| BackgroundCardiac arrest(CA)refers to the sudden discontinuation of the heart’s ejection function due to various causes and is the leading cause of death in adults worldwide.According to statistics,about 8-9 million people worldwide suffer from cardiac arrest every year.As a country with a large population,my country ranks first in the world in the number of cardiac arrests.Studies show that about 550,000 people in my country experience cardiac arrest every year.Although the continuous development of Cardiopulmonary resuscitation(CPR)in the past half century has increased the success rate of return of spontaneous circulation(ROSC)in CA patients to 28.2%,the survival rate of patients after ROSC is still only 10.4%.Therefore,how to improve the survival rate of cardiac arrest patients after successful resuscitation has become a key problem to be solved urgently.Post-cardiac arrest syndrome(PCAS)refers to a series of symptoms that occur after resuscitation of cardiac arrest patients.Systemic ischemia and reperfusion in resuscitated patients from CA lead to post-cardiac arrest syndrome,which may develop within days or weeks after the return of spontaneous circulation and mainly includes brain injury,myocardial dysfunction,generalized ischemia/Reperfusion injury and primary disease.Cardiac dysfunction after cardiac arrest is a key cause of high mortality after resuscitation.Studies have shown that 2/3 of resuscitated patients develop post-cardiac arrest myocardial dysfunction(PCAMD),and about 1/3 of CA patients die from PC AMD within 24 hours after ROSC,which is responsible for most deaths within 3 days after spontaneous circulation is restored.and causes of long-term mortality.Therefore,improving PCAMD is a key link in improving the prognosis of CA patients.Studies have shown that ischemia/reperfusion injury,asphyxia and hypoxia,and drug and mechanical damage during resuscitation are the main mechanisms that cause cardiac dysfunction after cardiac arrest.To date,several strategies have been demonstrated to reduce myocardial dysfunction after CA.Among them,remote ischemic adaptation(RIC)has emerged as one of the most promising strategies.RIC is a safe treatment strategy for a variety of ischemic diseases.It utilizes transient ischemia/reperfusion in distal organs to activate protective signaling pathways and ultimately protect the myocardium.The above scheme is engaged by a complex signaling cascade,including activation of sarcolemmal receptors,activation of intracellular enzymes,stabilization of mitochondrial structure,and inhibition of death signaling.Currently,distal ischemic adaptation has been successfully used in patients undergoing coronary revascularization and reperfusion after acute myocardial infarction.But the exact mechanism by which distal signals are transmitted from the periphery to the brain is unknown.Previous studies have shown that RIC caused by a transient ischemic attack of the limb can significantly improve myocardial function and survival after cardiopulmonary resuscitation(CPR).However,four or five cycles of RIC during CPR are difficult to implement in a clinical setting,placing a heavy burden on emergency physicians.Therefore,it is particularly important to explore more time-saving and efficient alternatives to RIC.Recent studies have shown that RIC plays an important role in myocardial protection and brain protection after CA/CPR.At present,the specific mechanism of action of RIC in improving PCAMD has not been revealed.Exosomes are membrane-bound vesicles secreted by various cells,with an average diameter of about 100 nm,originating from the endosomal system,existing in body fluids,and participating in various pathophysiological processes.Exosomes are currently considered to be an important mechanism of intercellular communication,carrying proteins,lipids,and genetic material for exchange between cells,and are involved in cardiovascular disease,immune response,pregnancy,central system disease,and cancer progression.Its role in regulating complex intracellular pathways makes it potentially useful in the treatment of various diseases.Furthermore,exosomes have important protective