Genistein药物后适应对大鼠缺血—再灌注心肌的保护作用及其机制研究
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摘要
研究背景:
尽管在冠状动脉阻塞后及早恢复缺血心肌的血液灌注对挽救心肌死亡是至关重要的,但大量临床和实验研究发现,在一定条件下,当组织、器官重新获得血液供应时,不但未使组织细胞原有的缺血性损害减轻或恢复,反而进一步产生了新的有害损伤,这种损伤被称为缺血再灌注损伤。它是高等动物机体组织缺血再灌注后发生的普遍现象。如心脏手术、冠脉搭桥、脏器血供梗塞后再通、器官移植以及休克脏器低灌流纠正后都可能发生再灌注损伤。缺血再灌注损伤发生的机制包括氧自由基生成增多、细胞内钙超载、急性炎症反应和血管内皮细胞自稳态破坏等等。Murry等首先观察到狗心肌在经历几次反复短暂的缺血再灌注后,可以提高心肌组织对随后持续严重的缺血再灌注损伤的耐受性,此现象被称为缺血预适应。缺血预适应可以显著减轻心肌缺血再灌注损伤,但其临床应用价值有很大的局限性。Zhao等在对狗的缺血再灌注研究中首次发现,在再灌注的早期对心脏进行数次极为短暂的冠脉再灌注/再缺血可以显著减少心梗面积,减轻缺血再灌注损伤,并将此现象称为缺血后适应。缺血后适应的保护效应不亚于缺血预适应,并为临床应用提供了可操作的空间。缺血后适应现象的发现及对其产生保护效应机制的研究,给我们一个启示,如果在再灌注早期,及时给予某种药物干预,该药物在体内发挥的作用与缺血后适应类似,那么,这种药物干预对缺血再灌注损伤的保护和治疗作用将不仅具有深远的理论意义,也将具有广泛的临床应用前景。在缺血预适应产生机制的研究过程中,曾经证明在缺血前给予某些药物,可以模拟缺血预适应的心肌保护效应,这种药物干预过程称为药物预适应。因此,再灌注前的药物干预过程相应地被称为药物后适应。
Genistein(GST)是1931年第一个从大豆中分离出的异黄酮类物质,它可以通过多种机制作用于靶组织,发挥多种生物学效应。它通过抗氧化和清除活性氧、抑制心肌钙超载、降血脂、影响血管反应性、抑制平滑肌细胞增殖、抑制心肌细胞凋亡、抗血栓形成等机制,对心血管疾病的预防和治疗产生积极的作用。心肌再灌注后迅速生成的活性氧类物质和细胞内Ca~(2+)超载是启动心肌缺血再灌注损伤病理过程的两个最重要的触发因素。Genistein具有抗氧化、清除活性氧和抑制心肌钙超载的作用,因此将其确定为减轻心肌缺血再灌注损伤的候选药物。我们将利用在体、离体大鼠心肌缺血再灌注模型检验Genistein可能具有的药物后适应心肌保护作用及其机制。
研究目的:
1、利用在体大鼠心脏局部缺血再灌注模型,通过测量大鼠心肌梗塞面积、心肌酶和心功能指标的变化,以及观测心肌组织病理形态学和超微结构的变化,明确Genistein对缺血再灌注损伤后的大鼠心肌是否存在药物后适应的保护效应。
2、通过观察Genistein对缺血再灌注损伤后的大鼠心肌组织、血清中NOS活力和NO含量变化的影响,以及对缺血再灌注损伤后心肌细胞凋亡的影响,结合在离体心脏局部缺血再灌注下,观测雌激素受体拮抗剂ICI 182780和酪氨酸磷酸酶抑制剂正钒酸钠对Genistein心肌保护效应的干预效果,分析Genistein产生药物后适应心肌保护效应的可能机制。
研究方法:
1、在体大鼠心脏局部缺血再灌注模型和Genistein药物后适应给药方式的建立
实验采用240～260g健康Wistar大鼠,按照丁延风等报道的方法建立在体大鼠心脏局部缺血再灌注模型。可逆性结扎左冠状动脉前降支,阻断左冠状动脉前降支血流,造成心肌缺血,心电图显示ST段抬高≥0.15 mV时为模型缺血成功,缺血30分钟。松开结扎线实现再灌注,再灌注3小时。实验分为假手术组、缺血再灌注组、Genistein 0.1 mg/kg组、Genistein 0.3 mg/kg组、Genistein 1.0 mg/kg组、Genistein 2.0 mg/kg组和Genistein 5.0mg/kg组等七组。各不同剂量Genistein组,从再灌注开始前20秒至再灌注第1分钟结束,经股静脉微量推注方式给药。
2、心脏梗死面积的测定
实验结束时,从测压管侧孔,经颈动脉迅速注入2%Even's blue 2ml,待大鼠口唇及足部蓝染后快速取出心脏,剔除左心室以外部分,将左心室垂直于心脏纵轴方向自心底向心尖平均切成六片环形的薄片,在37℃于1%TTC中避光孵育15 min后,用10%甲醛溶液固定24～48h。用扫描仪对心脏切面进行扫描,利用image-pro plus图像分析软件测定心肌危险区与梗塞区面积,对数据进行多组间单因素方差分析(ANOVA)统计分析。
3、心肌组织病理形态学和超微结构变化的观察
各组实验完成后,迅速取出心脏置于0℃生理盐水中,洗净心腔内残血,剪取冠状动脉前降支结扎线下约2mm处左室前壁组织(约0.2×0.2cm~2大小),经10%甲醛溶液固定后24～48h,常规石蜡包埋,4μm切片,常规HE染色,光镜(400×)下观察心肌组织病理形态学改变。留取的心肌左室前壁组织浸于2.5%戊二醛中,固定2～4小时,再用四氧锇酸固定2小时,丙酮脱水,环氧树脂618包埋,超薄切片,铀-铅双重染色,透射电镜(6000～20000×)下观察心肌组织超微结构改变。
4、血清、心肌组织中酶学指标的测定
实验结束时,经颈动脉测压管的侧孔,取动脉血1.5 ml,2000 r/min离心10 min,取上清,进行血清酶学指标测定;实验结束后,快速取出心脏,置于0℃生理盐水中,洗净心腔内残血,剪取冠状动脉前降支结扎线下约2mm处左室前壁组织(约0.2×0.2cm~2大小),制备10%的大鼠心肌组织匀浆,取心肌组织匀浆,10000 r/min离心10 min,取上清,进行心肌酶学指标测定。
