| Objective:To isolate the circulating microvesicles derived from myocardial ischemia preconditioning in rats,BCA and flow cytometry were used to identify content of protein and composition.Microarray was used to determine content and alterations in gene expression profiles in IPC-MVs and Sham-MVs.which provided insight into the roles of the specific miRNA that regulated specified genes in the ischemic injuries.Methods1.Establish ischemia preconditioning model in ratsHealthy Wistar Rats were divided into three groups randomly and then performed a thoracotomy before ligating left anterior descending coronary artery(LAD).?Sham group,rats were left untreated for 195 min.?I/R group,rats underwent ischemia for 30 min,then reperfusion for 120 min after being untreated for 45 min.? IPC-MVs group,rats performed with transient ischemic/reperfusion pretreatment which was three cycles of 5 min ischemia and 5 min reperfusion of LAD after being untreated for 15 min,and then followed ischemia for 30 min,reperfusion for 120 min.ECG was used to monitor heartbeats and blood pressure.Trypan blue and TTC were used to stain heart.2.Isolation and characterization of MVsHealthy Wistar rats were divided into two groups randomly,and then performed a thoracotomy before ligating left anterior descending coronary artery(LAD).A polyethylene tube was used to compress the LAD.?IPC-MVs group,rats performed with three cycles of 5 min ischemia and 5 min reperfusion of LAD after being untreated for 15 min.? Sham-MVs group,rats were left untreated for 45 min Blood was taken from the abdominal aorta after 45 min.Then MVs were obtained by centrifugation.Flow cytometry was used to detect the phenotypes and amount of MVs,BCA method was used for quantitative analysis.3.Establish myocardial ischemia/reperfusion model in ratsHealthy Wistar Rats were divided into two groups randomly,and then performed a thoracotomy before ligating left anterior descending coronary artery(LAD).?ham group,rats were left untreated for 145 min.? I/R group,rats underwent prolonged alchemist/reperfusion treatment which was ischemia for 30 min,and then reperfusion for 120 min after being untreated for 15 min.ECG was used to monitor heartbeats and blood pressure.Trypan blue and TTC were used to stain heart.4.IPC-MVs treat I/R injury ratsRats were divided into four groups randomly,Sham group(NS),I/R group(NS), IPC-MVs+I/R group(IPC-MVs)and Sham-MVs+I/R group(Sham-MVs).After ischemia 25 min,the rats underwent injecting of NS or MVs via femoral vein.MAP and EGG were monitored,infarct size and the activity of LDH were quantified.The occurrence of arrhythmia was recorded.5.microarray analysis and prediction of target genes of miRNARats were divided into two groups randomly,RNA was extracted from IPC-MVs and Sham-MVs,microarray was used to analysis the profile of miRNA in IPC-MVs and Sham-MVs.MiRNAorg,Targetscan and Pita database were used to predict the specific target genes.GO and KEGG were used to predicted their target genes and pathway.The miRNA-mRNA network was established and the functional miRNA-mRNA pairs were identified to explore the possibly central target genes.6.qRT-PCR verify the result of microarrayTotal RNA was extracted from IPC-MVs and Sham-MVs,qRT-PCR was used to verify whether the differently expressed miRNA was consistent with the result of the microarray.Results1.Establishment of IPC models in ratsCompared with I/R group,MAP and HR in IPC group were restored significantly.(59.92 ± 13.72 mmHg vs 53.29 ± 11.62 mmHg,P<0.05;320±14 beat/min vs 305±29 beat/min,P<0.05).The onset of ventricular premature contraction(VPC)and(VT)were late(14.48 ± 4.86 min vs 6.54 ± 1.19 min,P<0.001; 17.41 ± 6.63 min vs 7.33 ± 1.83,P<0.001,and the number was less(23 ± 15 vs 237± 78,P<0.001).The durations were shorter(12.89 ± 16.94 s vs 59.33 ± 41.27 s, P<0.01).The infract area was decreased significantly(20.10 ± 7.8%vs 42.98±4.7%,P<0.001).The IPC model in rats was established successfully.2.Isolation and characterization of MVsMVs were isolated by sequential centrifugations of the blood sample at 2,600 g for 15 minutes at room temperature,collected the supernatant and centrifuged at 10,000 g for 5 minutes to obtain platelet-free plasma(PFP).Take a small amount of PFP was stored at-80? after fixed with paraformaldehyde for flow cytometry,the remaining PFP centrifuged at 33,000 r/min for 148 min at 4?,the supernatant was removed to obtain