| Objective:Ischemic heart disease(IHD)is a major cause of mortality worldwide.Ischemia/reperfusion(I/R)injury induces myocardial infarction in the ischemia area.It is of great significance for better protection of myocardium and reduction of morbidity and mortality rates from IHD during pathophysiological process of I/R injury.Microvesicles(MVs),which also are known as microparticles,are released from cells during activation or apoptosis and played an important role in the development and progression of IHD.Our purpose of this study is to confirm the role of circulating MVs derived from myocardial ischemia in myocardial I/R injury in vivo in anesthetized rats and investigate the mechanisms.This study would provide necessary experimental basis for better understanding the role of MVs in I/R injury.Methods:1.Establishment of I/R model in vivo in rats and isolation and extraction of MVs in circulation from myocardial ischemiaHealthy male Wistar rats were divided into 2 groups randomly:(1)sham group,rats were left untreated for 165 min after a silk ligature was placed around the left anterior descending(LAD)coronary artery;(2)I/R group,rats were subjected to 30min ischemia/120 min reperfusion.A leadⅡelectrocardiogram(ECG)was monitored throughout the experiment and ventricular arrhythmia(VA)was recorded during ischemia period.Myocardial infarct size was determined by TTC staining.Healthy male Wistar rats were divided into 2 groups randomly:(1)sham group,rats were left untreated for 45 min after a silk ligature was placed around the LAD,blood was drawn from the abdominal aorta;(2)mycardial ischemia 30 minutes(I 30min)group,rats were subjected to 30 min of ischemia,and blood was drawn from the abdominal aorta at the instant.The blood was anticoagulated with 3.5%sodium citrate and platelet-free plasma(PFP)was obtained after two centrifugation steps were performed.Flow cytometry was used to count MVs in PFP.The remaining PFP was further ultercentrifuged to obtain MVs until subsequent use.2.The effects of MVs from myocardial ischemia on I/R injuryHealthy male Wistar rats were divided into 3 groups randomly:(1)sham group,rats were left untreated for 165 min after a silk ligature was placed around the LAD;(2)I/R group;(3)MVs group.All groups but not sham were subjected to 30 min ischemia/120 min reperfusion through ligation of LAD coronary artery.MVs the same level as circulation was infused intravenously via the femoral vein in MVs group.The same volume of NS was given to other two groups.All the agent treatments began at 25 min ischemia,totally 1 min for infusion time.A leadⅡECG was monitored throughout the experiment and VA was recorded during ischemia.The activity of serum lactate dehydrogenase(LDH)and creatine kinase-MB(CK-MB)were tested by spectrophotometer and microplate reader respectively.Myocardial infarct size was measured by TTC staining.Changes of myocardial morphology were observed after HE staining.3.The effects of MVs from myocardial ischemia on myocardial apoptosisHealthy male Wistar rats were divided into 3 groups randomly:(1)sham group;(2)I/R group;(3)MVs group.Processing method in all groups were the same as above.All groups but not sham were subjected to 30 min ischemia/120 min reperfusion through ligation of LAD coronary artery.MVs the same level as circulation was infused intravenously via the femoral vein.The same volume of NS was given to other two groups.All the agent treatments began at 25 min ischemia,totally 1 min for infusion time.Apoptosis of cardiomyocyte was assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assay.Activities of caspase 3,9 and 12 in myocardium was detected by microplate reader.The expression of Bcl-2,Bax,GRP78,SERCA2,PLB and p-PLB were determined by Western blot.Results:1.Establishment of I/R model in vivo in rats and isolation and counting of MVs in circulationIn I/R group,the dramatic elevation of ST-segment was observed.The onset of ventricular premature contraction(VPC)was 5.15±0.96 min with the number of237±48.The onset of ventricular tachycardia(VT)was 7.92±0.81 min and the duration was 0.56±0.20 min.The incidences of VPC,VT and ventricular fibrillation(VF)were 100%,100%and 81.5%,respectively.Infarct size(IS)and the ratio of IS versus area at risk(AAR)were 78.65±6.74 mg and 39.01±6.07%,respectively.As demonstrated by flow cytometry,after two steps of centrifugation,the signal of particles smaller than 1μm accounted for more than 99%signal of all.Compared with sham group,MVs derived from LAD 30 min ischemia were significantly increased((15.27±0.99)×10~5 events/μL vs.(12.69±0.52)×10~5 events/μL,P<0.001).2.The effects of MVs from myocardial ischemia on I/R injury in vivo in ratsIn I/R and MVs group,the of ST-segment during ischemia was elevated dramatically,after reperfusion,ST-segment was decreased gradually.There was no difference in the changes of ST-segment and VA between these two groups.Compared with sham group,the activities of serum LDH and CK-MB were obviously increased in the end of ischemia and after 120 min reperfusion(P<0.001)in other two group,moreover,compared with I/R group,MVs group marked decreased after 120min reperfusion.Compared with I/R group,IS and IS/AAR was significantly lower(47.61±8.36 mg vs.81.78±5.45 mg,22.49±3.66%vs.38.01±4.74%,respectively,P<0.001),and the damage of tissue was obviously alleviated in MVs group.3.The effects of MVs from myocardial ischemia on myocardial apoptosisTUNEL staining displayed there were more TUNEL-positive nuclei in other two groups,in contrast to sham group(P<0.001).Compared with I/R group,MVs was obviously attenuated,and apoptosis index(AI)was significantly reduced(22.14±1.89%vs.37.01±1.12%,P<0.001).The activities of caspase 3,9 and 12 in other tow groups were remarkable risen compared with sham group(P<0.001),however,compared with I/R group,MVs group was evidently fallen(20.59±2.38U/μg vs.36.10±3.24 U/μg,25.00±2.64 U/μg vs.35.10±3.15 U/μg,5.54±0.80RFU/mg vs.9.84±1.02 RFU/mg,respectively,P<0.001).Western blot indicated that the expression of Bax was markedly higher but Bcl-2,SERCA2 and p-PLB lower in other two groups especially in I/R group(P<0.001),in constrast to sham group.As a result,compared with I/R group,the ratio of Bcl-2/Bax and p-PLB/PLB were clearly increased in MVs group(P<0.001).Compared with sham group,the expression of GRP78 was clearly decreased in I/R group(0.34±0.04 vs.0.44±0.02,P<0.01),nevertheless,significantly increased in MVs group(0.68±0.07 vs.0.44±0.02,P<0.001).Conclusions:1.The model of I/R in vivo in rats was successfully established.A dramatically increased in circulating MVs derived from myocardial ischemia 30 min was observed.2.The protective effects of MVs at the circulating levels of MVs on myocardial I/R injury in vivo in rats by reducing IS and the radio of IS/AAR,decreasing the activities of LDH and CK-MB.3.MVs at the circulating levels of MVs exhibited protective effects against myocardial apoptosis in intrinsic pathway through up-regulating the expression of Bcl-2,GRP78,SERCA2 and p-PLB and down-regulating the expression of Bax. |
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