| Ischemic heart disease(IHD)is the leading cause of death,which is developed when there is a constriction in the blood and oxygen supply to the heart.The most effective treatment for IHD is early revascularization,but revascularization is often accompanied by myocardial ischemia/reperfusion injury,which often leads to myocardial stunning,arrhythmia and cell death.Therefore,it is critical to explore the underlying mechanism of ischemia/reperfusion.Therapeutic hypothermia can significantly reduce the infarct area caused by ischemia/reperfusion injury.It is an effective method to find related drugs to induce therapeutic hypothermia for myocardial protection.Objective: 1.To explore the role of T1 AM in ischemia-reperfusion injury.2.To study whether T1 AM can reduce myocardial ischemia/reperfusion injury via Akt/Fox O1 signaling pathway.3.To explore whether T1 AM can inhibit metabolism and apoptosis of myocardiocytes via Akt/Fox O1 signaling pathway.Methods: In vivo,the rectal temperature was recorded in rats with or without T1 AM.We divided all the rats into three groups: 1.Sham Group: The sham group was induced by opening chest without ligation of left coronary artery.2.Ishchemia/Reperfusion Group: Sprague-Dawley rats underwent transient ligation of the left anterior descending coronary artery for 30 min and 3 h followed by reperfusion.3.T1 AM Treatment Group: T1 AM at the dose of 50 mg/kg was given before left anterior descending coronary artery occlusion for 30 minutes and 3 h followed by reperfusionin rats in vivo.ECG was used to detect the change of ECG at different time points in each group.Left ventricular wall movement and ejection fraction were detected by echocardiography.Myocardial infarction area was observed by TTC staining.Ultrastructure of myocardial cells was observed by transmission electron microscopy.The changes of serum creatine kinase(CK),myocardial-associated isoenzyme of creatine kinase(CK-MB)and lactate dehydrogenase(LDH)were detected.The expression of Fox O1,Akt,p-Fox O1,PPAR?,Glut1,Bcl-2,Bax and Caspase 8 were detected by Western Blot.2.AC-16 cells were cultured in vitro.The cell experiments were divided into 4 groups: 1.AC-16 group:AC-16 cells were cultured with vehicle undernormoxia.2.T1 AM group:AC-16 cells were cultured with 2 ?M T1 AM undernormoxia.3.H/R group: AC-16 cells were cultured with hypoxia for 24 h and followed by reoxygen for 4 h.4.H/R+T1AM group: AC-16 cells were cultured with 2 ?M T1 AM in hypoxia for 24 h and followed by reoxygen for 4 h.Differentially expressed genes(DEGs)were detected by RNA-Seq.The common DEGs were analyzed by Venn diagram.The pathway of DEGs was enriched by KEGG biologicalpathway.The reliability of RNA-Seq was checked by q RT-PCR.3.The cell experiments in this part were divided into 7 groups: 1.AC-16 group: AC-16 cells were cultured with vehicle undernormoxia.2.T1 AM group:AC-16 cells were cultured with 2 ?M T1 AM undernormoxia.3.H/R group: AC-16 cells were cultured with hypoxia for 24 h and followed by reoxygenation for 4 h.4.H/R+0.5 ?M T1 AM group: AC-16 cells were cultured with 0.5 ?M T1 AM in hypoxia for 24 h and followed by reoxygenation for 4 h.5.H/R+2 ?M T1 AM group: AC-16 cells were cultured with 2 ?M T1 AM in hypoxia for 24 h and followed by reoxygenation for 4 h.6.H/R+5 ?M T1 AM group: AC-16 cells were cultured with 5 ?M T1 AM in hypoxia for 24 h and followed by reoxygen for 4 h.7.H/R+2 ?M T1AM+30 n M AS1842856 group: AC-16 cells were cultured with 2 ?M T1 AM and 30 n M AS1842856 in hypoxia for 24 h and followed by reoxygen for 4 h.The cell morphology was observed by inverted microscope.The glucose uptake of cells was measured by Glycogen Colorimetric Assay Kit.The lactate production of each group was measured by Lactate Colorimetric Assay Kit.The apoptosis and necrosis ratio of each group was measured by Annexin V-PI.The expression of Akt,Fox O1,p-Fox O1,PPAR ?,Glut1,Bcl-2,Bax and GCK protein in each group was detected by Western Blot.The gene expression of Akt,Fox O1,PPAR? and GCK in each group was detected by RT-q PCR method.Results:The temperature of rats decreased rapidly after T1 AM treatment,which could be as low as 32.68+1.08?.Compared with sham