effects on cardiovascular disease and ischemia/reperfusion injury in multiple organs.Therefore,we speculate that exosomes also have therapeutic effects on myocardial injury and survival in PCAMD.Therefore,combined with the current research progress at home and abroad,this project intends to use the rat cardiac arrest model and the primary cardiomyocyte hypoxia-reoxygenation(H/R)model to investigate the RIC-induced exosomes(RIC-exosomes).The therapeutic effect of PCAMD is expected to provide new methods and ideas for clinical treatment of PC AMD.ObjectivesThe main aims of the present study are:1.To investigate the therapeutic effect of RIC-exosome on PC AMD.2.To investigate the exact mechanism of myocardial protection in RIC-exosome.3.To investigate the specific mechanism of miR-542-5p exerting its anti-myocardial apoptosis effect.MethodsWe established a rat CA/CPR model by asphyxiation to explore the effects of RIC-induced exosomes on cardiac function,survival rate and myocardial injury in PCAMD rats.By extracting primary cardiomyocytes and performing hypoxia/reoxygenation(H/R)stimulation,the effect of RIC-exosome on cardiomyocyte injury was observed,and the influence of non-exosome factors was excluded by exosome inhibitors.Then we screened the differentially expressed miRNAs specifically expressed by RIC-exosome by miRNA chip technology,and verified their functions.Finally,we used a variety of biological techniques to explore the specific mechanism by which miRNAs regulate downstream target proteins and exert cardioprotective effects.(Ⅰ)In vivo Study1.Remote ischemic conditioning model establishmentThe rats were anesthetized with pentobarbital,the lower limbs were tightened with elastic bands,5 minutes of ischemia and 5 minutes of reperfusion were used as a cycle,and a total of 4 cycles were performed.After RIC,blood was collected from the apex of the rat with an anticoagulation tube,and the rat plasma was collected by centrifugation of the whole blood for exosome separation.2.Plasma exosome isolationAfter RIC,whole blood was collected from rats,plasma was collected at 1600 g*20 min,followed by 2000 g*30 min and 12000 g*45 min to remove cell debris,and the supernatant was collected and passed through a 220 μm filter.The remaining supernatant was ultracentrifuged at 110,000 g*90 min to get the pellet;the pellet was resuspended in PBS and then ultracentrifuged again at 110,000 g*70 min,and the pellet was the exosome.The exosomes were resuspended in PBS,negatively stained with uranyl acetate,and their morphology was observed under a transmission electron microscope.The expression of the marker protein marker was detected by Western blot,and particle size analysis(NTA)was used to perform multiplexing of exosomes identification.3.Asphyxia-induced CA/CPR model EstablishmentMale Wistar rats,weighing 400-450 g,were anesthetized with isoflurane,given tracheal intubation,ventilator-assisted ventilation,and femoral artery and femoral vein catheterization.Blood pressure,left ventricular pressure,and electrocardiogram were recorded using the PowerLab data acquisition and analysis system.The ventilator was turned off and the tracheal intubation was clamped to induce asphyxia.Cardiac arrest was defined as MAP<30mmHg.After 8.5 minutes of asphyxiation,the ventilator was started,and manual chest compressions(200 times/min)were given,and epinephrine injection(administered 1 minute after the initiation of CPR,2 μg/100 g,administered every 3 minutes).ROSC was defined as a return to sinus rhythm with a MAP≥60 mmHg for 10 minutes or more.Those who reach ROSC continue the experiment.If cardiopulmonary resuscitation continued for 10 minutes and the rat could not resume spontaneous circulation,it was considered that the resuscitation failed.4.Experimental groups and drug administration(1)Animal experiment 1:To verify the improvement effect of RIC-exosome on PCAMD.Rats were randomly divided into 4 groups after anesthesia,and each group was divided into 3 subgroups at 4 h,24 h,and 7 d after ROSC.The 4 h subgroup had 5 rats in each group,and the 24 h subgroup had 7 rats in each group.Rats,7 d subgroups with 10 rats in each group.Grouping:1)CA/CPR group:rats were intravenously injected with 500 μL of PBS after ROSC;2)RIC+CA/CPR group:rats were subjected to CA/CPR after RIC;3)CA/CPR+RIC-exosome group:rats were treated with ROSC 500 μL of RIC-exosome was injected intravenously;4)CA/CPR+Ctrl-exosome group:rats were injected with 500 μL of Ctrl-exosome after ROSC.The 4-h subgroup was sacrificed 4 h after ROSC,and the 24-h subgroup was sacrificed 24 h after ROSC.The 7-day subgroup was used to observe the cardiac function and 7-day survival of ROSC from 1 to 4 h.