注射用心肌肽对幼鼠未成熟心肌的保护作用及其机制的研究
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摘要
本次研究主要通过动物实验,探讨注射用心肌肽对幼大鼠未成熟心肌缺血再灌注损伤的保护作用及其机制。
第一部分幼鼠离体心脏Langendorff灌流模型的建立及注射用心肌肽对幼鼠离体心脏的心功能影响
目的:1建立稳定的幼大鼠离体心脏Langendorff灌流模型,2.观察注射用心肌肽对幼大鼠不同状态下的离体心脏心功能的影响。
方法: 50只SD幼鼠,随机分为5组,每组10只,离体心脏用Langendorff灌流、灌流用K-H缓冲液,正常对照组:心脏稳定20min后,再持续灌注150min;正常+注射用心肌肽组:同正常对照组、但在灌流液中加入注射用心肌肽50 mg/L;缺血再灌注损伤模型组分3组:心脏稳定20min后,停跳液灌三次使心脏停搏(缺血)90min,再恢复正常灌流(再灌注)60min,建立心肌缺血再灌注损伤模型,对照组(Control):灌流用K-H缓冲液、停搏用ST.Thomas'II停搏液,注射用心肌肽用药两组(CMP1组:停搏液加入注射用心肌肽100 mg/L;CMP2组:灌流液加入注射用心肌肽50 mg/L及停搏液加入注射用心肌肽100 mg/L)。监测不同组离体灌流心脏心率(HR,min-1)、收缩力(△T,g)及最大收缩速度(+dT/dtmax,g.s-1)和最大舒张速度(-dT/dtmax,g.s-1)、冠脉流量(CF,ml.min-1)、复跳时间(s)反映心功能,
结果:正常对照组离体心脏长时间灌流后心功能无明显变化,正常+注射用心肌肽组的心功能也无明显变化。停搏再灌流后,对照组心搏功能下降,冠脉流量减少,而两个用药组心搏功能仅轻微下降,冠脉流量减少较轻,且停跳液和灌流液中均加入注射用心肌肽的离体心脏的心功能更佳。
结论:幼鼠离体心脏Langendorff灌流模型的性能稳定,可用于下一步的实验研究。注射用心肌肽对正常心脏心功能无明显影响。注射用心肌肽可改善缺血心脏再灌注后的冠脉流量,提高心功能,对幼鼠未成熟心肌缺血再灌注损伤可能具有保护作用。
第二部分注射用心肌肽对幼鼠离体未成熟心肌缺血再灌注损伤保护作用及机制
目的:探讨注射用心肌肽对幼鼠离体灌流的心脏缺血再灌注损伤有无保护作用及机制。
方法: 30只SD幼鼠,随机分为3组,每组10只,离体心脏用Langendorff灌流、灌流用K-H缓冲液,停搏用ST.Thomas'II停跳液;心脏稳定20min后,停搏液停搏(缺血)90min,再恢复正常灌流(再灌注)60min,建立心肌缺血再灌注损伤模型,对照组(Control):灌流用K-H液、停搏用ST.Thomas'II停跳液,注射用心肌肽用药两组(CMP1组:停跳液加入注射用心肌肽100 mg/L;CMP2组:灌流液加入注射用心肌肽50 mg/L及停跳液加入注射用心肌肽100 mg/L)。监测离体灌流心脏的心功能、包括心率(HR,min-1)、收缩力(△T,g)及最大收缩速度(+dT/dtmax,g.s-1)和最大舒张速度(-dT/dtmax,g.s-1)、冠脉流量(CF,ml.min-1)、复搏时间(s),检测冠脉流出液中肌酸激酶同工酶(CK-MB)含量的变化,实验结束、取心尖部的心肌组织、光镜及透射电镜观察缺血再灌注损伤模型组中幼鼠心肌组织超微结构改变,生化检测心肌组织能量指标的变化,心肌组织超氧化物歧化酶(SOD)、丙二醛、NO的含量,检测心肌组织NOS、醛糖还原酶的活性,实时荧光定量PCR相对定量检测心肌组织iNOSmRNA、eNOSmRNA和Akr1b4 mRNA表达。
