Exosomes Induced By Remote Ischemic Preconditioning Attenuate Myocardial Reperfusion Injury Through Transferring MiR-24

BackgroundIschemic heart disease(IHD)is the leading cause of the death worldwide.Meanwhile,IHD costs a large number of medical resources.According to the latest data from the United States,the per capita costs of ST-elevation myocardial infarction in hospitalized and outpatients were US$245,000 and US$129,000 respectively.The key to save the lives of patients should restore coronary blood supply with thrombolysis and PCI striking treatment as soon as possible,but regretably,some studies have found that myocardial ischemia lasting for a period of time after the reperfusion will bring new injury to the heart tissue,thereby cause onset of secondary damage of myocardial ischemia-reperfusion injury(IRI).Animal studies show that nearly 50% of necrotic myocardium is caused by reperfusion injury.Clinical studies have also shown that ischemia-reperfusion injury can cause severe arrhythmia and aggravate myocardial injury,which seriously affects the prognosis.Therefore,the effective prevention and treatment of IRI is an important clinical problem for the treatment of IHD.The study found that multiple transient ischemic stimulation of the extremities away from the heart before cardiac lethal ischemia can significantly reduce myocardial infarct size in experimental animals.This phenomenon is called remote ischemic preconditioning(RIPC).However,the mechanism of RIPC is very complex.It is of great scientific significance to thoroughly investigate the RIPC mechanisms.Exosome is an endogenous particle that has recently attracted attention.It is a vesicle-like body,with the diameter of 30 to 120 nm,which is fused with the cell membrane and released from the cell’s inner vesicles.Exosomes were once known as cell debris with no biological activity,however,the subsequent studies have confirmed that exosomes from different cell sources can carry different biologically active molecules,such as proteins,m RNAs,mi RNAs,etc.and act as mediators of cell-to-cell communication,playing a part in regulating a variety of cell biological functions.In physiological conditions,cells can release a small amount of exosomes,but with certain internal and external stimuli(such as activation of signal pathways,stress,etc.),it can induce a sharp increase in the number of cells secreting exosomes.Ischemia and hypoxia are immense stimuli to tissues and organs of the body.Therefore,many kinds of cells secrete exosomes,which first enter the peripheral extracellular fluid and eventually converge in the blood.Exosomes are negatively charged(-26 m V)microparticles with a bilayer membrane structure,and express surface molecules such as CD9.These properties make it easy for them to be uptaken by IRI cardiomyocytes and deliver the bioactive molecules which they carry.Studies have shown that exosomes mediate the protective mechanisms of ischemic preconditioning on ischemia-reperfusion injury,but the exact mechanism is not clear.Our previous results showed that the amount of mi R-24 in exosomes after remote ischemic preconditioning increased significantly.Some studies have confirmed that mi R-24 can reduce the apoptosis of cardiomyocytes by down-regulating the expression of pro-apoptotic protein Bim.In summary,we speculate that a large number of exosomes are released into the blood circulation after RIPC.These exosomes carrying high concentrations of mi R-24 are taken up and absorbed by IRI cardiomyocytes.The mi R-24 can down-regulates the expression of the target protein Bim in cardiomyocytes,thereby exerting a myocardial protection function through multiple pathways.In order to confirm this speculation.The main content of this study is:firstly,establish a model of remote ischemic preconditioning in rats,extract exosomes in plasma after RIPC,verify the purity and concentration of exosomes in vitro,and further detect the expression of related mi RNAs in exosomes.Secondly,establish an oxidative stress injury model by treating H9c2 cells with hydrogen peroxide in vitro,and observe whether the exosomes induced by RIPC can reduce cell injury by the oxidative stress.Finally,establish a rat model of ischemia-reperfusion injury and inject the exosomes induced by RIPC into the myocardium to verify whether it can reduce the injury of the heart and improve the heart function.The first part: Establishment RIPC model and extraction and identification of exosomesObjective:To establish a model of remote ischemic preconditioning,extract exosomes and clarify the expression levels of related mi RNAs in exosomes.Method:1)The rat model of ischemic preconditioning was established by ligation of double hind limbs with a tourniquet.The laser Doppler flowmeter was