| Myocardial infarction(MI)is one of the serious threats to human health and lives,and is the main cause of death.After myocardial infarction,cardiomyocyte apoptosis and necrosis occur,causing cardiac dysfunction and ultimately developing into heart failure.Patients,with myocardial infarction,should be treated as soon as possible,by interventional therapy or coronary artery bypass grafting to achieve that blood flow in ischemic tissue again,which can effectively prevent the expansion of infarct size.But at the same time,drug therapy is significant for the patients.Therefore,we should study new effective drugs and their therapeutic targets from a multidimensional perspective,and then provide new strategies for the prevention and treatment of myocardial infarction with multiple targets and pathways.Histone deacetylases inhibitor(HDACI)regulates the expression of related genes at the transcriptional level by inhibiting histone deacetylases,and affects a series of biological reactions such as cell cycle,cell differentiation,and apoptosis.Sodium butyrate(NaBu),as one histone deacetylase inhibitor,plays a protective role in the treatment of myocardial infarction rats.Previous work in my laboratory showed that sodium butyrate can not only reduce the infarct size in the heart of rats with myocardial infarction,but also upregulate the expression of pyruvate kinase(PK)M2 isoform(PKM2),accelerate glucose metabolism,and increase the content of ATP in tissues.PKM2 is ubiquitous in most tissues other than brain and liver.PKM2 is the rate-limiting enzymes of glycolysis pathway in cytoplasm,capable of catalyzing ADP and Phosphoenolpyruvate(PEP)being converted to adenosine triphosphate(ATP)and pyruvate,respectively.Recent research shows that PKM2 can be transferred to the nucleus under certain stimuli,which can regulate the synthesis of some corresponding proteins.In tumor cells,EGFR can promote the transfer of PKM2 from the cytoplasm to the nucleus,combining with phosphorylated ?-catenin to form a transcriptional regulatory complex,which can accelerate cell cycle by up-regulating cyclin D1 and promoting cell proliferation.PKM2 can exert its phosphatase function in the nucleus.HIF-1? mediates the activation of STAT3 by PKM2,which can in turn up-regulates the expression of HIF-1?,thus forming a HIF-1?-PKM2-STAT3 feedback loop.In addition,recent studies have found that in NCI-H1299 lung cancer cells,PKM2 undergoes nuclear transfer under IGF-1 signaling and can induce STAT5 activation to exert its activity as transcription factor.At present,there are few studies on PKM2 in heart disease,especially its function in nucleus,which is still not reported.In summary,we hypothesize that sodium butyrate increases the anti-apoptotic capacity of the rat heart by up-regulating PKM2 nuclear translocation,and affecting STAT5 function in myocardial infarction rats.Objectives:The purpose of this experiment was to study the effect of sodium butyrate on cardiac tissue apoptosis in rats with myocardial infarction,to clarify the role of nuclear translocation of PKM2 during this process,and to explore the potential mechanism of sodium butyrate in protecting the hearts of rats with myocardial infarction.Methods:In this study,healthy male Wistar rats were used to establish the myocardial infarction model by ligating the left anterior descending coronary artery.Preoperative prophylaxis and postoperative maintenance therapy were given.The rats were treated normal saline,sodium butyrate or shikonin,one kind PKM2 inhibitor,respectively.The rats were sacrificed on 1d,2d,3d,7d and 14 d after operation respectively.The area of myocardial infarction was detected by TTC staining.The activities of AST,LDH and SOD in serum were detected by enzyme activity assay kit,and the concentrations of CK-MB and MDA were detected by ELISA kit.The changes of heart function in myocardial infarction rats were observed by echocardiography.HE staining was used to observe the structure of myocardium in rats.The ultrastructure of rat myocardial cells was observed by transmission electron microscope.Nucleoprotein and cytoplasmic protein in heart tissues of rats were isolated and extracted by kit.The expression of PKM2,STAT5,P-STAT5,Bcl-2,and Caspase-3 protein were detected by Western blot.Co-immunoprecipitation detected the interaction between PKM2 and STAT5 in rat heart.TUNEL staining was used to