采用Bionewtrans Pharmaciutical Biotechnology Co.,Ltd.的goat anti-rat cardiac TroponinⅠ试剂盒,用夹心酶联免疫吸附测试法(定量)检测血清中心肌肌钙蛋白Ⅰ(Tn-Ⅰ)含量;采用南京建成生物工程研究所的丙二醛(MDA)试剂盒,用TAB法测定心肌组织中的MDA含量;采用南京建成生物工程研究所的超氧化物歧化酶(SOD)试剂盒,用黄嘌呤氧化酶法测定心肌组织中的总超氧化物歧化酶(T-SOD)活力,对数据进行多组间单因素方差分析(ANOVA)统计分析。
5、心功能指标的监测
在实验过程中用RM6240B生物信号采集处理系统2.0e全程记录从缺血前到再灌注3小时结束后的心电图和左心室内压力波形。在缺血后30 min、再灌注10 min、再灌注30 min、再灌注60 min、再灌注120 min和再灌注180 min等时间点,测定HR、LVSP、LVDP、+dp/dtmax和-dp/dtmax等心功能指标与缺血前相比,计算得到HR、LVSP-LVDP、+dp/dtmax和-dp/dtmax的回复率数据,应用重复测量的方差分析模型进行对比分析。
6、血清NO含量和心肌组织NOS活力(分型)的测定
血清和心肌组织匀浆检测标本的制备同方法4。采用南京建成生物工程研究所的一氧化氮(NO)测定试剂盒,用化学法测定血清中NO含量;采用南京建成生物工程研究所的一氧化氮合酶(nitric oxide synthase,NOS)测定试剂盒(分型),测定心肌组织中的总NOS活力和iNOS活力,计算cNOS活力,对数据进行多组间单因素方差分析(ANOVA)统计分析。
7、心肌细胞凋亡的观测
采用德国Roche Diagnostics GmbH Mannheim公司的TUNEL凋亡检测试剂盒测定各实验组心肌组织中的凋亡细胞。留取的心肌左室前壁组织,经10%甲醛溶液固定后24～48h,常规石蜡包埋,4μm切片,用经多聚赖氨酸包被的玻片制作石蜡切片,严格按试剂盒说明书操作,制作TUNEL法凋亡检测石蜡切片,用荧光显微镜对切片上的凋亡细胞进行计数,计算凋亡指数,对数据进行多组间单因素方差分析(ANOVA)统计分析。
心肌组织匀浆检测标本的制备同方法4,利用美国Biological公司的Caspase-3凋亡检测试剂盒测定各实验组心肌组织中的Caspase-3活性,计算各组Caspase-3的比活性,对数据进行多组间单因素方差分析(ANOVA)统计分析。
8、离体大鼠心脏局部缺血再灌注模型和药物干预方式的建立
实验采用240-260g健康Wistar大鼠,利用Langendorff离体心脏灌流技术,按照Hausenloy等报道的方法建立离体大鼠心脏局部缺血再灌注模型。离体心脏从主动脉插管,悬挂于Langendorff装置上,行主动脉逆行灌注,恒定流速9 ml/min。可逆性结扎左冠状动脉前降支,阻断左冠状动脉前降支灌流,造成心肌缺血,心电图显示ST段抬高≥0.15 mV时为模型缺血成功,缺血30分钟。松开结扎线实现再灌注,再灌注3小时。应用工具药物雌激素受体拮抗剂ICI 182780和酪氨酸磷酸酶抑制剂正钒酸钠,观测离体大鼠心肌梗塞面积、心肌酶和心功能等指标变化。实验分为假结扎组、缺血再灌注组、Genistein组、ICI182780组、正钒酸钠组、Genistein+ICI 182780组和Genistein+正钒酸钠组等七组。在再灌注前1 min,分别用含有药物的灌注液给予干预,直到再灌注后9 min,共干预10 min,建立不同药物干预组。
研究结果:
1、局部缺血再灌注对大鼠在体心脏的损伤作用
缺血再灌注组心肌梗死区面积占危险区面积百分比、血清样品中心肌肌钙蛋白Ⅰ含量、心肌组织样品中MDA含量和SOD活力的测定值分别为47.25±1.89%、0.72±0.06 ng/ml、2.42±0.15 nmol/mgprot和23.38±1.67 U/mgprot,而假手术组各测定值分别为4.36±0.77%、0.22±0.01 ng/ml、1.27±0.10 nmol/mgprot和44.21±1.38 U/mgprot。与假手术组相比,缺血再灌注组显著增高了心肌梗死区面积、心肌肌钙蛋白Ⅰ和MDA含量,降低了心肌组织的SOD活力。缺血再灌注组的心率、LVSP-LVDP和-dp/dtmax回复率均显著低于假手术组。同时,缺血再灌注组的心肌组织表现出广泛坏死,肌纤维排列紊乱、断裂,部分心肌细胞核肿胀、固缩、破裂、溶解,线粒体形态异常,大量炎性细胞渗出等严重损伤的病理形态学和超微结构变化。以上结果显示局部缺血再灌注对在体心脏具有明显的损伤作用,同时也证实我们比较成功地建立了在体心脏缺血再灌注实验模型。
2、Genistein药物后适应对心肌梗死区面积的影响
Genistein 0.1 mg/kg组、0.3 mg/kg组、1.0 mg/kg组、2.0 mg/kg组和5.0 mg/kg组的心肌梗死区面积占危险区面积百分比分别为20.37±2.05%、16.93±2.26%、8.76±2.17%、28.46±2.34%和40.75±1.90%。与缺血再灌注组47.25±1.89%相比,分别显著减少了心肌梗死面积56.89%、64.17%、81.46%、39.77%和13.76%。但0.1～1.0 mg/kg组表现出剂量依赖性减少心梗面积,1.0～5.0 mg/kg组表现出剂量依赖性增加心梗面积。
3、Genistein药物后适应对心肌组织病理形态学和超微结构变化的影响