结果:。停搏再灌流后,对照组心搏功能下降,冠脉流量减少;而两个用药组心搏功能仅轻微下降,冠脉流量减少较轻。电镜观察可见对照组心肌肌原纤维断裂,线粒体肿胀,而用药组心肌组织结构较完整,线粒体未见明显肿胀变性。再灌注后,对照组CK-MB增加。与对照组比较,两个用药组的CK-MB含量低,Na+-K+ ATP酶、Ca2+-Mg2+ ATP酶、心肌总ATP酶活性相对较高,SOD活性也增高,丙二醛、NO含量低,心肌组织NOS、醛糖还原酶的活性下降,iNOS、Akr1b4 mRNA表达下调,其中CMP2组改变更为明显(P<0.01或P<0.05)。此外eNOS mRNA表达在CMP2组有升高(P<0.05),而在CMP1组中变化无统计学意义。
结论:注射用心肌肽可改善缺血心脏再灌注后的能量代谢,增加冠脉流量,提高心功能,对幼鼠未成熟心肌缺血再灌注损伤具有保护作用,其作用机制可能与抑制心肌细胞iNOS, Akr1b4 mRNA表达,减少NO生成,抑制醛糖还原酶的活性有关。停跳液和灌流液中均加入注射用心肌肽对未成熟心肌缺血再灌注损伤的保护作用效果更佳。
第三部分注射用心肌肽预处理减轻大鼠未成熟心肌缺血-再灌注损伤
目的:探讨注射用心肌肽预处理对幼鼠未成熟心肌缺血再灌注损伤的保护作用及其相应的作用机制。
方法:30只SD幼鼠,随机分为3组,每组10只,通过结扎幼鼠冠状动脉左前降支建立缺血再灌注动物模型,分为假手术组、缺血再灌注组和注射用心肌肽组,全自动生化仪检测血清酶学LDH、CK-MB、Elisa法检测血清肌钙蛋白Tn-T,生化检测心肌组织匀浆MDA、SOD、TNOS、iNOS、eNOS含量变化,免疫组化法检测NOS2(iNOS)、NOS3(eNOS)含量变化,光学显微镜观察心肌梗死范围和透射电镜检测心肌组织结构改变,TUNEL法观察心肌凋亡细胞,免疫组化法检测活性Caspase-3蛋白表达水平以及应用实时荧光定量PCR分析心肌组织iNOS、eNOS、Caspas-3 mRNA表达变化。
结果:注射用心肌肽组LDH、CK-MB、Tn-T的浓度低于缺血再灌注组,MDA、TNOS、iNOS含量以及Caspase-3、iNOSmRNA表达水平较假手术组增高,但低于缺血再灌注组,SOD的活性高于缺血再灌注组(P<0.01或P<0.05),差异有统计学意义。缺血再灌注组心肌片状出血、炎性细胞浸润,心肌细胞严变性坏死,心肌细胞凋亡显著;而注射用心肌肽组心肌梗死范围小于缺血再灌注组,心肌细胞变性坏死程度较轻,血管结构正常,心肌凋亡细胞水平介于假手术组和缺血再灌注组之间。
结论:注射用心肌肽预处理可以减轻幼鼠未成熟心肌缺血再灌注损伤、对未成熟心肌具有保护作用,其作用机制可能与减少氧自由基损伤及减少NO生成,抑制心肌细胞Caspase-3 mRNA和蛋白表达,从而减少细胞凋亡有关。
Part I: The establishment of Langendorff isolated heart perfusion model and the effect of Cardiomyopeptidin for injection on the cardiac function of the isolated hearts of young rats
Objective: 1. This experiment was designed to establish a stable Langendorff isolated heart perfusion model in the hearts of young rats; 2. to investigate the effect of cardiomyopeptidin for injection on the cardiac function of the different states isolated hearts of young rats.
