| Objective: To explore the protective effect of Annexin A1 tripeptide on myocardial ischemia reperfusion injury in diabetic rats,and to clarify whether mito KATP and Sirt3 are involved in the myocardial protective effect of Annexin A1 tripeptide on diabetic rats.Methods: SD rats of SPF grade male were selected,with a body weight of 300g-350 g and an age of 12-14 weeks,and were randomly divided into sham operation group(Sham group),myocardial ischemia reperfusion injury group(MIRI group),myocardial ischemia reperfusion injury + Annexin A1 tripeptide group(MIRI + ANX group),myocardial ischemia reperfusion injury + 5-hydroxydecanoate group(MIRI + 5-HD group)and myocardial ischemia reperfusion injury + Annexin A1 tripeptide +5-hydroxydecanoate group(MIRI + ANX + 5-HD group)according to the random number table method.Diabetic rats in the Sham group did not experience ischemia;The MIRI group,the diabetic rats were ligated left anterior descending coronary artery for40 min and then myocardial reperfusion injury for 3h;The MIRI + ANX group,the diabetic rats were ligated the left anterior descending coronary artery for 40 min,then myocardial reperfusion injury for 3h and the reperfusion immediately intravenous injection Anx A1 sp through the rat tail vein.The MIRI + 5-HD group,the diabetic rats were ligated the left anterior descending coronary artery for 40 min,then myocardial reperfusion injury for 3h and the reperfusion immediately intravenous injection 5-HD through the rat tail vein.The MIRI + ANX + 5-HD group,the diabetic rats were ligated the left anterior descending coronary artery for 40 min,then myocardial reperfusion injury for 3h and the reperfusion immediately intravenous injection Anx A1 sp and 5-HD through the rat tail vein.After 3h of myocardial reperfusion injury and 24 h of myocardial reperfusion injury: The contents of CK-MB and LDH in serum were detected by Elisa method,the contents of SOD and MDA in myocardial tissue were detected by Elisa method,the expression of ROS in myocardial tissue was detected by DHE method,and the myocardial infarction area was determined by TTC staining method,Transmission electron microscopy was used to observe myocardial ultrastructure and mitochondrial Flameng score,ATP kit to detect myocardial tissue ATP level,echocardiography to detect rat cardiac function,Western-blot method to detect Sirt3 and mitoKATP subunit Kir6.1 expression.Results:1、Myocardial injury markers in serum of rats in each group Myocardial reperfusion injury occurred for 3h and 24 h after the operation,Compared with the Sham group,CK-MB and LDH in the MIRI group increased(P<0.05).Compared with the MIRI group,CK-MB and LDH in the MIRI + ANX group decreased(P<0.05);CK-MB and LDH in the MIRI + 5-HD group increased(P<0.05).Compared with the MIRI + ANX group,CK-MB and LDH in the MIRI + ANX + 5-HD group increased(P<0.05).Compared with the MIRI + 5-HD group,CK-MB and LDH in the MIRI + ANX + 5-HD group decreased(P<0.05).Compared with MIRI + ANX+ 5-HD group,MIRI group had no significant difference in CK-MB and LDH contents(P>0.05).2、Myocardial oxidative damage indexes of rats in each group Myocardial reperfusion injury occurred for 3h and 24 h after the operation,Compared with the Sham group,MDA,ROS increased and SOD decreased in the MIRI group(P<0.05).Compared with the MIRI group,MDA,ROS decreased and SOD increased in the MIRI + ANX group(P< 0.05);MDA,ROS increased and SOD decreased in the MIRI + 5-HD group(P<0.05).Compared with the MIRI + ANX group,MDA,ROS increased and SOD decreased in the MIRI + ANX + 5-HD group(P<0.05).Compared with the MIRI + 5-HD group,MDA,ROS decreased and SOD increased in the MIRI + ANX + 5-HD group(P<0.05).Compared with MIRI + ANX+ 5-HD group,MIRI group had no significant difference in MDA,SOD and ROS(P>0.05).3、Measurement of myocardial infarction area of rats in each group Myocardial reperfusion injury occurred for 3h and 24 h after the operation,Compared with the Sham group,myocardial infarction area increased in the MIRI group(P<0.05).Compared with the MIRI group,myocardial infarction area decreased in the MIRI + ANX group(P<0.05);myocardial infarction area increased in the MIRI +5-HD group(P<0.05).Compared with the MIRI + ANX group,myocardial infarction area increased in the MIRI + ANX + 5-HD group(P<0.05).Compared with the MIRI+ 5-HD group,myocardial infarction area decreased increased in the MIRI + ANX +5-HD group(P<0.05).Compared with MIRI + ANX + 5-HD group,MIRI group had no significant difference in myocardial infarction area(P>0.05).4、Changes of mitochondrial ultrastructure and mitochondrial score in each group of rats(1)Observation of mitochondrial ultrastructure in each group under transmission electron microscope,In the Sham group,the mitochondria were