用光镜和电镜观察各组心肌组织病理形态学和超微结构的变化,经历30分钟缺血、3小时再灌注以后,心肌组织光镜下表现为广泛坏死,肌纤维排列紊乱、断裂,部分心肌细胞核溶解、消失,中性粒细胞大量渗出等变化;电镜下表现为心肌肌原纤维排列紊乱、大面积断裂、融合、消失,肌节结构不清,心肌细胞核肿胀、空亮,核膜不完整,核染色欠均匀,核染色质固缩、边聚,线粒体形态异常,肿胀,嵴排列紊乱,嵴断裂、消失形成大空泡,线粒体间糖原颗粒消失,双层细胞膜内包裹部分线粒体,与母细胞完全脱离,形成凋亡小体等变化。0.1～5.0 mg/kg各不同剂量Genistein组中,心肌组织损伤的病理形态学和超微结构变化随给药剂量不同而变化,呈现出0.1～1.0 mg/kg时损伤逐渐变轻,而1.0-5.0mg/kg时又逐渐加重。
4、Genistein药物后适应对心肌组织和血清中酶学指标的影响
0.1～5.0 mg/kg各不同剂量Genistein组和缺血再灌注组相比,血清样品中心肌肌钙蛋白Ⅰ含量、心肌组织样品中MDA含量均显著少于缺血再灌注组,心肌组织样品中SOD活力显著高于缺血再灌注组。0.1～1.0 mg/kg组表现出剂量依赖性减少心肌肌钙蛋白Ⅰ和MDA含量,增高SOD活力,1.0～5.0 mg/kg组表现出剂量依赖性增加心肌肌钙蛋白Ⅰ和MDA含量,降低SOD活力。
5、Genistein药物后适应对心功指标回复率的影响
Genistein 1.0 mg/kg组的心率、LVSP-LVDP、+dp/dtmax和-dp/dtmax等指标的回复率(%)分别为100.40%、109.69%、133.28%和116.03%,均显著高于缺血再灌注组,缺血再灌注组各指标的回复率分别为78.51%、77.96%、80.66%和79.32%。
6、Genistein药物后适应对心肌组织各型NOS活力和血清NO含量的影响
各不同剂量Genistein组和缺血再灌注组相比,血清样品中NO含量显著高于缺血再灌注组,其中,1.0 mg/kg组的NO含量最高;心肌组织样品中总NOS活力和cNOS活力显著高于缺血再灌注组,其中,1.0 mg/kg组的总NOS活力和cNOS活力最高;iNOS活力在各组之间没有差异,且iNOS活力比cNOS活力低得多。
7、Genistein药物后适应对心肌细胞凋亡的影响
缺血再灌注组的心肌损伤严重,凋亡指数高达28.58±2.37%,在0.1、1.0和5.0 mg/kg三个Genistein组中,凋亡指数分别为19.48±2.13%、10.55±1.93%和21.49±2.34%,其中,1.0 mg/kg组最低,各不同剂量Genistein组的凋亡指数与缺血再灌注组相比均有显著下降。设定假手术组心肌细胞Caspase-3比活性为1.00,缺血再灌注组与之相比,Caspase-3比活性增高至2.75±0.25,在0.1、1.0和5.0 mg/kg三个Genistein组中,Caspase-3比活性分别为1.51±0.15、1.28±0.10和1.71±0.13,其中,1.0 mg/kg组最低,各不同剂量Genistein组的Caspase-3比活性与缺血再灌注组相比均有显著下降。
8、雌激素受体拮抗剂ICI 182780和酪氨酸磷酸酶抑制剂正钒酸钠对Genistein药物后适应作用的干预
离体大鼠心脏局部缺血再灌注模型经不同药物干预后,ICI 182780组、正钒酸钠组、Genistein+ICI 182780组的心肌梗死区面积占危险区面积的百分比分别为48.92±1.63%、46.07±1.81%和29.02±1.65%,显著高于Genistein组10.35±1.74%,而Genistein+正钒酸钠组仅为7.45±1.40%,显著低于Genistein组。各药物干预组心脏灌流漏出液中T-SOD活力的变化趋势与心肌梗死区面积的变化趋势相反。离体心脏经过缺血30分钟、再灌注3小时后,心率回复率在各组之间不具有显著性差异;ICI 182780组、正钒酸钠组、Genistein+ICI 182780组的LVSP-LVDP、+dp/dtmax和-dp/dtmax各指标回复率显著劣于Genistein组;Genistein+ICI 182780组的各项心功能指标回复率数值,比较接近缺血再灌注组;Genistein+正钒酸钠组的各项心功能指标回复率,均与Genistein组非常接近,且数值上略好于Genistein组;ICI 182780组、正钒酸钠组的大部分心功能指标回复率与缺血再灌注组相比没有显著性差异。
研究结论:
1、利用在体大鼠心脏局部缺血再灌注模型证实,在再灌注早期静脉给予Genistein能有效减轻心肌缺血再灌注损伤。表现为心肌梗死面积减小,心肌肌钙蛋白Ⅰ和MDA含量下降,SOD活力升高,心功能指标回复率提高,心肌超微结构的破坏明显减轻,多方面地显示了Genistein药物后适应的心肌保护作用。
2、在0.1～5.0 mg/kg剂量范围内,Genistein都具有明显的心肌保护作用,但表现出双向的剂量依赖性。在0.1～1.0 mg/kg剂量范围内其保护效应呈剂量依赖性增强,而在1.0～5.0mg/kg剂量范围内呈剂量依赖性减弱。
3、通过心肌NOS活性与血清NO含量的测定,凋亡指数和Caspase-3比活性的测定,结合对雌激素受体拮抗剂和酪氨酸磷酸酶抑制剂对Genistein药物后适应作用干预的分析,提示Genistein药物后适应的心肌保护作用与增强心肌NOS活性、抑制细胞凋亡、和对雌激素受体的激动作用有关;它本身的酪氨酸蛋白激酶抑制作用,对心肌保护是不利的,这可能是大剂量范围内保护效应呈剂量依赖性减弱的重要原因。
Background:
Although the early restoration of blood flow to the ischemiac myocardium after coronary artery occlusion is critical to salvage myocytes from eventual death,accumulating clinical and experimental studies have demonstrated that reperfusion has additional deleterious injury on the ischemic myocardium that are not expressed during ischemia,which was referred to as ischemic reperfusion injury.It is the universal phenomenon in the higher animals when tissue and organ are subjected to ischemia-reperfusion.It often occurred in cardiac surgery,coronary bypass. infarct recanalization,organ transplantation and so on.The mechanisms responsible for ischemia reperfusion injury include increasing of oxygen free radicals,overloading of intracellular calcium,acute inflammatory response and damage of vascular endothelial cell auto-homeostasis, etc.Murry,et al.first discovered that repetitions of brief ischemia-reperfusion led to a pronounced protection against subsequent lethal ischemia.This phenomenon was known as ischemia preconditioning.Ischemic preconditioning can significantly reduce myocardial ischemia-reperfusion injury,but the clinical application is limited.In 2003,Zhao,et al.witnessed for the first time that repetitions short-term coronary ischemia and reperfusion at early period of reperfusion significantly reduced the infarct size and alleviated ischemia reperfusion injury.This phenomenon was called ischemic postconditioning.Postconditioning possesses a comparable protective effect as preconditioning and provides operation spaces for clinical application.After ischemic postconditioning phenomenon was recognized,the possible mechanisms of its protective effect were explored gradually.We were encouraged to testify the hypothesis that the administration of drug intervention at the early period of reperfusion can simulate the protective effect of postconditioning.The protective and remedial effect of this drug intervention will have not only profound significance of theoretical study,but also broad prospects of clinical application.The process of drug intervention before ischemia was known as pharmacological preconditioning.Therefore,the process of drug intervention before reperfusion was accordingly called pharmacological postconditioning.