Methods: 50 young healthy SD rats(aged 20±3 day and weighing 50-70g)were randomly divided into five groups, Normal control group (NC group ): the isolated heart were stable 20min, and then 150 min continuous perfusion ;Normal + cardiomyopeptidin group (NCMP group ): the same as normal control group, but K-H buffer solution were added with 50 mg/L cardiomyopeptidin for injection;Control group: the isolated rat hearts were perfused with K-H buffer and then arrested with cardioplegic solution; CMP1 group: the above ST.Thomas'II cardioplegic solution was added with 100 mg/L cardiomyopeptidin for injection; CMP2 group: the above K-H buffer and ST.Thomas'II cardioplegic solution were added with 50 mg/L and 100 mg/L cardiomyopeptidin for injection respectively. The isolated rat hearts were initially perfused with K-H buffer in 37℃Langendorff system for 20 min, following a procedure of perfusion with ST.Thomas'II cardioplegic solution at 4℃for 3min, and then arrest for 27min, with the Langendorff system kept in 32℃. This procedure was repeated three times. After arrested for 90min (undergone ischemia), the rat hearts were reperfused for 60min in normal temperature. The cardiac functional indexes were monitored, including heart rate, myocardial contractility and diastolic function, peak systolic and diastole myocardial velocities and coronary flow.
Results: In NC group, after prolonged perfusion, the cardiac function of isolated hearts had been no significant change; cardiomyopeptidin for injection had been no significant effect on normal isolated hearts; Compared with the control group, the decreased degree of the cardiac function indexes and coronary flow in the groups treated with cardiomyopeptidin for injection were much less, Administration of cardiomyopeptidin in both K-H buffer and ST.Thomas'II cardioplegic solution is better than adding cardiomyopeptidin for injection in cardioplegic solution only.
Conclusion: The Langendorff perfusion model of the rat’s isolated hearts was stable, can be used for next experiments. Cardiomyopeptidin for injection had no significant effect on the cardiac function of normal isolated hearts. Cardiomyopeptidin for injection may improve coronary flow and cardiac function after reperfusion, thus protected immature myocardial against ischemia-reperfusion injury in young rats.
Part II: Protective effects and its mechanisms of Cardiomypeptidin for injection on the isolated hearts of young rats from ischemia-reperfusion injury
Objective: This experiment was designed to investigate the effect of cardiomyopeptidin for injection on immature myocardium ischemia-reperfusion injury in the isolated hearts of young rats and its mechanism.
Methods: 30 young healthy SD rats(aged 20±3 day and weighing 50-70g)were randomly divided into three groups, with 10 rats in each group. Control group: the isolated rat hearts were perfused with K-H buffer and then arrested with ST.Thomas'II cardioplegic solution; CMP1 group: the above ST.Thomas'II cardioplegic solution was added with 100 mg/L cardiomyopeptidin for injection; CMP2 group: the above K-H buffer and ST.Thomas'II cardioplegic solution were added with 50 mg/L and 100 mg/L cardiomyopeptidin for injection respectively. The isolated rat hearts were initially perfused with K-H buffer in 37℃Langendorff system for 20 min, following a procedure of perfusion with ST.Thomas'II cardioplegic solution at 4℃for 3min, and then arrest for 27min, with the Langendorff system kept in 32℃. This procedure was repeated three times. After arrested for 90min (undergone ischemia), the rat hearts were reperfused for 60min in normal temperature. The cardiac functional indexes was monitored, including heart rate, myocardial contractility and diastolic function, peak systolic and diastole myocardial velocities and coronary flow; myocardial structure was observed through optical microscopy and Transmission Electron Microscopy; the content of Na+-K+ ATPase, Ca2+-Mg2+ ATPase, total ATPase, superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide(NO), total nitric oxide synthase (TNOS), inducible nitric oxide synthase (iNOS) and aldosereductase were measured in myocardium tissue; the relative expression levels of iNOS mRNA, eNOS mRNA and Akr1b4 mRNA in myocardial tissue were detected by relative real-time fluorescence quantitative PCR.