used to verify the success of the model establishment.2)The exosomes in the plasma before and after induction of RIPC were collected.The expression of CD63 and CD9 CD81 on the surface of exosomes was detected by WB,and the morphology and size of exosomes were detected by transmission electron microscopy.3)The concentration of plasma exosomes before and after the induction of RIPC was detected by using a nanoparticle tracking analyzer.RT-PCR was used to detect the expression levels of the related mi RNAs in plasma exosomes before and after the induction of RIPC.Result:1)The results of laser Doppler flow showed that the blood flow of the double hind limbs of the rats decreased significantly after the double hind limbs were ligated by the tourniquet(P<0.05).2)The results of WB showed that the surface markers of plasma exosomal proteins CD63,CD81 and CD9 were all expressed,and compared with those exosomes before RIPC,the above surface marker proteins were detected in the exosomes induction RIPC and the Expression levels of proteins increased significantly(P<0.05).The results of transmission electron microscopy showed that the morphology of exosomes collected was cup-shaped with a diameter of about 100 nm.3)Nanosight results showed that the number of exosomes after RIPC was significantly higher than that before RIPC(P<0.05).4)RT-PCR results showed that mi R-24 expression in exosomes induced by RIPC was significantly higher than that before RIPC(P<0.05).Conclusion:1)The number of exosomes induced by RIPC in rat plasma increased significantly.2)The expression level of mi R-24 in the plasma exosomes induced by RIPC increased significantly.The second part: Exosomes induced by RIPC attenuate H9c2 apoptosis induced by oxidative stress through transferring mi R-24 Objective:The purpose of this study was to establish an oxidative stress injury model of H9c2 cardiomyocytes and to explore the mechanism of protective effect of exosomes on H9c2 cells after oxidative stress treatment induced by RIPC.Method:1)The H9c2 cells oxidative stress injury model was established after 100 m M H2O2 treated for 6 h.The apoptosis of the cells was detected by flow cytometry,WB,etc.The expression of mi R-24 was detected by q RT-PCR.2)mi R-24 mimics,mi R-24 inhibitor,and two empty groups were transfected into H9c2 cells using Lipofectamine 3000,and treated with H2O2.The experiment was divided into six groups: Control group,H2O2 treated group,H2O2 treated + mi R-24 mimics group,H2O2 treated + mi R-24 mimics negative control group,H2O2 treated + mi R-24 inhibitor group and H2O2 treated + mi R-24 inhibitor negative group.Flow cytometry was used to detect the apoptosis of each group of cells.WB was used to detect the expression of pro-apoptotic protein Bim.3)The exosomes collected before and after RIPC were added to H2O2-treated H9c2 cells,and the experiments were divided into the following six groups: Control group,H2O2 treated group,H2O2 treated + EXO group,H2O2 treated + RIPC-EXO Group,H2O2 treated + RIPC-EXO + mi R-24 inhibitor group and H2O2 treated + RIPC-EXO + mi R-24 inhibitor-NC group.Then,the apoptosis of each group was detected by flow cytometry,the expression of pro-apoptotic protein Bim was detected by WB,and the expression level of mi R-24 in each group was detected at the same time.Result:1)The incidence of apoptosis of H9c2 cells after H2O2 treatment increased significantly(P<0.05),and the expression of mi R-24 decreased significantly in H9c2 cells.2)Compared with the H2O2 treated group,the survival rate of H9c2 cells in the H2O2-treated+mi R-24 mimics group increased significantly(P<0.05),and the expression levels of Bim and cleaved-caspase-3 proteins decreased significantly(P<0.05).Flow cytometry results showed that the apoptosis rate of the cells decreased significantly(P<0.05).The survival rate of H9c2 cells in the H2O2 treated +mi R-24 inhibitor group decreased significantly(P<0.05).Apoptosis such as Bim and cleaved-caspase-3 was detected in WB and the expression level of the proteins increased significantly(P<0.05),and the apoptosis was detected by flow cytometry.The results showed that the apoptosis rate of the cells increased significantly(P<0.05).3)Compared with H2O2 treated group,the survival rate and apoptosis rate of H9c2 cells in H2O2+EXO group did not change significantly(P>0.05),the expression level of mi R-24 did not change significantly(P>0.05).The survival rate of H9c2 cells in H2O2+RIPC-EXO group increased significantly(P<0.05).The expression levels of Bim and cleaved-caspase-3 proteins decreased significantly in WB(P<0.05).The apoptosis was detected by flow cytometry,and the apoptotic rate of the cells decreased significantly(P<0.05),and the expression level of mi R-24 increased significantly(P<0.05).However,compared