Urocortin I后处理抗心肌缺血再灌注损伤的线粒体机制研究
发布时间：2018-07-18 10:49
【摘要】：目的:观察UrocortinⅠ后处理对缺血/缺氧大鼠心脏功能、线粒体结构及功能的影响,探讨UrocortinⅠ后处理产生心肌保护作用的线粒体机制。方法:1.采用Langendorff离体心脏灌注装置建立大鼠心肌缺血再灌注损伤模型,SPF级健康雄性SD大鼠48只随机分为正常组(Nor组)、缺血再灌组(I/R组)、UrocortinⅠ后处理组(UcnⅠ组)、5-羟葵酸拮抗UrocortinⅠ组(5-HD+UcnⅠ组),每组12例。其中Nor组:Kerbs-Henseleit(K-H)液平衡20min后持续灌注135min。I/R组:平衡20min后灌注4℃停跳液,于32℃下全心缺血40min,再复灌95min。UcnⅠ组:缺血后复灌含UcnⅠ的K-H液30min,再续灌K-H液65min。5-HD+UcnⅠ组:UcnⅠ后处理前先给予含5-HD的K-H液5min,其余处理同UcnⅠ组。各组于平衡末及复灌末:(1)Powerlab/8SP数据采集系统记录:心率(HR)、左室发展压(LVDP)、左室舒张末压力(LVDEP)及最大dp/dt(+dp/dtmax)等心功能指标;(2)取左室心肌组织提取线粒体,汉莎氧电极测定线粒体3态呼吸、呼吸控制比(RCR)等呼吸功能及烟酰胺腺嘌呤二核苷酸氧化酶(NADH-OX)、琥珀酸氧化酶(Suc-OX)等呼吸酶活性变化;(3)透射电子显微镜(TEM)观察心肌超微结构改变。2.离体心脏灌注装置(MPA)分离成年大鼠心肌细胞,培养24h后进行细胞计数并随机分为正常组(Nor组)、缺氧/复氧组(I/R组)、UrocortinⅠ后处理组(UcnⅠ组)、5-羟葵酸拮抗UrocortinⅠ组(5-HD+UcnⅠ组)。其中Nor组:37℃培养箱中细胞持续培养135min;I/R组:细胞缺氧40min后复氧95min;UcnⅠ组:缺氧后复氧时先在含UcnⅠ的培养基中处理30min后再复氧60min;5-HD+UcnⅠ组:在给予UcnⅠ处理前先用特异性线粒体ATP敏感性钾通道(mito-KATP)拮抗剂5-HD处理5min,余处理同UcnⅠ组。各组于复氧末对比观察(1)激光共聚焦显微镜(LSCM)下线粒体膜电位(MMP)的变化;(2)CCK-8试剂盒检测细胞活力的改变。结果:1.心功能指标变化:与复灌末相比各组HR、LVDP、LVEDP、+dp/dtmax心功能缺血前均好于复灌末(P0.05);而缺血前各组心脏功能差异无统计学意义(P0.05);在复灌末:Nor组均优于其余各组(P0.05),而UcnⅠ组比I/R组和5-HD+UcnⅠ组心功能好(P0.05);I/R组LVEDP、+dp/dtmax较5-HD+UcnⅠ组差(P0.05),但两组间HR、LVDP比较差异无统计学意义(P0.05)。2.心肌线粒体呼吸功能和酶活性变化:缺血前各组呼吸功能及酶活性改变无统计学意义(P0.05);与复灌末相比,各组呼吸控制比(RCR)、烟酰胺腺嘌呤二核苷酸氧化酶(NADH-OX)、琥珀酸氧化酶等酶活性变化缺血前均好于复灌末(P0.05);在复灌末:Nor组线粒体呼吸功能及酶的活性均优于其余各组(P0.05),而UcnⅠ组呼吸酶活性及3态呼吸、呼吸控制比(RCR)较I/R组和5-HD+UcnⅠ组要好(P0.05),但I/R组较5-HD+UcnⅠ组差(P0.05);关于4态呼吸(State4)缺血前及复灌末组间、组内比较差异均无统计学意义(P0.05)。3.线粒体膜电位的变化:Nor组心肌细胞膜电位比例均高于其余各组(P0.01),而UcnⅠ组膜电位比例高于I/R组和5-HD+UcnⅠ组(P0.05),但5-HD+Ucn I组与I/R组比差异无统计学意义(P0.05)。4.心肌细胞活力测定:Nor组细胞活力高于其余各组(P0.05);UcnⅠ组细胞活力较5-HD+UcnⅠ组和I/R组高(P0.05),而5-HD+UcnⅠ组与I/R组比差异无统计学意义(P0.05)。结论:1.UcnⅠ后处理可改善离体心脏心肌收缩力、降低舒张末压,恢复缺血再灌注后的心脏功能。2.UcnⅠ后处理可通过保护缺血心肌线粒体3态呼吸、RCR呼吸功能及Suc-OX、NADH-OX、Cyt-OX呼吸酶的活性,维持呼吸链电子传递及氧化磷酸化正常进行,并稳定线粒体膜电位,减轻线粒体结构的损害而产生心肌保护效应。且该心肌保护效应与UcnⅠ后处理能开放mito-KATP通道有关。
[Abstract]:Objective: To observe the effects of Urocortin I post treatment on cardiac function, mitochondrial structure and function in ischemic / anoxic rats, and to explore the mitochondrial mechanism of myocardial protection after Urocortin I treatment. Methods: 1. the model of myocardial ischemia reperfusion injury in rats was established by Langendorff isolated cardiac perfusion device, and 48 healthy male SD rats of SPF grade were established. Only randomly divided into normal group (group Nor), ischemia-reperfusion group (group I/R), Urocortin I post treatment group (group Ucn I), 5- hydroxyl acid antagonistic group Urocortin I (group 5-HD+Ucn I), 12 cases in each group. Group 95min.Ucn I: 30min of K-H solution containing Ucn I after ischemia-reperfusion, and then reperfusion K-H liquid 65min.5-HD+Ucn I group: Ucn I was given K-H liquid 5min with 5-HD before reprocessing, and the rest treatment was in the same Ucn group. Each group was at the end of balance and the end of reperfusion: (1) recording of heart rate, left ventricular development pressure and left ventricular end diastolic pressure. And the maximum dp/dt (+dp/dtmax) index of cardiac function; (2) extracting mitochondria from left ventricular myocardium, measuring the respiratory function of mitochondria 3 State respiration, respiratory control ratio (RCR), nicotinamide adenine dinucleotide oxidase (NADH-OX), succinic oxidase (Suc-OX) and other respiratory enzyme activities by the Lufthansa oxygen electrode; (3) observation of transmission electron microscope (TEM) Myocardial ultrastructure changes.2. isolated cardiac perfusion device (MPA) isolated adult rat cardiac myocytes, and then cultured 24h to carry out cell count and randomly divided into normal group (group Nor), hypoxia / reoxygenation group (I/R group), Urocortin I post treatment group (Ucn I group), 5- hydroxyl acid antagonistic Urocortin I group (5-HD+Ucn I group). Nor group: cell holding in 37 C incubator Continuous culture of 135min; group I/R: reoxygenation 95min after anoxic 40min; group Ucn I: reoxygenation 60min in the medium containing Ucn I first in the culture medium containing Ucn I after hypoxia; group 5-HD+Ucn I was treated with specific mitochondrial ATP sensitive potassium channel (mito-KATP) antagonist before the treatment of Ucn I. The