Results: Compared with the control group, the decreased degree of the cardiac function indexes and coronary flow in the groups treated with cardiomyopeptidin for injection were much less. Cardiac myofibrillar fragmentation and mitochondrial swelling were observed in the control group, while in the CMP groups, the myocardial structure was near-complete, and only mild mitochondria swelling and degeneration could be seen. There was a less decline in the contents of Na+-K+ ATPase, Ca2+-Mg2+ ATPase and Total ATPase in the treatment groups, and a increase in the activity of SOD (P<0.01 or P<0.05). The concentration of NO and MDA produced after ischemia-reperfusion injury were much less in two CMP groups; moreover, the activity of iNOS and aldosereductase were inhibited, the expression levels of iNOSmRNA and Akr1b4mRNA were significantly down-regulated in two CMP groups. These changes were more prominent in the CMP 2 group (P<0.01 or P<0.05). Besides, the eNOS mRNA levels in CMP2 group was up-regulated (P<0.05).
Conclusion: Cardiomyopeptidin for injection may improve coronary blood flow and cardiac function, thus protect immature myocardial against ischemia-reperfusion injury in young rats. The mechanism may be associated with inhibited the expression of iNOS mRNA and Akr1b4 mRNA in cardiomyocytes, the inhibited activity of iNOS and aldosereductase, and the reduced NO production. Administration of cardiomyopeptidin for injection in both ST.Thomas'II cardioplegic solution and K-H buffer is better than adding cardiomyopeptidin for injection in cardioplegic solution only.
PartⅢCardiomypeptidin for injectionon preconditioning attenuates immature myocardial ischemia - reperfusion injury in young rats
Objective: This experiment was designed to investigate the Cardioprotective of cardiomyopeptidin for injectionon the immature hearts of young rats from ischemia-reperfusion injury and its mechanism.
Methods 30 young healthy SD rats(aged 20±3 day and weighing 50-70g)were randomly divided into three groups, with 10 rats in each group. Control group, sham-operated group and cardiomyopeptidin for injection group (CMP group). The serum Concentration of LDH, CK-MB were detected by CX7-type automatic biochemical analyzer, the serum Concentration of troponin Tn-T were detected by Elisa; the content of superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide(NO), total nitric oxide synthase (TNOS), inducible induse nitric oxide synthase (iNOS)and eNOS were measured in myocardium tissue; the expression level of NOS2 and NOS3 were detected by Immunohistochemical; the relative expression levels of iNOS, eNOS and Caspase-3mRNA in myocardial tissue were also detected by real-time fluorescence quantitative PCR; myocardial structure were observed through optical microscopy and Transmission Electron Microscopy; the expression level of active Caspase-3 protein was detected by Immunohistochemical; each groups Myocardial apoptosis was observed by TUNEL.
Results: Compared with the control group, the concentration of LDH, CK-MB, Tn-T was much lesser in CMP group; Compared with the sham-operated group, the content of MDA, SOD, TNOS, and iNOS were increased and the expression levels of iNOS mRNA and Caspase-3 mRNA were significantly down-regulated in CMP groups, but less than the control group,(P<0.01 or P<0.05. Several pieces of myocardial hemorrhage and inflammatory cell infiltration were observed in the control group, severe degeneration and necrosis of myocardial cells could be seen, apoptotic myocardial cells were significant increased; while in the CMP group, myocardial infarct size was reduced, myocardial pathological injuries were mitigated, only mild degeneration and necrosis of myocardial cells could be seen, the vascular structure was near-complete, the number of apoptotic myocardial cells was less than the control group; In the sham operation group, the structure of myocardial tissue were basically normal, there was a small amount of myocardial cell apoptosis.
Conclusion: Cardiomyopeptidin for injection preconditioning protected immature myocardium against ischemia-reperfusion injury in young rats. The mechanism may be associated with reduced Oxygen free radical (OFR) and NO production, and the inhibited expression of iNOS mRNA, Caspase-3mRNA and Caspase-3 protein in cardiomyocytes, thereby reduced apoptosis.
