| Objective:To observe the alteration of phenotypes and concentration of circulating microvesicles(MVs) from myocardial ischemic preconditioning(IPC) treated rats(IPC-MVs). To investigate the effects of IPC-MVs on ischemia/reperfusion(I/R)injury in rats, and the underlying mechanism of endoplasmic reticulum stress(ERS)-mediated myocardial apoptosis.Methods:1. Establishment of I/R injury and IPC models in vivo in ratsHealthy male Wistar rats were divided into three groups randomly: Sham, I/R and IPC group. Sham group: rats were left untreated after a silk ligature was placed around the left anterior descending(LAD) coronary artery. I/R group: rats were subjected to 30-min ischemia and 120-min reperfusion of LAD. IPC group: rats received three cycles of 5-min ischemia and 5-min reperfusion before 30-min ischemia and 120-min reperfusion of LAD. A lead ? electrocardiogram(ECG),systolic blood pressure(SBP), diastolic blood pressure(DBP) was monitored throughout the experiment and ventricular arrhythmia(VA) was recorded during ischemia. Myocardial infarct size was measured by trypan blue/TTC double staining.2. Isolation and characterization of IPC-MVsHealthy male Wistar rats were randomized into two groups: Sham and IPC group. Sham group: rats were left untreated for 30 min after a silk ligature was placed around the LAD. IPC group: rats received three cycles of 5-min ischemia and 5-min reperfusion of the LAD. The blood was drawn from abdominal aorta once the operation was finished, and was anticoagulated with sodium citrate. Platelet-free plasma(PFP) was isolated through two steps of centrifugation at room temperature from the peripheral blood, and IPC-MVs were isolated by ultracentrifugation from PFP until subsequent use. PFP was incubated with anti-CD61, anti-CD144,anti-CD45 and anti-Erythroid Cells, and added 1, 2 ?m latex beads to calibrate and absolutely count by flow cytometry.3. Effects of IPC-MVs on MAP, HR, ST-segment and necrosis in I/R injury ratsHealthy male Wistar Rats were divided into three groups randomly: Sham, I/R and IPC-MVs+I/R group. Sham group: rats were left untreated for 150 min after a silk ligature was placed around the LAD. I/R group: rats were subjected to 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs+I/R group: rats were subjected to 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg was infused via the femoral vein at 25 min during ischemia, while the same volume of 0.9 %normal saline(NS) was given to the other two groups. ECG, SBP, DBP was monitored throughout the experiment and VA was recorded during ischemia. The activity of plasma lactate dehydrogenase(LDH) was tested by Microplate Reader.Myocardial infarct size was measured by trypan blue/TTC double staining. Changes of myocardial morphology were observed via hematoxylin-eosin(HE) staining.4. Effects of IPC-MVs on myocardial apoptosis in I/R injury ratsGroups of experiment were same with Method 3. Apoptosis of cardiomyocytes was assessed by terminal deoxyribonucleotidyl transferase-mediated d UTP nick-end labeling(TUNEL) assay. Activity of Caspase 3 was detected by Microplate Reader.5. Effects of IPC-MVs on associated protein expression ofendoplasmic reticulum in I/R injury ratsGroups of experiment were same with Method 3. Activity of Caspase 12 in myocardial tissues was detected by Microplate Reader. Moreover, the levels of GRP78 and CHOP were determined by Western blot.Results:1. Establishment of I/R injury and IPC models in vivo in ratsThe I/R injury model in rats was established successfully. Compared with pre-ischemia, mean arterial blood pressure(MAP) was decreased and ST-segment was elevated at the beginning of ischemia(56.62±9.10 mm Hg vs 85.86±10.71 mm Hg;0.322±0.034 m V vs 0.012±0.031 m V, P<0.001). The ST-segment corresponding to ischemic injury was elevated significantly at the end of ischemia(0.500±0.060 m V).Compared with pre-ischemia, heart rate(HR) was decreased significantly at the end of reperfusion(285±22 beats/min vs 464±20 beats/min, P<0.001). The onset of ventricular premature contraction(VPC) was early(6.54±1.11 min), and the number was more(205±33). The onset of ventricular tachycardia(VT) was early(8.36±1.28min), and the durations were long(0.68±0.41 min). The incidences of VPC, VT and ventricular fibrillation(VF) were 100 %, 100 % and 75 %, respectively. The percentages of IS to area at risk(AAR) in myocardium were 43.04±6.26 %.Compared with I/R group, IPC group showed a significant improvement in above mentioned data. The elevation of