Objective: To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles(MVs) from myocardial ischemic preconditioning(IPC) treated rats(IPC-MVs), and to i...Objective: To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles(MVs) from myocardial ischemic preconditioning(IPC) treated rats(IPC-MVs), and to investigate the effects of IPC-MVs on ischemia/reperfusion(I/R) injury in rats. Methods: Myocardial IPC was elicited by three cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending(LAD) coronary artery. 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. PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 μm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure(MAP), heart rate(HR) and ST-segment of electrocardiogram(ECG) were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin(HE) staining. The activity of plasma lactate dehydrogenase(LDH) was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining. Results: 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(<1 μm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats(P<0.05, respectively). In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR(P<0.01), decreased ST-segment and LDH activity(P<0.05, P<0.01). The damage of myocardium was obviously alleviated and myocardial infarct size was significantly lowered after IPC-MVs treatment(P<0.01). Conclusion: The method of flow cytometry was successfully established to detect the phenotypes and concentration alteration of IPC-MVs, including PMVs, EMVs, LMVs and RMVs. Furthermore, circulating IPC-MVs protected myocardium against I/R injury in rats.展开更多
Emerging evidence indicates that ischemic preconditioning （IPC） induces autophagy which attenuates myocardial ischemia/reperfusion （I/R） injury. However, the precise mechanisms remain com- plex and unclear. The pr...Emerging evidence indicates that ischemic preconditioning （IPC） induces autophagy which attenuates myocardial ischemia/reperfusion （I/R） injury. However, the precise mechanisms remain com- plex and unclear. The present study was to investigate which autophagy pathway was involved in the cardioprotection induced by IPC, so that we can acquire an attractive treatment way for iscbemic heart disease. Adult male Sprague-Dawley （SD） rats were randomly divided into sham group, I/R group and IPC group. IPC was induced with three cycles of 5 min regional ischemia alternating with 5 m^n reper- fusion in a heart I/R model. Samples were taken from the center of the infracted heart and examined by using the electron microscopy, the terminal deoxynucleotidyl transferase-mediated nick end-labeling （TUNEL） method, Western blotting and co-immunoprecipitation （Co-IP）. A large number of autophagic vacuoles were observed in the cardiomyocytes oflPC group as compared with I/R group. LC3-II forma- tion, an autophagy marker, was up-regulated in IPC group as compared with FR group （P〈0.05）. Moreover, the interaction between Beclin 1 and Bcl-2 was significantly increased in IPC group as com- pared with I/R group （P〈0.01）. It was also found that IPC decreased I/R-induced apoptosis （P〈0.01）. These results suggest that IPC inhibits Beclin 1-dependent excessive autophagy in reperfusion phase and cooperates with anti-apoptosis pathway to diminish the cell death induced by the myocardial I/R injury.展开更多
Objective: To identify the protective effects of hypovolemic hypotension preconditioning on cardiopulmonary function after myocardial ischemia/reperfusion injury and to explore the possible mechanism.Methods: Twenty-f...Objective: To identify the protective effects of hypovolemic hypotension preconditioning on cardiopulmonary function after myocardial ischemia/reperfusion injury and to explore the possible mechanism.Methods: Twenty-four male white rabbits were randomly assigned to two groups. In the control group, ischemia/reperfusion animals(Group I/R, n=10) were subjected to thirty-minute occlusion of left anterior descending coronary artery and two-hour reperfusion. Animals in hypovolemic hypotension preconditioning group (Group HHP, n=14) experienced brief systemic ischemia preconditioning through blood withdrawl to lower blood pressure to 40%-50% of the baseline before myocardial ischemia/reperfusion. Hemodynamic parameters were recorded. Blood sample was taken to measure superoxide dismutase (SOD), malondialdehyde (MDA) and nitrogen monoxide (NO) changes with blood gas analysis. Myocardium specimens were sampled to examine apoptosis-related gene interleukin-1 