第一部分幼鼠离体心脏Langendorff灌流模型的建立及注射用心肌肽对幼鼠离体心脏的心功能影响
目的:1建立稳定的幼大鼠离体心脏Langendorff灌流模型,2.观察注射用心肌肽对幼大鼠不同状态下的离体心脏心功能的影响。
方法: 50只SD幼鼠,随机分为5组,每组10只,离体心脏用Langendorff灌流、灌流用K-H缓冲液,正常对照组:心脏稳定20min后,再持续灌注150min;正常+注射用心肌肽组:同正常对照组、但在灌流液中加入注射用心肌肽50 mg/L;缺血再灌注损伤模型组分3组:心脏稳定20min后,停跳液灌三次使心脏停搏(缺血)90min,再恢复正常灌流(再灌注)60min,建立心肌缺血再灌注损伤模型,对照组(Control):灌流用K-H缓冲液、停搏用ST.Thomas'II停搏液,注射用心肌肽用药两组(CMP1组:停搏液加入注射用心肌肽100 mg/L;CMP2组:灌流液加入注射用心肌肽50 mg/L及停搏液加入注射用心肌肽100 mg/L)。监测不同组离体灌流心脏心率(HR,min-1)、收缩力(△T,g)及最大收缩速度(+dT/dtmax,g.s-1)和最大舒张速度(-dT/dtmax,g.s-1)、冠脉流量(CF,ml.min-1)、复跳时间(s)反映心功能,
结果:正常对照组离体心脏长时间灌流后心功能无明显变化,正常+注射用心肌肽组的心功能也无明显变化。停搏再灌流后,对照组心搏功能下降,冠脉流量减少,而两个用药组心搏功能仅轻微下降,冠脉流量减少较轻,且停跳液和灌流液中均加入注射用心肌肽的离体心脏的心功能更佳。
结论:幼鼠离体心脏Langendorff灌流模型的性能稳定,可用于下一步的实验研究。注射用心肌肽对正常心脏心功能无明显影响。注射用心肌肽可改善缺血心脏再灌注后的冠脉流量,提高心功能,对幼鼠未成熟心肌缺血再灌注损伤可能具有保护作用。
第二部分注射用心肌肽对幼鼠离体未成熟心肌缺血再灌注损伤保护作用及机制
目的:探讨注射用心肌肽对幼鼠离体灌流的心脏缺血再灌注损伤有无保护作用及机制。
方法: 30只SD幼鼠,随机分为3组,每组10只,离体心脏用Langendorff灌流、灌流用K-H缓冲液,停搏用ST.Thomas'II停跳液;心脏稳定20min后,停搏液停搏(缺血)90min,再恢复正常灌流(再灌注)60min,建立心肌缺血再灌注损伤模型,对照组(Control):灌流用K-H液、停搏用ST.Thomas'II停跳液,注射用心肌肽用药两组(CMP1组:停跳液加入注射用心肌肽100 mg/L;CMP2组:灌流液加入注射用心肌肽50 mg/L及停跳液加入注射用心肌肽100 mg/L)。监测离体灌流心脏的心功能、包括心率(HR,min-1)、收缩力(△T,g)及最大收缩速度(+dT/dtmax,g.s-1)和最大舒张速度(-dT/dtmax,g.s-1)、冠脉流量(CF,ml.min-1)、复搏时间(s),检测冠脉流出液中肌酸激酶同工酶(CK-MB)含量的变化,实验结束、取心尖部的心肌组织、光镜及透射电镜观察缺血再灌注损伤模型组中幼鼠心肌组织超微结构改变,生化检测心肌组织能量指标的变化,心肌组织超氧化物歧化酶(SOD)、丙二醛、NO的含量,检测心肌组织NOS、醛糖还原酶的活性,实时荧光定量PCR相对定量检测心肌组织iNOSmRNA、eNOSmRNA和Akr1b4 mRNA表达。
结果:。停搏再灌流后,对照组心搏功能下降,冠脉流量减少;而两个用药组心搏功能仅轻微下降,冠脉流量减少较轻。电镜观察可见对照组心肌肌原纤维断裂,线粒体肿胀,而用药组心肌组织结构较完整,线粒体未见明显肿胀变性。再灌注后,对照组CK-MB增加。与对照组比较,两个用药组的CK-MB含量低,Na+-K+ ATP酶、Ca2+-Mg2+ ATP酶、心肌总ATP酶活性相对较高,SOD活性也增高,丙二醛、NO含量低,心肌组织NOS、醛糖还原酶的活性下降,iNOS、Akr1b4 mRNA表达下调,其中CMP2组改变更为明显(P<0.01或P<0.05)。此外eNOS mRNA表达在CMP2组有升高(P<0.05),而在CMP1组中变化无统计学意义。
结论:注射用心肌肽可改善缺血心脏再灌注后的能量代谢,增加冠脉流量,提高心功能,对幼鼠未成熟心肌缺血再灌注损伤具有保护作用,其作用机制可能与抑制心肌细胞iNOS, Akr1b4 mRNA表达,减少NO生成,抑制醛糖还原酶的活性有关。停跳液和灌流液中均加入注射用心肌肽对未成熟心肌缺血再灌注损伤的保护作用效果更佳。
第三部分注射用心肌肽预处理减轻大鼠未成熟心肌缺血-再灌注损伤
目的:探讨注射用心肌肽预处理对幼鼠未成熟心肌缺血再灌注损伤的保护作用及其相应的作用机制。
方法:30只SD幼鼠,随机分为3组,每组10只,通过结扎幼鼠冠状动脉左前降支建立缺血再灌注动物模型,分为假手术组、缺血再灌注组和注射用心肌肽组,全自动生化仪检测血清酶学LDH、CK-MB、Elisa法检测血清肌钙蛋白Tn-T,生化检测心肌组织匀浆MDA、SOD、TNOS、iNOS、eNOS含量变化,免疫组化法检测NOS2(iNOS)、NOS3(eNOS)含量变化,光学显微镜观察心肌梗死范围和透射电镜检测心肌组织结构改变,TUNEL法观察心肌凋亡细胞,免疫组化法检测活性Caspase-3蛋白表达水平以及应用实时荧光定量PCR分析心肌组织iNOS、eNOS、Caspas-3 mRNA表达变化。