Genistein,one of the isoflavones was first isolated from soybeans in 1931.It has diverse bioactivity on target tissue via different mechanisms.Genistein is beneficial to the prevention and treatment of cardiovascular diseases by anti-oxidation,scavenging reactive oxygen species, inhibiting myocardial calcium overload,hypolipidemic,affecting vascular reactivity,inhibiting smooth muscle cell proliferation,prohibiting cardiomyocyte apoptosis,anti-thrombosis formation,and so on.Reactive oxygen species(ROS)and calcium overloading generated right after reperfusion are two most important triggers to ischemia reperfusion injury.Genistein can resist oxidant,scavenge reactive oxygen species and inhibit myocardial calcium overload. Therefore,we choose Genistein as our drug candidate.We will utilize rat myocardial ischemia reperfusion in vivo and in vitro model to testify the potential pharmacological postconditioning protective effect of genistein to myocardium and its mechanisms.
Aim:
1.To establish the rat heart ischemia-reperfusion in vivo model,compare the differences in myocardial infarction,myocardial enzyme,heart function indexes,heart pathomorphology and ultrastructure between Genistein given and Genistein not given in early stage of reperfusion,and identify the pharmacological postconditioning protective effect of Genistein on rat's myocardial ischemia-reperfusion injury.
2.To observe the effect of Genistein on myocardial NOS activity,serumal NO content and cardiomyocyte apoptosis in rats' ischemia-reperfusion in vivo hearts,and study the interventional effects of estrogen receptor antagonist ICI 182,780 and tyrosine phosphatase inhibitor vanadate on cardioprotection of Genistein against heart ischemia-reperfusion injury in an in vitro model, we analyze the potential mechanisms of Genistein pharmacological postconditioning.
Materials and methods:
1.The establishment of the rat heart ischemia-reperfusion in vivo model and the definement of the drug injection methods in Genistein pharmacological postconditioning
Healthy male adult Wistar rats weighed 240-260 g were selected to establish the ischemia reperfusion model according to the method published in literature.After blocking the left anterior descending coronary artery reversibly,when electrocardiogram showed ST-segment elevation＞0.15 mV,we thought ischemia model was finished.The experiment was divided into such seven groups as the sham group,ischemia-reperfusion group,Genistein 0.1 mg/kg group, Genistein 0.3 mg/kg group,Genistein 1.0 mg/kg group,Genistein 2.0 mg/kg group and Genistein 5.0 mg/kg group.In the early stage of reperfusion,different doses of Genistein were given by an intravenous microinjection.