(2)Animal experiment 2:Inhibition of exosome secretion blocks the cardioprotective effect of RIC.Rats were randomly divided into 2 groups,and the rats were administered intraperitoneally once every other day and 4 times a week.Then the rats underwent RIC+CA/CPR.After ROSC,each group was divided into 2 subgroups at time points 4 h and 7 d after ROSC.The 4 h subgroup had 5 rats in each group,and the 7 d subgroup had 10 rats in each group.Grouping:1)GW4869+RIC+CA/CPR group:rats were intraperitoneally injected with GW4869 one week in advance,followed by RIC+CA/CPR;2)Vehicle+RIC+CA/CPR group:rats were intraperitoneally injected with DMSO one week in advance,followed by RIC+CA/CPR.The 4-hour subgroup was sacrificed 4 hours after ROSC to collect the material,and the 7-day subgroup was used to observe the cardiac function and 7-day survival of ROSC from 1 to 4 hours.(3)Animal experiment 3:To verify the improvement effect of overexpression of miR-542-5p on PCAMD.Rats were randomly divided into 2 groups after anesthesia,and each group was divided into 2 subgroups at 4 h and 7 d after ROSC.The 4 h subgroup had 5 rats in each group,and the 7 d subgroup had 10 rats in each group.Groups:1)AAV9-miR-542-5p+CA/CPR group:CA/CPR was performed 1 month after AAV9-miR-542-5p myocardial-specific overexpression virus was injected into the tail vein of rats;2)AAV9-Scramble+CA/CPR group:CA/CPR was performed 1 month after AAV9-Scramble myocardial specific control virus was injected into the tail vein of rats.The 4-hour subgroup was sacrificed 4 hours after ROSC to collect the material,and the 7-day subgroup was used to observe the cardiac function and 7-day survival of ROSC from 1 to 4 hours.(4)Animal experiment 4:It was verified that inhibition of miR-542-5p could reverse the improvement effect of RIC-exosome on PCAMD.Rats were randomly divided into 2 groups after anesthesia,and each group was divided into 2 subgroups at 4 h and 7 d after ROSC.The 4 h subgroup had 5 rats in each group,and the 7 d subgroup had 10 rats in each group.Grouping:1)CA/CPR+RIC-exosome+miR-542-5p antagomir group:rats were intravenously injected with RIC-exosome+miR-542-5p inhibitor after ROSC;2)CA/CPR+RIC-exosome+NC antagomir group:large RIC-exosome+control antagomir was injected intravenously after ROSC in mice.The 4-h subgroup was sacrificed 4 h after ROSC,and the 24-h subgroup was sacrificed 24 h after ROSC.The 7-day subgroup was used to observe the cardiac function and 7-day survival of ROSC from 1 to 4 h.5.Heart rate and MAP monitoringThe heart rate was monitored by surface electrocardiogram,and the mean arterial pressure was monitored by catheterization of the left femoral artery.6.Assessment of myocardial functionLeft ventricular ejection fraction(LVEF)and cardiac output(CO)at baseline and 1 h,2 h,3 h,and 4 h after ROSC were calculated by ultrasound.7.Cardiomyocyte Apoptosis DetectionThe rats were sacrificed 4 h after ROSC,and frozen sections of the left ventricle were taken.The myocardial apoptosis was detected by Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)method.8.The levels of myocardial enzymes in the plasma4 h after ROSC,whole blood was collected in EDTA anticoagulated blood collection tubes,centrifuged at 1400g at 4℃ for 15 minutes,and serum was collected.Serum levels of cTnT,CK-MB and LDH were detected by ELISA.9.Examination of mitochondrial morphologyThe rats were sacrificed 4 h after ROSC,and the left ventricle was removed and fixed with pentylene glycol.The morphology of mitochondria in rat cardiomyocytes was observed by transmission electron microscope,and the degree of mitochondrial