with H2O2+RIPC-EXO group,the cell viability of H2O2+RIPC-EXO+mi R-24 inhibitor group cells decreased significantly(P<0.05),and the expression levels of Bim and cleaved-caspase-3 increased significantly detected by WB(P<0.05).Apoptosis results detected by flow cytometry showed that the apoptosis rate of the cells increased significantly(P<0.05),and the expression level of mi R-24 decreased significantly(P<0.05).Conclusion:1)The apoptosis rate of H9c2 cells after treatment with H2O2 increased significantly,and the expression level of mi R-24 decreased significantly.2)The RIPC-induced exosomes can reduce the apoptosis of H9c2 cells after treatment with H2O2.Its protective mechanism is related to exosomes transfer of mi R-24 to down-regulate the expression of pro-apoptotic protein Bim in cells.The third part: Exosomes induced by RIPC attenuate cardiac ischemia/reperfusion injury in Rats through transferring mi R-24 Objective :This study intends to establish a rat model of ischemia-reperfusion injury,and to explore the protective mechanism of exosome induced RIPC in ischemiareperfusion injury.Methods:1)A rat model of cardiac ischemia-reperfusion injury was established by reversibly ligating the left descending artery for 45 minutes and then reperfusion for 24 hours.The establishment of a rat model of cardiac ischemia was confirmed by echocardiography.2)To observe whether RIPC treatment can improve myocardial ischemia-reperfusion injury,the rats were divided into the following four groups: sham group,RIPC group,I/R group and RIPC+I/R group,and then each group rats were detected by cardiac ultrasound.After the cardiac function of the rats was sacrificed,TTC staining was performed on the hearts.3)The exosomes induced by RIPC were injected into the myocardium and were divided into the following six groups according to the contents of the injection: Control group,I/R+PBS group,I/R+EXO group,I/R+RIPC-EXO group,I/R +RIPC-EXO+mi R-24 antagomir group and I/R +RIPC-EXO+mi R-24 antagomir-NC group.After 24 hours of reperfusion,the cardiac function of each group of rats was examined with echocardiography,and then the rats were quickly killed by deep anesthesia.The heart was subjected to TTC staining and myocardial cell apoptosis was assessed by TUNEL.The expression of cardiac mi R-24 was detected by RT-PCR.4)Rats were divided into the following four groups: sham group,Cd group(HIF-1a inhibitor injection group),RIPC group,Cd+RIPC group.Then the expression levels of HIF-1a in the limbs of each group and the concentrations of plasma exosomes in each group were observed.Results1)After the rat model of ischemia-reperfusion injury was established,echocardiography indicated that the cardiac ejection fraction decreased significantly(P<0.05),and the TTC staining results suggested that the ischemic area of the heart increased significantly(P<0.05).2)There was no significant difference in cardiac function and TTC staining results between the RIPC group and the sham group(P >0.05).Compared with the I/R group,the cardiac function was significantly improved in the RIPC+I/R group.P <0.05),cardiac TTC staining results showed that the infarct size of the heart was significantly reduced(P<0.05).3)Compared with the I/R+PBS group,there was no significant change in the cardiac ejection fraction of rats in the I/R+EXO group(P>0.05).TTC staining showed no significant decrease in the infarct size of the heart(P>0.05).The staining showed that the apoptosis rate of heart cells did not change significantly,but the heart ejection fraction of rats in the I/R + RIPC-EXO group increased significantly.The TTC staining results showed that the infarct size of the heart decreased significantly(P<0.05),and TUNEL staining showed the apoptosis rate of the cells was significantly lower(P<0.05).However,compared with the I/R+RIPC-EXO group,the heart ejection fraction of the rats in the I/R+RIPC-EXO+mi R-24 antagomir group decreased significantly(P<0.05),the TTC staining results showed that the myocardial infarct area increased significantly(P<0.05),the result of TUNEL staining suggested that the apoptosis rate of cardiac cells increased significantly(P<0.05).4)The level expression of HIF-1a protein in the limb of rats significantly increased after RIPC(P<0.05).Compared with the RIPC group,the concentration of plasma exosomes in the Cd+RIPC group was significantly reduced.(P<0.05).Conclusion:1)RIPC can reduce cardiac ischemia-reperfusion injury.2)RIPC-indcued exosomes could deliver mi R-24 to ischemia-reperfusion injured cardiomyocytes,thereby downregulating the expression of the pro-apoptotic protein Bim and attenuating cardiac ischemia-reperfusion injury.The increased expression of HIF-1a in the limb of rats after RIPC can promote the release of exosomes in plasma.