2.The determination of myocardial infarct size
When the experiment was over,through the side hole of the pressure pipe,2%Even's blue was injected rapidly into left ventricular by carotid.After rats' lips and foot became blue,the heart was taken out quickly from thorax.The atrium and right ventricular was removed.The left ventricular was averagely cut into six annular slices perpendicular to the longitudinal axis direction of heart from the bottom to the cusp,and the slices were incubated at 37℃in 1% TTC for 15 min,and then were fixed 24 to 48 h with 10%formaldehyde solution.The images of the heart were scanned by scanner,and then the dangerous area and infarct size were determined by image-pro plus image analysis software.The date was analyzed by the one-way ANOVA of the multi-groups.
3.The observation of the pathomorphological and ultrastructural changes of myocardial tissue
When the experiment was over,the heart was taken out quickly from thorax,and was washed with the saline solution at 0℃.The myocardial tissue of left ventricular anterior wall under the ligation thread of anterior descending coronary artery about 2 mm was scissored to the size of about 0.2×0.2 cm~2.After being fixed for 24 to 48 h in 10%formaldehyde solution, followed conventional paraffin-embedded,it was sectioned to 4μm sections.Followed conventional HE staining,the sections were observed by the optical microscope(400×)for pathomorphology.The myocardial tissue of left ventricular anterior wall was fixed in 2.5% glutaraldehyde for 2 to 4 h and in osmium tetroxide acid for 2 h one by one.After acetone dehydration,618 epoxy-embedded,it was sectioned to ultrathin sections.Followed uranium-lead double staining,the ultrathin sections were observed by the TEM(6000～20000×)for ultrastructure.
4.The determination of myocardial and serumal enzyme indexes
The experiment was over,through the side hole of the pressure pipe,1.5 ml artery blood was drawn out.After the blood was centrifugated for 10 min at 2000 rpm,the supernatant was collected,and the serumal enzyme indexes were measured.10%myocardial tissue homogenate was prepared with the myocardial tissue of left ventricular anterior wall.The homogenate was centrifugated for 10 min at 10000 rpm.The supernatant was collected,and then the myocardial enzyme indexes were measured.
By sandwich enzyme-linked immunosorbent assay test(quantitative),the serumal cardiac troponin I(Tn-I)content was detected with the goat anti-rat cardiac troponin I kit,which was purchased from Bionewtrans Pharmaciutical Biotechnology Co.,Ltd.The myocardial MDA content was detected by TAB test with the maleic dialdehyde detection kit,which was purchased from Nanjing Jiancheng Bioengineering Institute.The myocardial SOD activity was detected by xanthine oxidase method with the superoxide dismutase detection kit,which was purchased from Nanjing Jiancheng Bioengineering Institute.The date was analyzed by the one-way ANOVA of the multi-groups.
5.The monitoring of the heart function indexes
During the experiment,the ECG and left ventricular pressure waveform were entirely recorded using RM6240B biological signal acquisition and processing system 2.0e from before ischemia beginning to after ischemia-reperfusion being finished for 3 h.The data of HR,LVSP, LVDP,+dp/dtmax and -dp/dtmax in 30 min after the ischemia and in 10 minutes,30 min,60 min,120 min,180 min after the reperfusion were compared with before ischemia,then the recovery rate of the HR,LVSP-LVDP,+dp/dtmax and -dp/dtmax at each time point were calculated.The date of recovery rate was analyzed by a variance analysis model of repeated measures.
6.The determination of serumal NO content and myocardial NOS activity
The preparation of serum and myocardial specimens for NO and NOS detections were defined as method 4.The serumal NO content was detected by chemic method with the nitric oxide detection kit,which was purchased from Nanjing Jiancheng Bioengineering Institute.The activities of each type NOS in myocardial tissue were detected with the nitric oxide synthase detection kit(typing),which was purchased from Nanjing Jiancheng Bioengineering Institute. The data was analyzed by the one-way ANOVA of the multi-groups.
7.The observation of the cardiomyocyte apoptosis
The cardiomyocyte apoptosis was detected with in situ cell death detection kit,which was purchased from Germany Roche Diagnostics Mannheim GmbH Company.Myocardial tissue specimens were prepared to TUNEL paraffin sections strictly in accordance with the manual method.Apoptotic cells in TUNEL paraffin sections were counted by a fluorescence microscopy, and apoptosis index was calculated.The data was analyzed by the one-way ANOVA of the multi-groups.
