| Background and objective Ischaemic heart disease and the resulting heart failure remain the leading causes of death and disability in worldwide.So novel therapies are required to protect the heart against the detrimental effects of acute ischaemia reperfusion injury(IRI).Exosome is a vesicular body which can besecreted actively to the extracell by cells.It is about 30 nm to 100nm in diameter.It can transport proteins,RNA or small RNA(microRNA,miRNA)and target to the cells.It is one of the important ways of intercellular signal communication.Studies have shown that exosome may play an important role in myocardial protection in cardiovascular disease.However,the role of exosome in myocardial protection mediated by ischemic preconditioning(ischemic preconditioning,IPC)has not been reported.Therefore,the purpose of this study is to investigate the effects and mechanism of exosome extracted from serum after IPC treatment on myocardial IRI,in order to provide the new mechanism and targets of myocardial protection of IPC.Methods 1.Isolation,extraction and identification of exosome Male SD rats weighing 250~300 g were used in this study.Rats were randomly(random number)divided into 2 groups of 4 animals each.In ischemia preconditioning group(IPC),left anterior descending branch of coronary artery was ligated by 3 cycles of 5 min ischemia-5 min reperfusion after thoracotomy.In control group(CON),rats were only punctured and not ligated.After preconditoning,ExoQuick~TMM kit was used to extract serum exosome and marked as Exo-IPC or Exo-CON.The morphological characteristics of exosome were observed by transmission electron microscope,the marker proteins were detected by Western blot and the concentration of exosome was measured by BCA.2.Animal experiment Another thirty male SD rats were randomly(random number)divided into five groups:sham group,ischemia reperfusion group(IR),IR+Exo-IPC,IR+Exo-CON,solvent control group(IR+PBS).Except the sham group,all other groups were treated by 30 min ischemia and 120min reperfusion in vivo animal experiments.Exo-IPC,Exo-CON and PBS were given intravenously in IR+Exo-IPC group,IR+Exo-CON group and IR+PBS group respectively,15 min before ischemia.At the end of reperfusion,venous blood samples were taken for determination of cardiac troponin I(cTnI)level.Hearts were stained by TTC for the measurement of left ventricular area(LV),right ventricular area(RV),infarct size(IS)and area at risk(AAR)as well as IS/AAR ratio.In vitro animal experiments,the rat model of isolated heart ischemia reperfusion injury was established by the method of Langendorff isolated heart perfusion.With the exception of continuous perfusion of K-H solution in Sham group,all other groups underwent 40 min ischemia and 90 min reperfusion.IR+Exo-IPC group,IR+Exo-CON and IR+PBS group was perfused with K-H solution containing Exo-IPC,Exo-CON or PBS 15 min before the end of the experiment.Coronary effluents were collected at baseline,5 min and 10 min after reperfusion,and lactate dehydrogenase(LDH)activity was detected by chemical colorimetry.After 90 min of reperfusion,the rat heart was taken out,and AAR,IS and IS/AAR were measured by TTC staining.3.Cell experiment H9c2 cardiomyocytes were randomly divided into control group(CON group),hypoxic reoxygenation group(HR group),HR Exo-IPC group,HR Exo-CON group,inhibitor group(HR Exo-IPC LY group)and inhibitor control group(HR LY group).Except the cells in CON group for normal culture in DMEM/F12 cell culture medium,all other groups were treated with hypoxia for 5h and reoxygenation for 1h.In HR+Exo-IPC group,HR+Exo-CON group and HR+LY group,Exo IPC,Exo-CON,LY294002 were incubated H9c2 cardiomyocytes for 12 h and then treated with HR.After treatment,the cell viability was detected by CCK-8 assay,the cell apoptosis was detected by flow cytometry and Western blot was used to detect the protein expression levels of?-actin,p-Akt,p-GSK-3?,GSK-3?,Bcl2 and Bax.Results 1.Under the transmission electron microscope,the exosome was round vesicle with an average diameter of 30~100 nm,and Western-blot demonstrated that the relative quantitative expression of HSP60,CD63 was increased in Exo-IPC compared with Exo-CON(P<0.05).The concentration of exosome in Exo-IPC was increased more than that in Exo-CON(P<0.05).2.In vivo animal experiments,IS,IS/AAR ratio and cTnI level were significantly decreased in IR+Exo-IPC group as compared to IR group(P<0.05).On the contrary,there was no significant difference about these changes between IR+Exo-CON group and IR+PBS group compared with IR group.In vitro animal experiments,compared with IR group,LDH activity was decreased after reperfusion for 5 min and 10 min,IS and IS/AAR decreased in IR Exo-IPC group(P<0.05,).On the contrary,there were no significant differences among IR+PBS group,IR+Exo-CON group and IR group.3.Compared with HR group,the viability of cardiomyocytes was increased,the apoptosis rate was significantly decreased,and the protein expression levels of p-Akt/Aktr,p-GSK-3?/GSK-3?and Bcl2/Bax were significantly increased in HR+Exo-IPC group(P<0.05).There was no significant difference about these changes between HR+Exo-CON group and HR group(P>0.05).Compared with HR+Exo-IPC group,the cardiomyocytes viability were significantly decreased,the apoptosis rate was significantly increased,and the protein expression levels of p-Akt/Akt,p-GSK-3?/GSK-3?and Bcl2/Bax were significantly decreased in HR+Exo-IPC+LY group.The difference was statistically significant(P<0.05).There was no significant difference between HR+Exo-IPC+LY group and HR+LY group(P>0 05).Conclusion 1.IPC can induce the increase of serum exosome release in rats.This endogenous exosome can alleviate myocardial IRI,and inhibit cardiomyocyte apoptosis,thus producing protective effect.2.The mechanism of cardiac protection induced by IPC may be related to the activation of PI3K/Akt/GSK-3?signaling pathway. |
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