MVs.Total IPC-MVs and different phenotypes,including platelet-derived MVs(PMVs),endothelial cell-derived MVs(EM Vs),leucocyte-derived MVs(LMVs)and erythrocyte-derived MVs(RMVs)were identified by flow cytometry.Particles smaller than 1 ? m were MVs,and the phenotype of MVs were identified by corresponding antibody.Compared with Sham group,the numbers of IPC-PMVs(2415 ±225 events/?L vs 1835 ± 119 events/?L,P<0.001)and IPC-RMVs(16091357 events/?L vs 1051 ± 183 events/?L,P<0.05)were significantly increased in circulation of IPC treated rats.Total number(4125 ±863 events/?L vs 3392 ± 154events/?L),IPC-EMVs(473 ± 79 events/?L vs 415 ±4 events/?L)and IPC-LMVs(115 ± 29 events/?L vs 95 ± 17 events/?L)did not significantly differ between these two groups.3.Establishment of I/R injury models in ratsCompared with Sham group,HR in I/R group was decreased progressively,at reperfusion 120,HR and MAP were decreased significantly.(315±26 beat/min vs 389 ± 22 beat/min,P<0.001;58.91 ± 12.23 mmHg vs 83.34 ± 12.30 mmHg,P<0.001)The ST-segment was elevated significantly at the end of ischemia(0.176 ± 0.044 mV vs 0.045 ± 0.016 mV,P<0.001).The first onset of VPC was 7.44 ± 1.19 min,and the number was 193?75.The first onset of VT was 7.54 ± 1.06 min,and the durations were 58.75 ± 34.85 s.The percentages of IS to area at risk(AAR)in myocardium were 46.00 ± 6.4%.The I/R injury model in rats was established successfully.4.IPC-MVs protect against myocardial I/R injury in ratsCompared with Sham group,HR in I/R group was decreased progressively,at reperfusion 120,HR and MAP were decreased significantly.(263±28 beat/min vs 358±46 beat/min,P<0.01;45.50 ± 3.84 mmHg vs 92.27 ± 11.61 mmHg,P<0.001)The ST-segment was elevated significantly at the end of ischemia(0.212 ± 0.095 mV vs 0.023 ± 0.004 mV,P<0.001).The percentages of IS to AAR in myocardium were 44.79 ± 9.2%.Compared with I/R group,MAP and HR in IPC-MVs group were restored significantly.(74.14 ± 8.20 mmHg vs 61.57 ± 8.93 mmHg,P<0.05;302 ± 23 beat/min vs 263 ± 28 beat/min,P<0.05).LDH activity in IPC-MVs group decreased significantly at 120 min reperfusion(10.46± 12.804 U/mL vs 15.832±2.102 U/mL,P<0.01).The extent of infarct size was determined 120 min after reperfusion and resulted in a significant decrease in IPC-MVs group(24.68 ± 7.5%vs 44.79 ± 9.2%,P<0.01),but not in Sham-MVs group(42.14 ± 3.6%vs 44.79 ± 9.2%)when Compared with I/R group,while there is no significant difference between AAR.5.Profile of miRNA in IPC-MVs and Sham-MVsCompared with Sham-MVs group,5 differentially and over 2 fold change expressed miRNA in IPC-MVs group(P<0.01,FER<0.05),of total,4 miRNA(miR-1-3p,miR-378b,miR-133a-3p,miR-133b-3p)were up-regulated with fold changes lager than two,and 1 miRNA(miR-702-3p)was down-regulated with fold changes less than 40 in the IPC-MVs group.GO analysis showed that the 5 miRNA in biological process were most likely to participate in negative regulating of cell apoptosis,regulating of calcium ion,regulating of blood pressure and participating in hypoxia response induced by ischemia and hypoxia.Based on the results of KEGG,miRNA target genes were involved in a variety of cardiovascular related pathways,aldosterone-regulated sodium reabsorption,leukocyte transendothelial migration,vasopressin regulates water reabsorption,sphingolipid signaling pathway,phosphatidylinositol signaling system,HIF-1 signaling pathway.Based on the miRNA-mRNA network,the key targets were Pcdha4;Acvrll;Ndrg1;Ddi2;Syt1;Vamp2;Tagln2;Ifit2;Slc6a7;Bcl212;Hsd17b11;Sv2a;Sulfi;Hivep2?6.qRT-PCRThe results of miR-1-3p,miR-133a-3p,miR-133b-3p were consistent with the expression patterns in microarray.However miR-378b and miR-702-3p were opposite,miR-378b was down-regulated with 0.67-fold change in the IPC-MVs,while miR-702-3p was up-regulated with above 2 fold change.Conclusion:1.IPC model in rats were established successfully.2.IPC-MVs in circulating blood were successfully isolated,and the number of MVs derived from platelets and erythrocyte were significantly increased.3.7 mg/kg IPC-MVs could effectively alleviate myocardial I/R injury.4.Compared with Sham-MVs group,5 differentially expressed miRNA in IPC-MVs group.miR-1-3p,miR-702-3p,miR-133a-3p and miR-133b-3p were up-regulated,miR-378b was down-regulated5.miRNA target genes were involved in apoptosis,sphingolipid signaling pathway,and HIF-1 signaling pathway. |
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