group,echocardiography showed that the activity of left ventricular wall movement was weakened and EF value was significantly reduced(P<0.001).TTC staining showed that the infarctsize of myocardium was significantly increased.Transmission electron microscopy showed mitochondrial swelling in Ischemia/reperfusion group.The level of CK,CK-MB and LDH in serum were significantly increased in myocardial ischemia/reperfusion rats(P<0.001).Compared with myocardial ischemia/reperfusion group,echocardiography showed that the activity of left ventricular wall movement was increased,ejection fraction and left ventricular short axis contraction rate were significantly increased(P <0.001,P<0.005).TTC staining showed that the infarctsize of myocardium was significantly reduced.Transmission electron microscopy showed that mitochondrial swelling was attenuated in T1 AM group.The level of CK,CK-MB and LDH in serum was significantly reduced.Compared with H/R group,Western Blot showed that the expression of Fox O1,PPAR?,Bcl-2 were decreased in T1 AM treated AC-16 cells exposed to H/R injury,but the expression of p-Fox O1,Glut1,Bax were increased in T1 AM.2.In vitro,the viability of AC-16 cells was 62.47%+3.49% after exposed to hypoxia/reoxygen injury compared with AC-16 group(P<0.001).Lower concentration of T1 AM could significantly improve the viability of hypoxic/reoxygenated cardiomyocytes.Compared with cardiomyocytes exposed to hypoxia/reoxygen injury,2.22 ?M T1 AM can significantly increase the viability(29.02%+4.34%,P<0.001).Higher concentration of T1AM(20 ?M,60 ?M)could induce cell toxicity.Therefore,T1 AM under concentration of 5 ?M was used in the subsequent experiment.The RNA-Seq analysis showed that there were 142 up-regulated genes and 68 down-regulated genes in H/R group with or without T1 AM.In normoxia group,89 up-regulated genes and 9 down-regulated genes were identified with or without T1 AM.1215 up-regulated genes and 452 down-regulated genes were identified in H/R injury group compared with normoxia group.135 genes were identified by Venn diagram.The results of KEGG pathway analysis indicated that the DEGs are mainly enriched in HIF-1 signaling pathway,Fox O signaling pathway,p53 signaling pathway,amino acid synthesis signaling pathway,arachidonic acid metabolism,?-linolenic acid metabolism signaling pathway,protein digestion and absorption signaling pathway.3.Higher concentration of T1 AM displays inhibition of AC-16 cells with IC50 value of approximately 62 ?M.Inverted microscope showed that most of the cells exposed to H/R injury lost its proper shape and the percentage of dead cells was increased.However,2 ?M T1 AM can protect AC-16 cells from H/R injury.Seahorse XF Analyzers showed that oxygen consumption rate(OCR)of AC-16 cells was decreased by T1 AM.However,Fox O1 inhibitor,AS1842856,can increase the decreased OCR by T1 AM.Compared with normoxia,the glucose uptake,the lactate production of cells and the apoptosis of the cells were increased by H/R exposure.T1 AM decreased the glucose uptake,the lactate production of cells and the apoptosis of the cells exposed to H/R.However,this phenomenon could be inversed by Fox O1 inhibitor,AS1842856.Western Blot showed that the expression of Akt,p-Fox O1,Gut1 and Bax could be decreased and the expression of PPAR?,Fox O1 and Bcl-2 could be increased by T1 AM.However,Fox O1 inhibitor,AS1842856,significantly decreased the expression of Fox O1(P<0.01),PPAR?(P<0.05),Bcl-2(P<0.005)and increased the expression of p-Fox O1(P<0.05),Bax(P<0.005),Glut1(P<0.01),but it does not have effects on the expression of Akt.q RT-PCR showed that the gene expression of Akt could be decreased and the expression of PPAR? and Fox O1 could be increased by T1 AM.What is more,Fox O1 inhibitor,AS1842856,significantly decreased the expression of Fox O1(P<0.01)and PPAR?(P<0.01).Conclusion: 1.T1 AM can alleviate myocardial ischemia reperfusion injury.2.Cells exposed to H/R affects numerous genes.T1 AM can reduce H/R induced cell injury via metabolism.3.T1 AM can inhibit cell metabolism and apoptosis in myocardial ischemia reperfusion injury via Akt/Fox O1 signaling pathway. |
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