| Objective:To explore the effect and mechanism of salt induced kinase 2(SIK2)on myocardial ischemia-reperfusion injury in rats through autophagy.Methods: SD male rats,weighing 200g～220g,were randomly divided into sham operation group(Sham group),myocardial ischemia-reperfusion model group(I/R group),myocardial ischemia-reperfusion model group+Bosutinib group(I/R+Bosutinib group)(10mg/kg),5 rats in each group.The cardiac function(LVEF,FS,IVSDd,LVPWDd)of rats was detected by echocardiography.The pathological changes such as myocardial fiber disorder and myocardial cell injury were observed by hematoxylin eosin staining(HE).The expression of SIK2,LC3 B,Beclin-1,p62 autophagy related proteins and the expression of p-mTOR,mTOR,p-ULK1,ULK1 related pathway proteins were detected by protein immunoblotting(WB)in the myocardial tissues of rats in each group.Autophagy and mitochondrial damage were observed by transmission electron microscopy(TEM).Results:1.Establishment and evaluation of myocardial ischemia-reperfusion injury model in rats:Compared with normal rats,ST segment of ECG of rats in coronary artery ligation group was significantly elevated,indicating that myocardial infarction model was successful.HE showed that part of myocardial cells in I/R group had edema,karyolysis,nuclear fragmentation,and there were cavities in the middle of the disordered arrangement of myocardial fibers.Compared with I/R group,I/R+Bosutinib group has a little acute inflammatory cell infiltration,which generally shows that the pathological changes of myocardial tissue cells are reduced.2.Changes of SIK2 expression in myocardial ischemia-reperfusion injury:WB results showed that compared with the Sham group,the expression of SIK2 protein in the myocardial tissue of the I/R group increased(P<0.05).3.Model construction and identification of myocardial ischemia-reperfusion group in SIK2 inhibitor group:WB results showed that compared with normal rats,the expression of SIK protein in rat heart tissue was significantly inhibited when the rats were given bosutinib at a dose of 10 mg/kg(P<0.05).4.Echocardiography to detect cardiac function in rats:Compared with Sham group,LVEF and FS in I/R group decreased significantly(P<0.05);Compared with the I/R group,LVEF and FS in the I/R+Bosutinib group increased(P<0.05),and LVPWDd and IVSDd values in the three groups had no significant difference(P>0.05).5.Changes in the expression levels of autophagy-related markers in myocardial tissue:Compared with Sham group,the expression of Beclin-1,LC3-Ⅱ/LC3-Ⅰ protein in I/R group increased;The expression of p62 protein decreased(P<0.05).Compared with the I/R group,the expression of Beclin-1,LC3-Ⅱ/LC3-Ⅰ protein in the I/R+Bosutinib group decreased,and the expression of p62 protein increased(P<0.05).6.Changes in expression levels of mTOR/ULK1-related signaling pathways:Compared with Sham group,the expression of p-mTOR protein in I/R group decreased significantly,and the expression of p-ULK1(Ser757)protein increased(P<0.05);Compared with the I/R group,the expression of p-mTOR protein in the I/R+Bosutinib group increased,and the expression of p-ULK1(Ser757)protein decreased(P<0.05);There was no significant difference in mTOR and ULK1 protein expression among the three groups(P>0.05).7.Observation of autophagy in cardiomyocytes of rats in each group by transmission electron microscope:The results showed that myocardial cells in I/R group had obvious swelling,mitochondrial cristae disorder,a large number of autophagic vesicles,and more autophages were formed.Compared with the I/R group,the myocardial cell swelling,vacuolization,necrosis and autophagy formation in the I/R+Bosutinib group were reduced.Conclusion:SIK2 is involved in the occurrence and development of cardiac I/R injury.Overexpression of SIK2 during cardiac I/R can aggravate cardiac I/R injury.Inhibition of SIK2 can reduce excessive autophagy,thereby alleviating myocardial I/R injury,and proves that SIK2 promotes and regulates autophagy through mTOR/ULK1 signal pathway. |
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