structure damage was semi-quantitatively analyzed by Flameng score.10.Measurement of inflammatory factorsThe rats were sacrificed 4 h after ROSC,serum and myocardial tissue were collected,and the levels of circulating interleukin-1β(IL-1β)and interleukin-10(IL-10)were detected by ELISA.11.Cardiac tissue protein expression detectionRats were sacrificed 4 h after ROSC,and total protein was extracted from left ventricular homogenate.20 μg of tissue protein was taken,and the protein expression in myocardium was detected by Western blot.12.Survival analysisAfter 4 h of ROSC,the cannula was removed,and the wound was sutured.Return to the cage and observe closely for 7 days.13.Bioinformatics AnalysisThe RIC-exosome and Ctrl-exosome were analyzed by miRNA chip to compare the differential expression of miRNA between the two groups,and qPCR was used to verify the differentially expressed miRNA.14.Statistical analysisWhen the measurement data conforms to the normal distribution and the variance is homogeneous,it is expressed as the mean ± standard error(Means ± SEM),and the t-test is used for statistics.Numbers(second quartile,third quartile)were expressed,and the Mann-Whitney test was used,and GraphPad Prism 8 software was used for statistical analysis of the data,including normal distribution test,homogeneity of variance test,t test and Mann-Whitney test et al,P<0.05 was considered statistically significant.(Ⅱ)In vitro study1.Primary cardiomyocytes H/R cell model establishmentFrom 1-3day old Wistar rats,the heart was cut into pieces,and the remaining blood cells were washed.The minced myocardial tissue was digested with collagenase Ⅱ at 37℃ for 1 hour,and then digested again for 1 hour after replacing the collagenase II.The cell pellet was obtained after two supernatants were collected and centrifuged.The pellet was resuspended in medium and passed through a 100 μm mesh,and then the supernatant was placed in a cell culture flask for differential adhesion for 90 minutes.The remaining supernatant was cardiomyocytes and adherent cells were fibroblasts.Cardiomyocytes were counted with a cytometer,and seeded in cell culture flasks at a density of 1*106 for subsequent experiments.The primary cardiomyocytes cultured in a normal environment(DMEM low-glucose medium,95%O2+5%CO2)were placed in a sugar-free hypoxic environment(DMEM low-sugar medium,1%O2+94%N2+5%C02)for 12 h,the cells were cultured in a normal environment for 4 h,and then perfused after simulating recovery.2.Experimental groups and drug administration(1)Cell experiment 1:To verify the effect of RIC-exosome on PCAMD.Cells were divided into 4 groups:1)Normoxia group:always cultured in normal environment;2)H/R+Vehicle group:cells were hypoxia without glucose for 12 h,added 100 μL PBS,and continued to culture in normal environment for 4 h;3)H/R+RIC-exosome group:cells were hypoxia without glucose for 12 h,100 μL RIC-exosome was added,and cultured in normal environment for 4 h;4)H/R+Ctrl-exosome group:cells were hypoxia without glucose for 12 h,and 100 μL Ctrl-exosome was added,and continue to culture for 4 h.(2)Cell Experiment 2:Inhibition of exosome secretion blocks the cardioprotective effect of RIC.Rats were randomly divided into 2 groups,respectively intraperitoneal injection of GW4869 and DMSO(4 times a week),plasma was collected after RIC to separate exosomes.The cells were divided into 2 groups:1)H/R+RIC-exosomeGW4869 group:cells without glucose and hypoxia for 12 h,added RIC-exosomeGW4869,and continued to culture in normal environment for 4 h;2)H/R+RIC-exosomeVehicle group:cells without After glucose hypoxia for 12 h,RIC-exosome was added and cultured in normal environment for 4 h.(3)Cell experiment 3:To verify the protective effect of miR-542-5p on H/R-stimulated cardiomyocyte injury.The cells were divided into 4 groups:1)miR-542-5p mimic+H/R:cells were transfected with miR-542-5p mimic 48 h in advance,followed by glucose-free hypoxia for 12 h,and cultured in normal environment for 4 h;2)NC mimic+H/R:cells were transfected with NC mimic 48 h in advance,followed by glucose-free hypoxia for 12 h,and continued to culture in normal environment for 4 h;3)miR-542-5p inhibitor+H/R+RIC-exosome:cells were transfected with miR 48 h in advance-542-5p inhibitor.After adding RIC-exosome,cells were cultured without glucose and hypoxia for 12 h and continued to culture in normal environment for 4 h;4)NC inhibitor+H/R+RIC-exosome:cells were transfected with NC inhibitor 48 h in advance.After adding RIC-exosome,the cells were hypoxic and glucose-free for 12 h,and cultured in a normal environment for 4 h.