结果:注射用心肌肽组LDH、CK-MB、Tn-T的浓度低于缺血再灌注组,MDA、TNOS、iNOS含量以及Caspase-3、iNOSmRNA表达水平较假手术组增高,但低于缺血再灌注组,SOD的活性高于缺血再灌注组(P<0.01或P<0.05),差异有统计学意义。缺血再灌注组心肌片状出血、炎性细胞浸润,心肌细胞严变性坏死,心肌细胞凋亡显著;而注射用心肌肽组心肌梗死范围小于缺血再灌注组,心肌细胞变性坏死程度较轻,血管结构正常,心肌凋亡细胞水平介于假手术组和缺血再灌注组之间。
结论:注射用心肌肽预处理可以减轻幼鼠未成熟心肌缺血再灌注损伤、对未成熟心肌具有保护作用,其作用机制可能与减少氧自由基损伤及减少NO生成,抑制心肌细胞Caspase-3 mRNA和蛋白表达,从而减少细胞凋亡有关。
Part I: The establishment of Langendorff isolated heart perfusion model and the effect of Cardiomyopeptidin for injection on the cardiac function of the isolated hearts of young rats
Objective: 1. This experiment was designed to establish a stable Langendorff isolated heart perfusion model in the hearts of young rats; 2. to investigate the effect of cardiomyopeptidin for injection on the cardiac function of the different states isolated hearts of young rats.
Methods: 50 young healthy SD rats(aged 20±3 day and weighing 50-70g)were randomly divided into five groups, Normal control group (NC group ): the isolated heart were stable 20min, and then 150 min continuous perfusion ;Normal + cardiomyopeptidin group (NCMP group ): the same as normal control group, but K-H buffer solution were added with 50 mg/L cardiomyopeptidin for injection;Control group: the isolated rat hearts were perfused with K-H buffer and then arrested with cardioplegic solution; CMP1 group: the above ST.Thomas'II cardioplegic solution was added with 100 mg/L cardiomyopeptidin for injection; CMP2 group: the above K-H buffer and ST.Thomas'II cardioplegic solution were added with 50 mg/L and 100 mg/L cardiomyopeptidin for injection respectively. The isolated rat hearts were initially perfused with K-H buffer in 37℃Langendorff system for 20 min, following a procedure of perfusion with ST.Thomas'II cardioplegic solution at 4℃for 3min, and then arrest for 27min, with the Langendorff system kept in 32℃. This procedure was repeated three times. After arrested for 90min (undergone ischemia), the rat hearts were reperfused for 60min in normal temperature. The cardiac functional indexes were monitored, including heart rate, myocardial contractility and diastolic function, peak systolic and diastole myocardial velocities and coronary flow.