final contrast observation (1) the change of the grain bulk membrane potential (MMP) under laser confocal microscopy (LSCM); (2) the change of cell viability by CCK-8 kit. Results: 1. changes of cardiac function index: HR, LVDP, LVEDP, +dp/dtmax in each group were better than that of the end of reperfusion (P0.05) before the end of reperfusion, but there was no difference in cardiac function before ischemia. Meaning (P0.05); at the end of reirrigation, group Nor was better than the other groups (P0.05), while group Ucn I was better than group I/R and 5-HD+Ucn I (P0.05); LVEDP in group I/R and 5-HD+Ucn (P0.05) in group I/R, but there was no significant difference in myocardial mitochondrial respiratory function and enzyme activity in the two groups: respiratory work before ischemia The changes of energy and enzyme activity were not statistically significant (P0.05). Compared with the end of reperfusion, the changes of respiratory control ratio (RCR), nicotinamide adenine dinucleotide oxidase (NADH-OX), succinic acid oxidase and other enzymes were better than those of the end of reperfusion (P0.05). At the end of reperfusion, the mitochondrial respiratory function and enzyme activity in the Nor group were better than those in the other groups (P0.05). In Ucn I group, respiratory enzyme activity and 3 State respiration, respiratory control ratio (RCR) were better than group I/R and 5-HD+Ucn I group (P0.05), but I/R group was less than 5-HD+Ucn I group (P0.05). There was no significant difference between group I/R and 5-HD+Ucn I group (State4) before and after reperfusion (P0.05).3. mitochondrial membrane potential change: the ratio of membrane potential ratio in the myocardial cell of Nor group Compared with the other groups (P0.01), the membrane potential ratio in group Ucn I was higher than that in group I/R and group 5-HD+Ucn I (P0.05), but there was no significant difference between 5-HD+Ucn I group and I/R group (P0.05).4. cardiomyocyte viability measurement: Nor group cell viability was higher than that of other groups (P0.05). There was no significant difference in the comparison between the group and the I/R group (P0.05). Conclusion: the post-treatment of 1.Ucn I can improve the cardiac contractility of the isolated heart, reduce the end diastolic pressure, and restore the cardiac function after the ischemia-reperfusion after the.2.Ucn I treatment can protect the 3 State respiration of the mitochondria of ischemic myocardium, the activity of RCR breathing and the activity of Suc-OX, NADH-OX, Cyt-OX respiratory enzyme, and maintain the reactivation of the respiratory tract. The absorption of chain electron transfer and oxidative phosphorylation is normal, and the mitochondrial membrane potential is stabilized, and the damage of mitochondria structure can be reduced to produce myocardial protection effect, and the protective effect of the myocardium is related to the opening of the mito-KATP channel after Ucn I treatment.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R654.2
本文编号：2131670
[Abstract]:Objective: To observe the effects of Urocortin I post treatment on cardiac function, mitochondrial structure and function in ischemic / anoxic rats, and to explore the mitochondrial mechanism of myocardial protection after Urocortin I treatment. Methods: 1. the model of myocardial ischemia reperfusion injury in rats was established by Langendorff isolated cardiac perfusion device, and 48 healthy male SD rats of SPF grade were established. Only randomly divided into normal group (group Nor), ischemia-reperfusion group (group I/R), Urocortin I post treatment group (group Ucn I), 5- hydroxyl acid antagonistic group Urocortin I (group 5-HD+Ucn I), 12 cases in each group. Group 95min.Ucn I: 30min of K-H solution containing Ucn I after ischemia-reperfusion, and then reperfusion K-H liquid 65min.5-HD+Ucn I group: Ucn I was given K-H liquid 5min with 5-HD before reprocessing, and the rest treatment was in the same Ucn group. Each group was at the end of balance and the end of reperfusion: (1) recording of heart rate, left ventricular development pressure and left ventricular end diastolic pressure. And the maximum dp/dt (+dp/dtmax) index of cardiac