(4)Cell experiment 4:IGF2R overexpression can reverse the cardioprotective effect of miR-542-5p.The cells were divided into 3 groups:1)Empty vector group+NC mimic+H/R group:cells were transfected with plasmid empty vector and NC mimic 48 h in advance,then cultured in sugar-free hypoxia for 12 h,and in normal environment for 4 h;2)Empty vector group+miR-542-5p mimic+H/R group:cells were transfected with plasmid empty vector and miR-542-5p mimic 48 h in advance,then cultured in sugar-free hypoxia for 12 h,and in normal environment for 4 h;3)IGF2R overexpression plasmid+miR-542-5p mimic+H/R group:cells were transfected with IGF2R overexpression plasmid and miR-542-5p mimic 48 h in advance,and then cultured in glucose-free hypoxia for 12 h and in normal environment for 4 h.3.Measurement of cardiomyocyte apoptosisThe apoptosis of four groups was detected by flow cytometry.4.Protein expression detectionAfter protein extraction from the treated cardiomyocytes,Western blot was used to detectthe protein expression of primary cardiomyocytes.5.Cardiomyocyte activity detectionCardiomyocytes were seeded in a 96-well plate at an appropriate density,and the activityof cardiomyocytes was detected by CCK8.6.Measurement of myocardial cell damageThe LDH activity of cardiomyocytes was detected by ELISA to reflect the degree ofmyocardial cell injury.7.Dual-luciferase reporter gene assayThe binding site of miRNA-542-5p and IGF2R was predicted by bioinformatics tools,and a mutant plasmid of IGF2R binding site was constructed.Firefly luciferase was used as the reporter gene and sea cucumber luciferase was used as the internal reference gene.HEK293T cells were treated according to(1)IGF2R-WT(wild type)plasmid+NC mimic,(2)IGF2R-WT plasmid+miRNA-542-5p mimic,(3)IGF2R-MUT(mutant)plasmid+NC mimic,(4)IGF2R-MUT plasmid+miRNA-542-5p mimic was grouped for transfection,and the fluorescence intensity was observed by fluorescence microplate reader.8.Statistical analysisSame as animal experiments.Results1.Baseline and procedural characteristics for the animal studyIn the 4 animal experiments,the animal baseline and model data were not significantly different from the control group,including body weight,heart rate,MAP,CO,EF,duration of cardiac arrest,duration of CPR,and ROSC(%).2.RIC significantly up-regulated the exosome content in plasmaWestern blot detection of exosome marker proteins CD9,CD63 and TSG101 showed that RIC significantly increased the exosome content in plasma.NTA particle size analysis also showed that in the same amount of plasma,the exosome content in the plasma of rats stimulated by RIC was higher.3.PKH26-labeled exosomes were taken up by cardiomyocytesPKH26-labeled exosomes are shown as red fluorescence.Fluorescence results showed that exosomes could be taken up by myocardial tissue 1h after injection,peaked at 24 h,and decreased slightly at 72 h.4.RIC-exosome ameliorated cardiac dysfunction after CA/CPRUltrasound results showed that compared with the CA/CPR group,the cardiac function of the CA/CPR+RIC-exosome group was significantly improved 1-4 h after ROSC.5.RIC-exosome improves the 7-day survival rate after ROSCSurvival analysis showed that RIC-exosome significantly improved the 7-day survival rate after ROSC in CA/CPR rats.6.RIC-exosome reduces myocardial enzyme levels after CA/CPRIn animal experiment 1,the ELISA results showed that the serum levels of cTnT,CK-MB and LDH in the CA/CPR group were significantly increased at 24 h after ROSC.However,the serum levels of cTnT,CK-MB and LDH in the CA/CPR+RIC-exosome group were significantly lower than those in the CA/CPR group at 24 h after ROSC.7.RIC-exosome suppressed cardiomyocyte apoptosis after CA/CPRIn animal experiment 