Results: In NC group, after prolonged perfusion, the cardiac function of isolated hearts had been no significant change; cardiomyopeptidin for injection had been no significant effect on normal isolated hearts; Compared with the control group, the decreased degree of the cardiac function indexes and coronary flow in the groups treated with cardiomyopeptidin for injection were much less, Administration of cardiomyopeptidin in both K-H buffer and ST.Thomas'II cardioplegic solution is better than adding cardiomyopeptidin for injection in cardioplegic solution only.
Conclusion: The Langendorff perfusion model of the rat’s isolated hearts was stable, can be used for next experiments. Cardiomyopeptidin for injection had no significant effect on the cardiac function of normal isolated hearts. Cardiomyopeptidin for injection may improve coronary flow and cardiac function after reperfusion, thus protected immature myocardial against ischemia-reperfusion injury in young rats.
Part II: Protective effects and its mechanisms of Cardiomypeptidin for injection on the isolated hearts of young rats from ischemia-reperfusion injury
Objective: This experiment was designed to investigate the effect of cardiomyopeptidin for injection on immature myocardium ischemia-reperfusion injury in the isolated hearts of young rats and its mechanism.
Methods: 30 young healthy SD rats(aged 20±3 day and weighing 50-70g)were randomly divided into three groups, with 10 rats in each group. Control group: the isolated rat hearts were perfused with K-H buffer and then arrested with ST.Thomas'II cardioplegic solution; CMP1 group: the above ST.Thomas'II cardioplegic solution was added with 100 mg/L cardiomyopeptidin for injection; CMP2 group: the above K-H buffer and ST.Thomas'II cardioplegic solution were added with 50 mg/L and 100 mg/L cardiomyopeptidin for injection respectively. The isolated rat hearts were initially perfused with K-H buffer in 37℃Langendorff system for 20 min, following a procedure of perfusion with ST.Thomas'II cardioplegic solution at 4℃for 3min, and then arrest for 27min, with the Langendorff system kept in 32℃. This procedure was repeated three times. After arrested for 90min (undergone ischemia), the rat hearts were reperfused for 60min in normal temperature. The cardiac functional indexes was monitored, including heart rate, myocardial contractility and diastolic function, peak systolic and diastole myocardial velocities and coronary flow; myocardial structure was observed through optical microscopy and Transmission Electron Microscopy; the content of Na+-K+ ATPase, Ca2+-Mg2+ ATPase, total ATPase, superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide(NO), total nitric oxide synthase (TNOS), inducible nitric oxide synthase (iNOS) and aldosereductase were measured in myocardium tissue; the relative expression levels of iNOS mRNA, eNOS mRNA and Akr1b4 mRNA in myocardial tissue were detected by relative real-time fluorescence quantitative PCR.
Results: Compared with the control group, the decreased degree of the cardiac function indexes and coronary flow in the groups treated with cardiomyopeptidin for injection were much less. Cardiac myofibrillar fragmentation and mitochondrial swelling were observed in the control group, while in the CMP groups, the myocardial structure was near-complete, and only mild mitochondria swelling and degeneration could be seen. There was a less decline in the contents of Na+-K+ ATPase, Ca2+-Mg2+ ATPase and Total ATPase in the treatment groups, and a increase in the activity of SOD (P<0.01 or P<0.05). The concentration of NO and MDA produced after ischemia-reperfusion injury were much less in two CMP groups; moreover, the activity of iNOS and aldosereductase were inhibited, the expression levels of iNOSmRNA and Akr1b4mRNA were significantly down-regulated in two CMP groups. These changes were more prominent in the CMP 2 group (P<0.01 or P<0.05). Besides, the eNOS mRNA levels in CMP2 group was up-regulated (P<0.05).