beta converting enzyme (ICE) mRNA. Results: Cardiac mechanical function and lung gas exchange remained stable in Group HHP with a significant increase in NO level; while in Group I/R without preconditioning, cardiopulmonary dysfunction was present after 2 h reperfusion associated with a significant reduction in NO formation and an increase in MDA (P<(0.001)). There was negative expression of ICE mRNA in the two groups.Conclusions: Hypovolemic hypotension preconditioning significantly improves cardiopulmonary function and increases NO formation and the protective benefit associated with hypovolemic hypotension preconditioning of the heart may be regulated through NO mediated mechanism.展开更多
Background Whether plasma can transfer the protective effect（s） of remote ischemic preconditioning （RIPC） between animals remains unresolved. We therefore investigated the effects of plasma collected 48 hours afte...Background Whether plasma can transfer the protective effect（s） of remote ischemic preconditioning （RIPC） between animals remains unresolved. We therefore investigated the effects of plasma collected 48 hours after transient limb ischemia on blood pressure recovery during myocardial ischemia reperfusion （IR） in homogenic rats. Methods Plasma was collected from Lewis rats, and the donor rats were randomly assigned to 2 groups： transient limb ischemia and control （n = 8 each）. Transient limb ischemia was achieved by four cycles of 5-minute ischemia and 5-minute reperfusion by noninvasive ligation and deligation of the both legs using elastic rubber bands after anesthesia. In the control group, no ligation was performed. Forty-eight hours later, whole blood was collected, and the plasma spun off. Study Lewis rats underwent 30-minute left anterior descending coronary artery occlusion followed by 180-minute reperfusion, and were randomly assigned to 2 groups （group A and group B, n = 24 each）, each further subdivided into 3 subgroups （n = 8 each）. The subgroups of group A received normal saline （group A1）, plasma of control rats （group A2）, plasma of transient limb ischemia rats （group A3） respectively at 1 hour before IR; the subgroups of group B received normal saline （group B1）, plasma of control rats （group B2）, plasma of transient limb ischemia rats （group B3） respectively at 24 hours before IR. BIOPAC systems were used to measure hemodynamics of rats during myocardial ischemia- reperfusion. Results Systolic blood pressure （SBP） after IR in group B3 was different from that in groups B1 and B2 （B3 vs. B1, ,~=-0.007; B3 vs. B2, P =0.039） at the beginning of reperfusion and 30 minutes after reperfusion. SBP was higher in group B3 than in groups B1 and B2 at the beginning of perfusion （B3 vs. B1, P=-0.010; B3 vs. B2, P=-0.002） and 30 minutes after reperfusion （B3 vs. B1, P=-0.001; B3 vs. B2, P=0.001）. SBP did not differ among subgroups A1, A2 and A3. Diastolic blood pressure and heart rate did not change in group A or group B. Conclusions The transfusion of plasma collected 48 hours after transient limb ischemia into homogenic rats 24 hours before IR can improve the SBP recovery during reperfusion. This may suggest that cardioprotective effect of late phase of RIPC is transferable via plasma.展开更多
目的:探讨κ-阿片受体是否参与缺血后处理的对抗心肌缺血/复灌(I/R)损伤和心肌细胞缺氧/复氧(H/R)损伤的作用及其机制。方法:采用离体大鼠心脏Langendorff灌流模型,全心停灌30 m in、复灌120 m in复制I/R模型,测定心室力学指标和复灌各...目的:探讨κ-阿片受体是否参与缺血后处理的对抗心肌缺血/复灌(I/R)损伤和心肌细胞缺氧/复氧(H/R)损伤的作用及其机制。方法:采用离体大鼠心脏Langendorff灌流模型,全心停灌30 m in、复灌120 m in复制I/R模型,测定心室力学指标和复灌各时点冠脉流出液中乳酸脱氢酶(LDH)含量。实验结束测定心肌组织form azan含量。酶解分离的心肌细胞采用缺氧60 m in、复氧60m in复制H/R模型,测定心肌细胞存活率。结果:在离体心脏模型上,与I/R组相比,缺血后处理组心肌组织的form azan含量明显增高,复灌期间冠脉流出液中LDH含量明显降低,同时缺血后处理明显改善心室力学指标,缓解冠脉流量的减少;在分离心肌细胞模型上,缺氧后处理明显提高心肌细胞存活率。κ-阿片受体阻断剂nor-b inaltorph im ine(nor-BN I)和线粒体ATP敏感性钾通道(m itoKATP)阻断剂5-hydroxydecanoate(5-HD)在离体大鼠心脏模型和分离心肌细胞模型上均能明显减弱缺血后处理的作用。同时在心肌细胞模型上,与H/R组相比,κ-阿片受体激动剂U 50488H明显提高心肌细胞存活率,其作用可被m itoKATP阻断剂5-HD所阻断。结论:缺血后处理具有抗心肌缺血/复灌损伤的作用,这种保护作用可能与其激活κ-阿片受体和m itoKATP有关。展开更多
基金supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China(20101202110005)the Natural Science Foundation of Tianjin(11JCZDJC18300)the Research Foundation of Tianjin Municipal Education Commission(20110106)