detect apoptosis.Results:(1)The result of TTC staining showed that there was almost no infarction in myocardium of Sham group.The infarct size of rat hearts in MI group increased significantly at 1d,2d,3d,7d,14d(P <0.001).Compared with MI group,the myocardial infarct size in MI+NaBu group was decreased(P <0.05).Compared with Sham group,AST activity of serum in MI group increased at 1 day and peaked at 1 day(P <0.001),while CK-MB concentration increased at 2d and LDH activity increased significantly at 3d and 7d(P <0.05).Compared with MI group,the AST activity in MI+NaBu group decreased,most obviously at 7d(P <0.001).Both of CK-MB concentration and LDH activity were dropped(P <0.05).Compared with Sham group,the MI group's MDA concentration increased to varying degrees and peaked at 3d and 7d(P <0.05),while the activity of SOD decreased in different days(P <0.05).Compared with MI group,the concentration of MDA in MI+NaBu group declined(P <0.05),most obviously on the 3rd and 14 th day(P <0.001),and SOD activity increased in different levels.Echocardiography showed that in MI group,ejection fraction decreased,compared with Sham group(P <0.01).Compared with MI group,ejection fraction of MI+NaBu group increased considerably on 1d and 7d(P <0.01).(2)HE staining showed that in Sham group myocardial tissue arranged neatly,with clear boundaries.Compared with Sham group,myocardial tissue in MI group arranged disorderly,with unclear boundaries,and obvious edema and infiltration of a large number of inflammatory cell.Compared with MI group,the severity of myocardial injuries was improved to a great extent in MI+NaBu group.We used transmission electron microscope to find that the myocardial fibers of the Sham group were dense and tidy,with clear light and dark bands and boundaries.A large number of mitochondria were arranged along the myocardial fibers,and the myocardial fibers around the nucleus were arranged regularly.Compared with Sham group,myofilament dissolved,ruptured and disappeared in MI group,with unclear boundary between light and dark zone.Some mitochondria cavitated and the arrangement of myocardial fibers around the nucleus were especially disordered.Compared with MI group,myocardial fibers in MI+NaBu group were relatively neat,myofilaments were slightly dissolved,and mitochondria arranged relatively neatly and clearly.(3)The results of nucleus-cytoplasm separation showed that compared with Sham group,the content of PKM2 in nucleoprotein increased significantly at 3 days in MI group(P <0.01).Compared with MI group,the content of PKM2 in nucleoprotein of MI+NaBu group increased at different levels except for 3d,most obviously at 7d(P <0.001).Compared with MI+NaBu group,the levels of PKM2 in nuclear protein of MI +NaBu+Shikonin group decreased significantly except 3 days(P <0.05).(4)Immunoprecipitation results showed that there was binding between PKM2 and STAT5 in rat heart tissue.Western Blot results showed that compared with Sham group,the expression of P-STAT5 and Bcl-2 decreased remarkably(P <0.001)but the cleaved-Caspase-3 significantly increased(P <0.01)in MI group;Compared with MI group,total P-STAT5 and Bcl-2 in cell,and P-STAT5 and STAT5 in the nucleus were increased(P<0.05),but cleaved-Caspase-3 decreased(P <0.05)in MI+Na Bu group.Compared with MI+NaBu group,P-STAT5 content in MI+Na Bu+Shikonin group decreased significantly(P <0.05),along with Bcl-2 and P-STAT5,but the degree of cleavage of Caspase-3 increased slightly.There was no obvious difference in the contents of STAT5 and phosphorylated STAT5 in the cytoplasm between groups.(5)TUNEL staining showed that there was almost no apoptosis in myocardium of Sham and Sham+NaBu group.Compared with Sham group,the number of apoptotic cells in myocardium of MI group increased remarkably.While the number of apoptotic cells in MI+NaBu group decreased dramatically,compared with MI group.The apoptotic cells increased again in MI+NaBu+Shikonin group,compared with MI+NaBu group.Conclusions:(1)Sodium butyrate can remain cardiocyte's structure,prevent the apoptosis of cardiac tissue,and improve cardiac function of rats with myocardial infarction;(2)Sodium butyrate showed its anti-apoptotic activity by promoting the translocation of PKM2 into nucleus to affect the phosphorylation of STAT5 and up-regulate the expression of Bcl-2. |
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