Conclusion: Cardiomyopeptidin for injection may improve coronary blood flow and cardiac function, thus protect immature myocardial against ischemia-reperfusion injury in young rats. The mechanism may be associated with inhibited the expression of iNOS mRNA and Akr1b4 mRNA in cardiomyocytes, the inhibited activity of iNOS and aldosereductase, and the reduced NO production. Administration of cardiomyopeptidin for injection in both ST.Thomas'II cardioplegic solution and K-H buffer is better than adding cardiomyopeptidin for injection in cardioplegic solution only.
PartⅢCardiomypeptidin for injectionon preconditioning attenuates immature myocardial ischemia - reperfusion injury in young rats
Objective: This experiment was designed to investigate the Cardioprotective of cardiomyopeptidin for injectionon the immature hearts of young rats from ischemia-reperfusion injury and its mechanism.
Methods 30 young healthy SD rats(aged 20±3 day and weighing 50-70g)were randomly divided into three groups, with 10 rats in each group. Control group, sham-operated group and cardiomyopeptidin for injection group (CMP group). The serum Concentration of LDH, CK-MB were detected by CX7-type automatic biochemical analyzer, the serum Concentration of troponin Tn-T were detected by Elisa; the content of superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide(NO), total nitric oxide synthase (TNOS), inducible induse nitric oxide synthase (iNOS)and eNOS were measured in myocardium tissue; the expression level of NOS2 and NOS3 were detected by Immunohistochemical; the relative expression levels of iNOS, eNOS and Caspase-3mRNA in myocardial tissue were also detected by real-time fluorescence quantitative PCR; myocardial structure were observed through optical microscopy and Transmission Electron Microscopy; the expression level of active Caspase-3 protein was detected by Immunohistochemical; each groups Myocardial apoptosis was observed by TUNEL.
Results: Compared with the control group, the concentration of LDH, CK-MB, Tn-T was much lesser in CMP group; Compared with the sham-operated group, the content of MDA, SOD, TNOS, and iNOS were increased and the expression levels of iNOS mRNA and Caspase-3 mRNA were significantly down-regulated in CMP groups, but less than the control group,(P<0.01 or P<0.05. Several pieces of myocardial hemorrhage and inflammatory cell infiltration were observed in the control group, severe degeneration and necrosis of myocardial cells could be seen, apoptotic myocardial cells were significant increased; while in the CMP group, myocardial infarct size was reduced, myocardial pathological injuries were mitigated, only mild degeneration and necrosis of myocardial cells could be seen, the vascular structure was near-complete, the number of apoptotic myocardial cells was less than the control group; In the sham operation group, the structure of myocardial tissue were basically normal, there was a small amount of myocardial cell apoptosis.
Conclusion: Cardiomyopeptidin for injection preconditioning protected immature myocardium against ischemia-reperfusion injury in young rats. The mechanism may be associated with reduced Oxygen free radical (OFR) and NO production, and the inhibited expression of iNOS mRNA, Caspase-3mRNA and Caspase-3 protein in cardiomyocytes, thereby reduced apoptosis.
引文
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[2] Durandy Y.Pediatric myocardial protection [J].Curr Opin Cardiol, 2008, 23(2): 85-90.
[3] Kaneko M,Matsumoto Y,Hayashi H,et al.Oxygen free radicals and calcium homeostasis in the heart [J]. Mol Cell Biochem,1994,135:99-105.
[4] Lipschultz SE, Rifai N, Sallan SE, et al. Predictive value of cardiac troponin T in pediatric patients at risk for myocardial injury [J].Circulation, 1997, 96:2641-2648.
[5] Manukhina EB, Mashina SY, Smirin BV, et,al. Role of nitric oxide in adaptation to hypoxia and adaptive defense [J].Physiol Res,2000,49:89-97.
[6] Shami PJ, Sauls DL, Weinberg JB. Schedule and concentration-dependent induction of apoptosis in leukemia cells by nitric oxide [J]. Leukemia1998,12:1461-1466.
[7] Moro MA, Almeida A, Bloanos JP, et al Mitochondrial respiratory chain and free radical generation in stroke [J].Free Radic Biol Med,2005,39:1291-1304.
[8]Manukhina EB, Downey HF, Mallet RT. Role of nitric oxide in cardiovascular adaptation to intermittent hypoxia [J]. Exp Biol Med,2006,231:343-365.
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