two groups of rats:2.1 The expression levels of PI3K-Akt signaling pathway related proteins in the two groups of rats:Compared with group A,the expression level of Akt protein in group B was significantly decreased(608.8±43.37 vs 435.4±50.68),(P<0.05);Compared with group A,p-Akt protein expression in group B was significantly decreased(378.66±75.21 vs 135.6±36.79),(P<0.05);This indicates that LY294002 can effectively inhibit the expression of Akt and phosphorylated Akt in the PI3K-Akt signaling pathway.2.2 The expression levels of myocardial mitochondrial autophagy-related proteins in the two groups of rats:Compared with group A,the expression level of LC3-II protein in group B decreased significantly(309.5±44.78 vs 184.2±83.49),(P<0.05);compared with group A,the expression level of Beciln-1 protein in group B decreased significantly(422.7±20.56vs 209.5±67.12),(P<0.05);3.Changes in the microstructure of myocardial cells and formation of autophagosomes in the two groups of rats:3.1 Changes in the microstructure of myocardial cells in the two groups of rats:Under light microscopy,the TUNEL staining of myocardial tissue shows long strips of pink myocardial fibers,blue normal cells,and brown or tan apoptotic cells.The sections of group A showed regular myocardial fibers with small space,A large number of blue normal nuclei and A few scattered brown apoptotic nuclei.The myocardial fibers of rats in group B were enlarged and deformed,disorderly arranged,with large intercellular space,and a large number of brown apoptotic cells and inflammatory cells could be seen..3.2 Changes in mitochondrial ultrastructure and autophagosome formation of myocardial cells in the two groups of rats:Swelled mitochondria were observed in group A rats,with different sizes and disordered arrangement,and occasionally A few vacuolated autophagosomes were observed.Under electron microscope,the number of normal mitochondria in group B was significantly reduced,a large number of deformed and edematous mitochondria were visible,the structure of mitochondrial bilayer membrane was blurred or disappeared,the mitochondrial ridges were deformed and fused to varying degrees,and a large number of vacuolated autophagosomes were visible.Compared with group A,the number of autophagosomes in group B was significantly decreased(23 VS 15),(P<0.05)..4.Apoptosis of myocardial cells in the two groups of rats:Compared with group A,brown-brown apoptotic cells were widely distributed in group B.Compared with group A,the myocardial apoptosis index of group B was higher(0.215±0.004 vs 0.327±0.003),(P<0.05).Conclusion:1.The PI3K-Akt signaling pathway may be one of the key pathways for exercise preconditioning to reduce the apoptosis of ischemic myocardial cells.2.Exercise preconditioning can increase the expression of mitochondrial autophagy-related proteins LC3-II and Beciln-1 protein,increase mitochondrial autophagy,and reduce cell apoptosis. |
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