| Objective:To isolate the circulating microvesicles(MVs) derived from myocardial ischemic preconditioning(IPC) treated rats(IPC-MVs) and observe the microstructure of IPC-MVs by transmission electron microscope. To investigate the effects of IPC-MVs on myocardial ischemia/reperfusion(I/R) injury in rats and explore the underlying mechanism which is related to mitochondrialmediated myocardial apoptosis pathway was studied.Methods:1. Establishment of myocardial I/R and IPC model of rats in vivoHealthy male Wistar rats were divided into 3 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; IPC group, the rats were subjected to three cycles of 5-min ischemia and 5-min reperfusion of the LAD, then were subjected to 30-min ischemia and 120-min reperfusion. ECG was monitored throughout the operation and ventricular arrhythmia was recorded during ischemia period. Myocardial infarct size was detected by TTC staining.2. Isolation of IPC-MVs and observation of the microstructure of IPC-MVsHealthy male Wistar rats were left untreated for 15 min after a silk suture was placed around the LAD coronary artery. Rats were subjected to 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 anti-coagulated with sodium citrate.Platelet-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 ?. IPCMVs were stained by 2 % phosphotungstic acid and the microstructure was observed by transmission electron microscope(TEM).3. The effects of IPC-MVs on MAP, HR, ST-segment and myocardial tissue necrosis in I/R injury ratsHealthy male Wistar rats were divided into 3 groups randomly: sham, I/R and IPC-MVs+I/R group. Sham group, rats were left untreated for 148 min after a silk ligature was placed around the LAD coronary artery; I/R group, rats were subjected to30-min ischemia and 120-min reperfusion; IPC-MVs+I/R group, rats received 30-minischemia and 120-min reperfusion of the LAD. IPC-MVs 7 mg/kg was infused via the femoral vein within 1 min at 25 min during ischemia, while the same volume of NS was given to the other two groups. ECG was monitored throughout the operation.Plasma activity of lactate dehydrogenase(LDH) was tested by colorimetry.Myocardial infarct size was detected by TTC staining. Morphological changes of myocardium were observed by HE staining.4. The effects of IPC-MVs on myocardial apoptosis in I/R injury ratsExperiment operations were same with Method 3. Apoptosis of myocardial cells were detected with TUNEL assay. The activity of caspase 3 in myocardium was assayed with spectrophotometry.5. The effects of IPC-MVs on mitochondria associated protein in I/R injury ratsExperiment operations were same with Method 3. Expression of Bcl-2 and Bax protein was examined by Western blot.Results:1. The myocardial I/R and IPC model of rats in vivo were successfully establishedIn I/R group, compared with pre-ischemia, the ST-segment was elevated significantly at the end of ischemia(0.495±0.059 m V vs 0.041±0.036 m V, P<0.001).The incidences of ventricular premature contraction(VPC) and ventricular tachycardia(VT) were both 100 %. The incidence of ventricular fibrillation(VF) was75 %. The onset of VPC was 6.65±1.02 min, the number was 205±43. The onset of VT was 8.24±1.17 min, and the duration was 0.68±0.14 min. Infarct size(IS) was94.85±20.15 mg, and the ratio of IS/AAR was 42.04±6.26 %.Compared with I/R group, IPC group revealed a remarkable improvement of above mentioned data. The elevation of ST-segment was significantly decreased at the end of ischemia(0.384±0.032 m V vs 0.495±0.059 m V, P<0.001). The incidences of VT and VF were decreased(50 % vs 100 %, P<0.05; 25 % vs 75 %, P<0.05), the onset of VPC and VT was delayed(15.51±2.52 min vs 6.65±1.02 min, P<0.001;17.59±1.34 min vs 8.24±1.17 min, P<0.001), the duration of VT was shortend(0.29±0.18 min vs 0.68±0.14 min, P<0.001). The ratio of IS/AAR was also decreased(20.45±4.18 % vs 42.04±6.26 %, P<0.001).2. Isolation of IPC-MVs and observation of the microstructure of IPC-MVsIPC-MVs were isolated by ultracentrifugation, then re-suspended in NS and stored at-80? for the experiments. To quantify the protein content of IPC-MVs, a BCA protein assay kit was used. The protein content of IPC-MVs was 4.46 mg/m L.The isolated IPC-MVs were subjected to transmission electron microscopy revealing small, rounded vesicles(100-1000 nm) surrounded by a bilayered membrane. Some of them had a distinct electron dense appearance while others displayed an electron lucent interior.3. IPC-MVs pretreatment ameliorates HR, ST-segment and reduces myocardial tissue necrosis in I/R injury ratsThe value change tendency of MAP, HR and ST-segment in I/R and IPCMVs+I/R groups was similar. Compared with I/R group, IPC-MVs increased HR(361±15 beats/min vs 344±14 beats/min, P<0.05), and decreased ST-segment at the end of 120-min reperfusion in myocardial I/R injury rats(0.023±0.012 m V vs0.082±0.055 m V, P<0.05). There was no significant difference in the change of MAP between two groups. Compared with sham group, the activity of plasma LDH was markedly increased both at the end of 30-min ischemia and 120-min reperfusion in other two groups. Compared with I/R group, the activity of plasma LDH significantly decreased at the end of 120-min reperfusion in IPC-MVs+I/R group(11.345±0.756U/m L vs 13.035±0.977 U/m L, P<0.01). IPC-MVs alleviated the damage of tissues in I/R injury rats significantly. Compared with I/R group, IS(62.28±8.75 mg vs86.24±7.45 mg, P<0.01) and IS/AAR(28.73±4.02 % vs 40.68±3.24%, P<0.01) was obviously lower.4. IPC-MVs pretreatment inhibits myocardial apoptosis in I/R injury ratsCompared with I/R group, myocardial apoptosis index(19.98±1.51 vs35.75±1.20 %), as well as the activity of caspase 3(52.43±2.55 vs 70.25±3.21 U/?g,P<0.001) was markedly decreased in IPC-MVs treated rats.5. IPC-MVs pretreatment elevates the ratio of Bcl-2/Bax in I/R injury ratsCompared with I/R group, the expression of Bcl-2 was significantly increased(0.81±0.11 vs 0.51±0.24, P<0.01), the expression of Bax was decreased(0.38±0.15 vs 0.60±0.13, P <0.01), the ratio of Bcl-2/Bax was markedly increased after IPCMVs treatment(1.54±0.25 vs 0.86±0.17, P<0.001).Conclusions:1. The myocardial I/R and IPC model of rats was successfully established in vivo.2. The circulating IPC-MVs were isolated by ultracentrifugation and displayed a spherical shape under transmission electron microscopy.3. 7 mg/kg IPC-MVs was injected into I/R injury rats via the femoral veinwhich could increase the HR, decrease the ST-segment and plasma activity of LDH at the end of 120-min reperfusion, reduce myocardial infarct size, and alleviate myocardial tissue necrosis.4. 7 mg/kg IPC-MVs was injected into I/R injury rats via the femoral vein which shown protective effects on myocardial apoptosis by decreasing the myocardial apoptotic index and caspase 3 activity.5. 7 mg/kg IPC-MVs could up-regulate the expression of Bcl-2, and downregulate the expression of Bax, and decrease the caspase 3 activity in I/R injury rats.It was shown that the inhibition of mitochondrial-mediated myocardial apoptosis might be involved in the cardioprotective mechanisms of IPC-MVs. |
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