function; (2) extracting mitochondria from left ventricular myocardium, measuring the respiratory function of mitochondria 3 State respiration, respiratory control ratio (RCR), nicotinamide adenine dinucleotide oxidase (NADH-OX), succinic oxidase (Suc-OX) and other respiratory enzyme activities by the Lufthansa oxygen electrode; (3) observation of transmission electron microscope (TEM) Myocardial ultrastructure changes.2. isolated cardiac perfusion device (MPA) isolated adult rat cardiac myocytes, and then cultured 24h to carry out cell count and randomly divided into normal group (group Nor), hypoxia / reoxygenation group (I/R group), Urocortin I post treatment group (Ucn I group), 5- hydroxyl acid antagonistic Urocortin I group (5-HD+Ucn I group). Nor group: cell holding in 37 C incubator Continuous culture of 135min; group I/R: reoxygenation 95min after anoxic 40min; group Ucn I: reoxygenation 60min in the medium containing Ucn I first in the culture medium containing Ucn I after hypoxia; group 5-HD+Ucn I was treated with specific mitochondrial ATP sensitive potassium channel (mito-KATP) antagonist before the treatment of Ucn I. The final contrast observation (1) the change of the grain bulk membrane potential (MMP) under laser confocal microscopy (LSCM); (2) the change of cell viability by CCK-8 kit. Results: 1. changes of cardiac function index: HR, LVDP, LVEDP, +dp/dtmax in each group were better than that of the end of reperfusion (P0.05) before the end of reperfusion, but there was no difference in cardiac function before ischemia. Meaning (P0.05); at the end of reirrigation, group Nor was better than the other groups (P0.05), while group Ucn I was better than group I/R and 5-HD+Ucn I (P0.05); LVEDP in group I/R and 5-HD+Ucn (P0.05) in group I/R, but there was no significant difference in myocardial mitochondrial respiratory function and enzyme activity in the two groups: respiratory work before ischemia The changes of energy and enzyme activity were not statistically significant (P0.05). Compared with the end of reperfusion, the changes of respiratory control ratio (RCR), nicotinamide adenine dinucleotide oxidase (NADH-OX), succinic acid oxidase and other enzymes were better than those of the end of reperfusion (P0.05). At the end of reperfusion, the mitochondrial respiratory function and enzyme activity in the Nor group were better than those in the other groups (P0.05). In Ucn I group, respiratory enzyme activity and 3 State respiration, respiratory control ratio (RCR) were better than group I/R and 5-HD+Ucn I group (P0.05), but I/R group was less than 5-HD+Ucn I group (P0.05). There was no significant difference between group I/R and 5-HD+Ucn I group (State4) before and after reperfusion (P0.05).3. mitochondrial membrane potential change: the ratio of membrane potential ratio in the myocardial cell of Nor group Compared with the other groups (P0.01), the membrane potential ratio in group Ucn I was higher than that in group I/R and group 5-HD+Ucn I (P0.05), but there was no significant difference between 5-HD+Ucn I group and I/R group (P0.05).4. cardiomyocyte viability measurement: Nor group cell viability was higher than that of other groups (P0.05). There was no significant difference in the comparison between the group and the I/R group (P0.05). Conclusion: the post-treatment of 1.Ucn I can improve the cardiac contractility of the isolated heart, reduce the end diastolic pressure, and restore the cardiac function after the ischemia-reperfusion after the.2.Ucn I treatment can protect the 3 State respiration of the mitochondria of ischemic myocardium, the activity of RCR breathing and the activity of Suc-OX, NADH-OX, Cyt-OX respiratory enzyme, and maintain the reactivation of the respiratory tract. The absorption of chain electron transfer and oxidative phosphorylation is normal, and the mitochondrial membrane potential is stabilized, and the damage of mitochondria structure can be reduced to produce myocardial protection effect, and the protective effect of the myocardium is related to the opening of the mito-KATP channel after Ucn I treatment.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R654.2
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