slightly swollen,the double-layer membrane structure was relatively complete,the mitochondrial cristae structure was visible,the muscle fibers were neatly arranged,the z-line and the light and dark bands were clear.In MIRI group,mitochondria were swollen,membrane structure disappeared,mitochondrial cristae became shorter and shallower,electron density in matrix decreased significantly,muscle fiber arrangement was slightly disordered,and a few were dissolved and broken.In the MIRI + ANX group,the structure of most of the mitochondria was basically normal,and there was no obvious disorder in the arrangement.The mitochondria were still swollen,and the basic knot of the mitochondrial cristae existed.The myocardial fibers were improved compared with the MIRI group.In the MIRI + 5-HD group,most of the mitochondria were dissolved and disappeared,and the remaining mitochondria had unclear structure,the membrane was dissolved,and the interior was vacuolated.In the MIRI + ANX + 5-HD group,mitochondria were swollen or even ruptured,the integrity of the inner and outer membranes was lost,the mitochondrial cristae disappeared,the arrangement of muscle fibers was disordered,and the z-line and the light-dark band were blurred.(2)Mitochondrial Flameng score of rats in each group Myocardial reperfusion injury occurred for 3h and 24 h after the operation,Compared with the Sham group,the Flameng score increased in the MIRI group(P<0.05).Compared with the MIRI group,the Flameng score decreased in the MIRI +ANX group(P<0.05);the Flameng score increased in the MIRI + 5-HD group(P<0.05).Compared with the MIRI + ANX group,the Flameng score increased in the MIRI + ANX + 5-HD group(P<0.05).Compared with the MIRI + 5-HD group,the Flameng score decreased in the MIRI + ANX + 5-HD group(P<0.05).Compared withMIRI + ANX + 5-HD group,MIRI group had no significant difference in the Flameng score(P>0.05).5、Determination of ATP levels of rats in each group Myocardial reperfusion injury occurred for 3h and 24 h after the operation,Compared with the Sham group,the ATP level decreased in the MIRI group(P<0.05).Compared with the MIRI group,the ATP level increased in the MIRI + ANX group(P<0.05);the ATP level decreased in the MIRI + 5-HD group(P<0.05).Compared with the MIRI + ANX group,the ATP level decreased in the MIRI + ANX + 5-HD group(P<0.05).Compared with the MIRI + 5-HD group,the ATP level increased in the MIRI + ANX + 5-HD group(P<0.05).Compared with MIRI + ANX + 5-HD group,MIRI group had no significant difference in the FLameng score(P>0.05).6、Expression of Sirt3 and mito KATP channel subunit Kir6.1 of rats in each group Myocardial reperfusion injury occurred for 3h and 24 h after the operation,Compared with the Sham group,the expression of Sirt3 and Kir6.1 decreased in the MIRI group(P<0.05).Compared with the MIRI group,the expression of Sirt3 and Kir6.1 increased in the MIRI + ANX group(P<0.05);the expression of Sirt3 and Kir6.1 decreased in the MIRI + 5-HD group(P<0.05).Compared with the MIRI +ANX group,the expression of Sirt3 and Kir6.1 decreased in the MIRI + ANX + 5-HD group(P<0.05).Compared with the MIRI + 5-HD group,the expression of Sirt3 and Kir6.1 increased in the MIRI + ANX + 5-HD group(P<0.05).Compared with MIRI +ANX + 5-HD group,MIRI group had no significant difference in the expression of Sirt3 and Kir6.1(P>0.05).7、Echocardiogram of rats in each group Myocardial reperfusion injury occurred for 3h and 24 h after the operation,Compared with the Sham group,EF,FS,LVAWd,LVAWs,LVPWd,LVPWs decreased and LVIDd,LVIDs increased in the MIRI group(P<0.05).Compared with the MIRI group,EF,FS,LVAWd,LVAWs,LVPWd,LVPWs increased and LVIDd,LVIDs decreased in the MIRI + ANX group(P<0.05);EF,FS,LVAWd,LVAWs,LVPWd,LVPWs decreased and LVIDd,LVIDs increased in the MIRI + 5-HD group(P<0.05).Compared with the MIRI + ANX group,EF,FS,LVAWd,LVAWs,LVPWd,LVPWs decreased and LVIDd,LVIDs increased in the MIRI + ANX + 5-HD group(P<0.05).Compared with the MIRI + 5-HD group,EF,FS,LVAWd,LVAWs,LVPWd,LVPWs increased and LVIDd,LVIDs decreased in the MIRI + ANX + 5-HD group(P<0.05).Compared with MIRI + ANX + 5-HD group,MIRI group had no significant difference in EF,FS,LVAWd,LVAWs,LVPWd,LVPWs LVIDd and LVIDs(P >0.05).8、Colocalization of SIRT3 with COXIV in myocardial tissue slides via fluorescence microscope Myocardial reperfusion injury occurred for 3h and 24 h after the operation,SIRT3 expression appeared to be localized to mitochondria in Cardiomyocytes as demonstrated by co-staining with mitochondrial complex IV.Conclusions1.Annexin A1 tripeptide may regulate Sirt3 expression through mito KATP,maintain mitochondrial function,and alleviate myocardial ischemia-reperfusion injury in diabetic rats. |
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