| [Objective]:Myocardial ischemia-reperfusion injury（MIRI）refers to the pathological process of progressive aggravation of myocardial tissue injury after acute coronary obstruction.It has been proved that atrial natriuretic peptide（ANP）and zinc ion have protective effects on myocardial ischemia/reperfusion injury.However,whether zinc homeostasis is involved in the protection of myocardial ischemia-reperfusion injury by ANP has not been elucidated.Therefore,the effects of zinc homeostasis on the protection of myocardial ischemia-reperfusion injury by ANP were observed and the mechanism of its effect was discussed by using the model of ischemia-reperfusion injury in ex-vivo and in vivo.[Methods]:（1）The traditional Langendorff isolated heart perfusion device was improved and integrated in three parts:the liquid storage tank,the serpentine tube and the hanging heart device,and the traditional balloon pressure measuring device was improved and innovated.（2）SD rats were divided into six groups,which were sham operation group（Sham）,ischemia-reperfusion group（I/R）,ischemia-reperfusion+atrial natriuretic peptide group（I/R+ANP）,ischemia-reperfusion+atrial natriuretic peptide+zinc chelating agent group（I/R+ANP+TPEN）,ischemia-reperfusion+zinc ion group(I/R+Zn2+).The isolated I/R model was prepared by ligating the anterior descending coronary artery（LAD）by using Langendorff in ex-vivo perfusion device.The I/R model was prepared by ligating the anterior descending coronary artery（LAD）under isoflurane inhalation anesthesia.The following experiments were carried out:the changes of the myocardial infarction area in ex-vivo/in vivo were observed by EB+TTC double staining method;The expression of related protein in ex-vivo and in vivo samples was observed by Western blotting;The mitochondrial structure of isolated cardiac myocytes in rats was observed by transmission electron microscopy;The concentration of zinc in ex-vivo and in vivo heart tissue was determined by Inductively coupled ion emission spectrometer（ICPOES）.（3）12 SD rats were randomly selected,6 in ex-vivo and 6 in vivo.The in ex-vivo I/R model was made by Langendorff isolated heart perfusion device.The perfusate was taken every 30 minutes during the whole experiment.The in vivo I/R model was established under isoflurane inhalation anesthesia.The in vivo I/R model was established under isoflurane inhalation anesthesia.The tail vein blood was collected once in the early stage of operation,once in the stage of myocardial infarction,once in the stage of reperfusion 1 h and 2 h respectively,and centrifuged for determination.（4）Plasma samples of 264 patients with ischemic related diseases were collected,including control group,ST segment elevation myocardial infarction group,non ST segment elevation myocardial infarction group,unstable angina pectoris group and heart failure group.[Results]:（1）Through the improvement and innovation of the traditional Langendorff isolated heart perfusion device,a new type of glass device including liquid storage tank,serpentine tube and heart hanging device was obtained;by using the Y-shaped structure of venous indwelling needle,a new type of device for measuring left ventricular pressure was obtained.（2）In the isolated rat model of ischemia-reperfusion injury,compared with the stable phase,the secretion of ANP in the perfusate increased slightly in the myocardial infarction phase（P>0.05）,gradually increased at 1 h of reperfusion phase,and reached the peak at 2h of reperfusion phase（P<0.05）;Compared with the stable phase,ANP secretion in plasma decreased slightly in myocardial infarction phase and increased slowly in reperfusion phase（P>0.05）.（3）The results of EB+TTC staining showed that compared with I/R group,ANP significantly resisted reperfusion induced myocardial infarction,and the infarct size decreased significantly（P<0.001）.At the same time,compared with I/R group,the myocardial infarct size of pretreatment Zn2+group was also significantly reduced（P<0.001）.（4）In sham group,I/R group,I/R+ANP group and I/R+Zn2+group,the structure of myocardial fibers in sham group was normal and orderly,the bright and dark bands were clearly visible,the morphology of mitochondria was complete and the steep structure was clear.In I/R group,the mitochondria swelled obviously,the myocardial fiber structure was disordered and incomplete,the structure was greatly damaged,some regional fibers were broken,some mitochondria were steep,and the structure became fuzzy and unclear.In pretreatment ANP group and Zn2+group,the structure of myocardial fibers was normal and orderly,the bright and dark bands were clearly visible,the morphology of mitochondria was complete,and the steep structure was clear.（5）ANP（40 pg/ml）was pretreated for 30 min at 20 min after myocardial infarction.In vivo,ANP（2 ng/ml）was injected into the caudal vein 20 min after myocardial infarction to observe the effect of ANP on the concentration of zinc ion in myocardial tissue after ischemia-reperfusion.The results showed that in the isolated I/R model,compared with the sham operation group,the concentration of zinc ion decreased significantly（P<0.01）and increased significantly（P<0.001）in the ANP preconditioning group;Compared with the sham operation group,the concentration of zinc ion decreased slightly（P>0.05）,but increased significantly in ANP pretreatment group（P<0.05）.（6）The effects of ANP and TPEN+ANP on myocardial infarct size were observed after pretreatment with zinc chelator TPEN（10μmol/L）for 5 min and ANP for 30 min.The results showed that compared with I/R group,ANP pretreatment significantly reduced myocardial infarction area（P<0.001）,but TPEN reversed the effect of ANP on myocardial infarction area（P<0.01）.Similarly,compared with the I/R group,the infarct size of pretreatment Zn2+group was significantly reduced（P<0.001）,but the infarct size of pretreatment Zn2+and TPEN group was significantly increased（P<0.05）.（7）Western blot results showed that compared with sham group,I/R group significantly increased the expression of ZnT8 in ex-vivo and in vitro（P<0.05 in ex-vivo/in vivo）,while pretreatment with ANP significantly decreased the expression of ZnT8.At the same time,TPEN could reverse the effect of ANP on the decrease of ZnT8 expression in ex-vivo（P<0.001）.However,both in ex-vivo and in vivo results showed that the I/R model did not increase but decreased the expression of ZnT1,and ANP could increase the level of ZnT1 in myocardial tissue in vitro model,while TPEN could not reverse the effect of ANP on increasing the expression of ZnT1 in myocardial tissue（P>0.05 in ex-vivo/in vivo）.At the same time,both in ex-vivo and in vivo models showed that the expression of Zip2 was significantly decreased during reperfusion（P<0.01 in ex-vivo;P<0.05 in vivo）,and pretreatment with ANP could significantly increase the expression of Zip2 in myocardial tissue,while TPEN reversed the effect of ANP on the increased expression of Zip2 in reperfusion myocardial tissue（P<0.01 in ex-vivo;P<0.05 in vivo）.[Conclusions]:ANP regulates the expression of zinc transporter ZnT8 and Zip2 and regulates the concentration of zinc ions in myocardium to protect myocardial ischemia reperfusion injury,ZnT1 may not be involved in the mechanism of ANP protection and reperfusion by zinc ion. |
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