文摘Objective: To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles(MVs) from myocardial ischemic preconditioning(IPC) treated rats(IPC-MVs), and to investigate the effects of IPC-MVs on ischemia/reperfusion(I/R) injury in rats. Methods: Myocardial IPC was elicited by three cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending(LAD) coronary artery. 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. PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 μm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure(MAP), heart rate(HR) and ST-segment of electrocardiogram(ECG) were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin(HE) staining. The activity of plasma lactate dehydrogenase(LDH) was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining. Results: 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(<1 μm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats(P<0.05, respectively). In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR(P<0.01), decreased ST-segment and LDH activity(P<0.05, P<0.01). The damage of myocardium was obviously alleviated and myocardial infarct size was significantly lowered after IPC-MVs treatment(P<0.01). Conclusion: The method of flow cytometry was successfully established to detect the phenotypes and concentration alteration of IPC-MVs, including PMVs, EMVs, LMVs and RMVs. Furthermore, circulating IPC-MVs protected myocardium against I/R injury in rats.
基金supported by the Pathology Laboratory, Immunology Laboratory, General Surgery Laboratory and Animal Laboratory of Tongji Medical College,Huazhong University of Science and Technology
文摘Emerging evidence indicates that ischemic preconditioning （IPC） induces autophagy which attenuates myocardial ischemia/reperfusion （I/R） injury. However, the precise mechanisms remain com- plex and unclear. The present study was to investigate which autophagy pathway was involved in the cardioprotection induced by IPC, so that we can acquire an attractive treatment way for iscbemic heart disease. Adult male Sprague-Dawley （SD） rats were randomly divided into sham group, I/R group and IPC group. IPC was induced with three cycles of 5 min regional ischemia alternating with 5 m^n reper- fusion in a heart I/R model. Samples were taken from the center of the infracted heart and examined by using the electron microscopy, the terminal deoxynucleotidyl transferase-mediated nick end-labeling （TUNEL） method, Western blotting and co-immunoprecipitation （Co-IP）. A large number of autophagic vacuoles were observed in the cardiomyocytes oflPC group as compared with I/R group. LC3-II forma- tion, an autophagy marker, was up-regulated in IPC group as compared with FR group （P〈0.05）. Moreover, the interaction between Beclin 1 and Bcl-2 was significantly increased in IPC group as com- pared with I/R group （P〈0.01）. It was also found that IPC decreased I/R-induced apoptosis （P〈0.01）. These results suggest that IPC inhibits Beclin 1-dependent excessive autophagy in reperfusion phase and cooperates with anti-apoptosis pathway to diminish the cell death induced by the myocardial I/R injury.
文摘Objective: To identify the protective effects of hypovolemic hypotension preconditioning on cardiopulmonary function after myocardial ischemia/reperfusion injury and to explore the possible mechanism.Methods: Twenty-four male white rabbits were randomly assigned to two groups. In the control group, ischemia/reperfusion animals(Group I/R, n=10) were subjected to thirty-minute occlusion of left anterior descending coronary artery and two-hour reperfusion. Animals in hypovolemic hypotension preconditioning group (Group HHP, n=14) experienced brief systemic ischemia preconditioning through blood withdrawl to lower blood pressure to 40%-50% of the baseline before myocardial ischemia/reperfusion. Hemodynamic parameters were recorded. Blood sample was taken to measure superoxide dismutase (SOD), malondialdehyde (MDA) and nitrogen monoxide (NO) changes with blood gas analysis. Myocardium specimens were sampled to examine apoptosis-related gene interleukin-1 beta converting enzyme (ICE) mRNA. Results: Cardiac mechanical function and lung gas exchange remained stable in Group HHP with a significant increase in NO level; while in Group I/R without preconditioning, cardiopulmonary dysfunction was present after 2 h reperfusion associated with a significant reduction in NO formation and an increase in MDA (P<(0.001)). There was negative expression of ICE mRNA in the two groups.Conclusions: Hypovolemic hypotension preconditioning significantly improves cardiopulmonary function and increases NO formation and the protective benefit associated with hypovolemic hypotension preconditioning of the heart may be regulated through NO mediated mechanism.