ST-segment was decreased at the end of ischemia(0.403±0.043 m V vs 0.500±0.060 m V, P<0.01, respectively). HR was restored significantly at the end of reperfusion(327±11 beats/min vs 285±22beats/min, P<0.01). The onsets of VPC(16.49±9.49 min vs 6.54±1.11 min, P<0.01)and VT(15.23±2.40 min vs 8.36±1.28 min, P<0.001) were delayed. The number of VPC was decreased(15±23 vs 205±33, P<0.001, respectively). The durations of VT were shortened(0.12±0.18 min vs 0.68±0.41 min, P<0.05, respectively), and incidences of VT and VF were decreased(37.5 % vs 100 %; 0 % vs 75 %, P<0.01,respectively). The myocardial infarct size(IS/AAR) was also reduced(18.76±4.18 %vs 43.04±6.26 %, P<0.001, respectively). The IPC model in rats was established successfully.2. Isolation and characterization of IPC-MVsPlatelet-free plasma(PFP) was obtained from blood samples by two steps of centrifugation in 2,600 g, 15 min and 10,000, 5 min at room temperature. IPC-MVs were isolated by ultracentrifugation from PFP at 33,000 rpm, 148 min, 4 ?. Total IPC-MVs and different phenotypes, including platelet-derived MVs(PMVs),endothelial cell-derived MVs(EMVs), leucocyte-derived MVs(LMVs) and erythrocyte-derived MVs(RMVs) were all isolated which were identified membrane vesicles smaller than 1 ?m with corresponding antibody positive. Compared with Sham group, the numbers of PMVs(1094±181 events/?L vs 719±115 events /?L,P<0.05), EMVs(475±42 events/?L vs 388±34 events/?L, P<0.05) and RMVs(1539±212 events/?L vs 1200±171 events/?L, P<0.05) were significantly increased in circulation of IPC treated rats. Total number(4380±745 events/?L vs 3453±307events/?L) and LMVs(117±20 events/?L vs 91±11 events/?L) did not significantly differ between these two groups.3. Protective effects of IPC-MVs on HR, ST-segment and necrosis in I/R injury rats.Compared with I/R group, IPC-MVs increased HR at the end of 120-min reperfusion in I/R injury rats(331±9 beats/min vs 305±13 beats/min, P<0.01,respectively). ST-segment during ischemia was elevated dramatically and decreased gradually after reperfusion in I/R and IPC-MVs+I/R groups. IPC-MVs decreased ST-segment at the end of 120-min reperfusion compared with I/R group(0.043±0.027 m V vs 0.097±0.056 m V, P<0.05, respectively). There was no difference in the changes of MAP and VA between these two groups. Compared with Sham group,LDH activity was distinctly increased at the end of 30-min ischemia and 120-min reperfusion both in I/R and IPC-MVs+I/R groups(P<0.001). Moreover, compared with I/R group, the LDH activitiy was obviously decreased after 120-min reperfusion in IPC-MVs+I/R group(10.98±1.01 vs 12.91±1.11 U/m L, P<0.01, respectively).IPC-MVs treatment decreased IS/AAR % significantly(29.63±3.90 vs 40.50±2.54 %,P<0.01, respectively), and the damage of myocardium was obviously alleviated in IPC-MVs+I/R group.4. Anti-apoptotic effects of IPC-MVs on myocardial I/R injury in ratsCompared with I/R group, TUNEL staining displayed that apoptosis index(AI)of cardiomyocytes was significantly reduced in IPC-MVs+I/R group(22.98±1.61 vs34.14±1.26 %, P<0.001, respectively). IPC-MVs treatment substantially reduced the activity of Caspase 3 compared with I/R group(54.35±3.75 U/?g vs 71.23±4.35 U/?g,P<0.001, respectively).5. IPC-MVs against myocardial I/R injury in rats through attenuation of ERSThe activity of Caspase 12 was reduced to 43 % with IPC-MVs treatment compared with I/R group(P<0.05). Western blot indicated that the expression of GRP78 and CHOP were clearly decreased in IPC-MVs+I/R group(0.47±0.07 % vs0.77±0.10 %, P<0.001; 0.68±0.07 % vs 0.89±0.10 %, P<0.01, respectively).Conclusion:1. I/R injury and IPC models in vivo in rats were established successfully.2. IPC-MVs were identified the membrane vesicles smaller than 1 ?m, and released from different cells including platelets, endothelial cells, leukocytes and erythrocytes. The levels of PMVs, EMVs and RMVs were significantly increased in IPC-MVs.3. 7 mg/kg IPC-MVs restored HR, decreased ST-segment, and alleviated necrosis of I/R injury rats by decreasing the plasma activity of LDH, and reducing myocardial infarct size.4. 7 mg/kg IPC-MVs exhibited protective effects on myocardial apoptosis in I/R injury rats by decreasing the myocardial apoptotic index and Caspase 3 activity.5. The attenuation of ERS-induced apoptosis might be involved in the cardioprotective mechanisms of IPC-MVs through decreasing Caspase 12 activity,and down-regulating the expression of GRP78 and CHOP. |
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