1,TUNEL results showed that the ratio of myocardial cell apoptosis in the CA/CPR+RIC-exosome group was significantly lower than that in the CA/CPR group.In cell experiments,the administration of RIC-exosome significantly alleviated the apoptosis of primary cardiomyocytes caused by H/R.Western blot results showed that compared with the Normoxia group,the expression of Cleaved caspase-3 increased 4 h after H/R,and RIC-exosome administration significantly inhibited the apoptosis caused by H/R.8.RIC-exosome fails to reduce myocardial mitochondrial structural damage and inflammation levels after CA/CPRIn animal experiment 1,the results of myocardial tissue electron microscopy indicated that the mitochondrial structure of myocardial cells in the CA/CPR group was more damaged at 4 h after ROSC,such as swelling and mitochondrial cristae rupture.The semi-quantitative analysis of the severity of mitochondrial structural damage was performed using the Flameng score,and it was found that mitochondria were significantly damaged 4 h after ROSC,while the degree of mitochondrial structural damage in rat cardiomyocytes in the CA/CPR+RIC-exosome group was significantly higher than that of CA/CPR 4 h after ROSC.There were no significant differences in the CPR group.ELISA results showed that the levels of IL-1β and IL-10 in serum and myocardial tissue homogenate of CA/CPR+RIC-exosome group were not significantly different from those of CA/CPR group.9.RIC-exosome significantly alleviated H/R-induced cardiomyocyte injuryExosome uptake was observed after 6 hours of co-incubation of PKH26-labeled exosomes with primary cardiomyocytes.Cardiomyocyte apoptosis was detected by flow cytometry,and the results showed that RIC-exosome significantly reduced H/R-induced cardiomyocyte apoptosis.Western blot detection of apoptosis protein indicators Cleaved caspase 3,Bax and Bcl-2 also reached the same conclusion.Cell viability was detected by CCK8,and LDH activity was detected by ELISA.The results showed that RIC-exosome could significantly improve the decrease of cell viability caused by H/R and reduce cell damage.10.GW4869 reversed RIC-induced cardioprotectionAs an exosome inhibitor,the application of GW4869 significantly reduced RIC-induced plasma exosome content while reversing RIC-induced cardioprotection.In vitro experiments also confirmed that administration of GW4869 could reverse the anti-cardiomyocyte apoptosis effect of RIC-exosome.11.miR-542-5p was significantly upregulated in RIC-exosomeThe results of miRNA microarray analysis and qPCR validation indicated that the content of miRNA-542-5p was significantly up-regulated in RIC-exosomes derived from rats and humans.And mature miRNA-542-5p was transported from skeletal muscle to cardiac muscle using RIC-exosome as a carrier.12.miRNA-542-5p exerted cardioprotective effect in PCAMDRat myocardium-specific overexpression of miRNA-542-5p can improve cardiac function and 7-day survival rate in rats after ROSC,and reduce cardiomyocyte apoptosis;simultaneous injection of miRNA-542-5p antagomir with RIC-exosome can reverse the myocardium of RIC-exosome Protective effects.13.miRNA-542-5p reduced cardiomyocyte apoptosis by inhibiting IGF2R expressionThe results of bioinformatics analysis and mRNA sequencing indicated that IGF2R was a downstream target gene of miRNA-542-5p.The results of Western blot and luciferase reporter gene confirmed that miRNA-542-5p could inhibit the expression of IGF2R,thereby inhibiting the expression of apoptotic genes downstream of IGF2R and exerting anti-myocardial apoptosis effect.Overexpression of IGF2R reversed the anti-apoptotic effect of miRNA-542-5p in cell experiments.Conclusion1.Immediately after ROSC,intravenous injection of RIC-exosome can improve cardiac dysfunction after cardiac arrest,reduce myocardial cell death,and improve the survival rate after ROSC in rats.2.miRNA-542-5p was specifically up-regulated in RIC-exosome and exerted cardioprotective effect in PCAMD.3.miRNA-542-5p exerts anti-apoptotic effect on cardiomyocytes by inhibiting the expression of IGF2R and its downstream genes. |
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