The preparation of myocardial detection specimens was defined as method 4.The myocardial Caspase-3 activity was detected with Caspase-3 activity detection kit,which was purchased from American Biological Company.The ratios of Caspase-3 activity in each group were calculated.The data was analyzed by the one-way ANOVA of the multi-groups.
8.The establishment of the rat heart ischemia-reperfusion in vitro model and the definement of the drug intervention methods in Genistein pharmacological postconditioning
Healthy Wistar male rats weighed 240-260 g were selected to establish the ischemia reperfusion in vitro model by Langendorff isolated heart perfusion technology according to the method in literature.After blocking the left anterior descending coronary artery reversibly,when electrocardiogram showed ST-segment elevation≥0.15 mV,we thought ischemia model finished. Using estrogen receptor antagonist ICI 182,780 and tyrosine phosphatase inhibitor vanadate as tools drug,the area of myocardial infarction,activities of myocardial enzyme and heart function indexes in isolated rat myocardium were observed.The model rats was divided into such seven groups as sham group,ischemia-reperfusion group,Genistein group,ICI 182780 group,vanadate group,Genistein + ICI 182780 group and Genistein + vanadate groups.The fusion fluid containing drug was used to intervene at 1 min before reperfusion until 9 min after reperfusion,a total of 10 min intervention,and the different drug intervention groups were established.
Result:
1.The damage effect of regional ischemia-reperfusion on rat hearts in vivo
The percentage of myocardial infarct size to the ischemic area at risk,the cardiac troponin I levels of serumal samples,the MDA levels of myocardial tissue samples and the activity of SOD of myocardial tissue samples of the ischemia-reperfusion group were 47.25±1.89%,0.72±0.06 ng/ml,2.42±0.15 nmol/mgprot and 23.38±1.67 U/mgprot,respectively,and the dates of the sham group were 4.36±0.77%,0.22±0.01 ng/ml,1.27±0.10 nmol/mgprot and 44.21±1.38 U/mgprot.The ischemia-reperfusion group significantly increased the area of myocardial infarction,cardiac troponin I and MDA levels,and reduced SOD activity than the sham group. These results indicated that ischemia-reperfusion had obvious injury effect on rat in vivo heart, and it also proved that we were successful in establishing the myocardial ischemia-reperfusion in vivo model.
2.The effect of Genistein pharmacological postconditioning on myocardial infarction size
The percentage of myocardial infarct size to the ischemic area at risk of Genistein 0.1 mg/kg group,0.3 mg/kg group,1.0 mg/kg group,2.0 mg/kg group and 5.0 mg/kg group were respectively 20.37±2.05%,16.93±2.26%,8.76±2.17%,28.46±2.34%and 40.75±1.90%.They were significantly reduced myocardial infarction area than the ischemia-reperfusion group, which was 47.25±1.89%.In 0.1～1.0 mg/kg group,there were a dose-dependent decrease of myocardial infarction area,but in 1.0～5.0 mg/kg group,it showed a dose-dependent increase.
3.The effect of Genistein pharmacological postconditioning on the pathomorphological and ultrastructural changes of myocardial tissue
By the optical microscopy and TEM,the pathomorphology and ultrastructure of the myocardial tissue after ischemia or reperfusion for 30 minutes and 3 hours were observed.By the optical microscopy,the myocardial tissue showed extensive necrosis and severe muscle fiber fracture,myocardial cells dissolution and disappearance,neutrophils exudation,and so on.Under the TEM,it showed myofibrils disarrangement,large areas of myofibrils fracture,amalgamation, disappearance,sarcomere structure unclear,myocardial nuclear swelling,the nuclear membrane rupture,nuclear chromatin asymmetry,condensation,margination,mitochondrial morphological abnormalities and swelling,the ridge disarrangement,fracture,disappearance,forming cavity, glycogen granules between the mitochondrial vanishment,some mitochondria parceled by double membrane,and completely desquamated from the mother cells,forming apoptotic bodies. In the different dosage Genistein groups,with the difference of doses,myocardial pathomorphological and ultrastructural changes were discriminating,it was showed that the injury became gradually lighter in 0.1～1.0 mg/kg group,however,gradually more serious in 1.0～5.0 mg/kg group.
4.The effect of Genistein pharmacological postconditioning on myocardial and serumal enzyme indexes
In 0.1～5.0mg/kg dose range,the cardiac troponin I levels and MDA levels of the different dosage Genistein groups were significantly less than ischemia-reperfusion group,and the activity of SOD of myocardial tissue samples were significantly higher than ischemia-reperfusion group. In 0.1～1.0 mg/kg group,there were a dose-dependent decrease of the cardiac troponin I levels and MDA levels,and a dose-dependent increase of the SOD activity,but in 1.0-5.0 mg/kg group, it was the opposite.