文摘Background Whether plasma can transfer the protective effect（s） of remote ischemic preconditioning （RIPC） between animals remains unresolved. We therefore investigated the effects of plasma collected 48 hours after transient limb ischemia on blood pressure recovery during myocardial ischemia reperfusion （IR） in homogenic rats. Methods Plasma was collected from Lewis rats, and the donor rats were randomly assigned to 2 groups： transient limb ischemia and control （n = 8 each）. Transient limb ischemia was achieved by four cycles of 5-minute ischemia and 5-minute reperfusion by noninvasive ligation and deligation of the both legs using elastic rubber bands after anesthesia. In the control group, no ligation was performed. Forty-eight hours later, whole blood was collected, and the plasma spun off. Study Lewis rats underwent 30-minute left anterior descending coronary artery occlusion followed by 180-minute reperfusion, and were randomly assigned to 2 groups （group A and group B, n = 24 each）, each further subdivided into 3 subgroups （n = 8 each）. The subgroups of group A received normal saline （group A1）, plasma of control rats （group A2）, plasma of transient limb ischemia rats （group A3） respectively at 1 hour before IR; the subgroups of group B received normal saline （group B1）, plasma of control rats （group B2）, plasma of transient limb ischemia rats （group B3） respectively at 24 hours before IR. BIOPAC systems were used to measure hemodynamics of rats during myocardial ischemia- reperfusion. Results Systolic blood pressure （SBP） after IR in group B3 was different from that in groups B1 and B2 （B3 vs. B1, ,~=-0.007; B3 vs. B2, P =0.039） at the beginning of reperfusion and 30 minutes after reperfusion. SBP was higher in group B3 than in groups B1 and B2 at the beginning of perfusion （B3 vs. B1, P=-0.010; B3 vs. B2, P=-0.002） and 30 minutes after reperfusion （B3 vs. B1, P=-0.001; B3 vs. B2, P=0.001）. SBP did not differ among subgroups A1, A2 and A3. Diastolic blood pressure and heart rate did not change in group A or group B. Conclusions The transfusion of plasma collected 48 hours after transient limb ischemia into homogenic rats 24 hours before IR can improve the SBP recovery during reperfusion. This may suggest that cardioprotective effect of late phase of RIPC is transferable via plasma.
文摘目的:探讨κ-阿片受体是否参与缺血后处理的对抗心肌缺血/复灌(I/R)损伤和心肌细胞缺氧/复氧(H/R)损伤的作用及其机制。方法:采用离体大鼠心脏Langendorff灌流模型,全心停灌30 m in、复灌120 m in复制I/R模型,测定心室力学指标和复灌各时点冠脉流出液中乳酸脱氢酶(LDH)含量。实验结束测定心肌组织form azan含量。酶解分离的心肌细胞采用缺氧60 m in、复氧60m in复制H/R模型,测定心肌细胞存活率。结果:在离体心脏模型上,与I/R组相比,缺血后处理组心肌组织的form azan含量明显增高,复灌期间冠脉流出液中LDH含量明显降低,同时缺血后处理明显改善心室力学指标,缓解冠脉流量的减少;在分离心肌细胞模型上,缺氧后处理明显提高心肌细胞存活率。κ-阿片受体阻断剂nor-b inaltorph im ine(nor-BN I)和线粒体ATP敏感性钾通道(m itoKATP)阻断剂5-hydroxydecanoate(5-HD)在离体大鼠心脏模型和分离心肌细胞模型上均能明显减弱缺血后处理的作用。同时在心肌细胞模型上,与H/R组相比,κ-阿片受体激动剂U 50488H明显提高心肌细胞存活率,其作用可被m itoKATP阻断剂5-HD所阻断。结论:缺血后处理具有抗心肌缺血/复灌损伤的作用,这种保护作用可能与其激活κ-阿片受体和m itoKATP有关。