5.The effect of Genistein pharmacological postconditioning on the heart function indexes
The recovery rates of the HR,LVSP-LVDP,+dp/dtmax and -dp/dtmax in Genistein 1.0 mg/kg group were respectively 100.40%,109.69%,133.28%and 116.03%,and they were significantly higher than in ischemia-reperfusion group,which were respectively 78.51%, 77.96%,80.66%and 79.32%.
6.The effect of Genistein pharmacological postconditioning on the serumal NO content and myocardial NOS activity
The NO contents detected in different doses of Genistein groups were significantly higher than in ischemia-reperfusion group,the NO content detected in Genistein 1.0 mg/kg group was the highest.The activities of T-NOS and cNOS of myocardial tissue samples detected in the different doses of Genistein groups were significantly higher than in ischemia-reperfusion group, the activity of T-NOS and cNOS detected in Genistein 1.0 mg/kg group was the highest.There were no differences in activity of iNOS detected among the groups,and the activity of iNOS was much lower than cNOS.
7.The effect of Genistein pharmacological postconditioning on the cardiomyocyte apoptosis
Ischemia-reperfusion injury of ischemia-reperfusion group was more serious,in which the apoptosis index went up to 28.58±2.37%.Apoptosis index in 0.1,1.0 and 5.0 mg/kg genistein treated groups were respectively 19.48±2.13%,10.55±1.93%and 21.49±2.34%,and the 1.0 mg/kg genistein groups treated showed the lowest apoptosis index.Apoptosis index in the different dose of Genistein treated groups were significantly lower than in ischemia-reperfusion group.
After the ratio of Caspase-3 activity in sham group was set at 1.00,the ratios of Caspase-3 activity in other groups were calculated,which increased to 2.75±0.25 in ischemia-reperfusion group and which decreased tol.51±0.15,1.28±0.10 and 1.71±0.13 in 0.1,1.0,5.0 mg/kg groups.The ratios of Caspase-3 activity in Genistein given groups were significantly lower than in ischemia-reperfusion group.
8.The intervention effect of estrogen receptor antagonist ICI 182,780 and tyrosine phosphatase inhibitor vanadate on cardioprotection against ischemia-reperfusion injury by Genistein pharmacological postconditioning
After ischemia-reperfusion,the percentage of myocardial infarct size to the ischemic area at risk of isolated rat heart in ICI 182780,vanadate group and Genistein + ICI 182780 group were respectively 48.92±1.63%,46.07±1.81%and 29.02±1.65%,and they were significantly higher than in Genistein group,which was 10.35±1.74%.It was significantly lower in Genistein + vanadate group than in Genistein given group,which only was 7.45±1.40%.The varying tendency of SOD activity in heart perfusate in each group was completely opposite to the varying tendency of myocardial infarction area.There was no significant difference in the recovery rates of the HR between the groups after reperfusion for 180 min.The recovery rates of LVSP-LVDP,+dp/dtmax and -dp/dtmax in ICI 182780 group,vanadate group and Genistein + ICI 182780 group were significantly lower than in Genistein group.The recovery rates in Genistein+ICI 182780 group was almost as high as in ischemia-reperfusion group.The recovery rates in Genistein + vanadate group was almost as high as in Genistein group.Most of the recovery rates in ICI 182780 group and vanadate group did not differ from in ischemia-reperfusion group.
Conclusion:
1.Using the heart ischemia-reperfusion model of rat in vivo,it was identified that genistein could effectively reduce myocardial ischemia-reperfusion injury by intravenous injection in early reperfusion,which was mainly manifested in the reduction of the myocardial infarction size, lower activity of cardiac troponin I and MDA,higher activity of SOD,the improvement of the recovery of the heart function,and the marked attenuation of destruction of myocardial ultrastructure,indicating genistein has cardioprotection effect against ischemia-reperfusion injury by the pharmacological postconditioning.
2.Generally,genistein showed protective effects on myocardial ischemia-reperfusion injury in 0.1～5.0 mg/kg dose range.However,this protective effect showed two opposite trends in different dose range:a dose-dependent increasing in 0.1～1.0 mg/kg dose range,while a dose-dependent weakening in 1.0～5.0 mg/kg dose range.
3.The effects of genistein on the myocardial NOS activity,serumal NO content, cardiomyocyte apoptosis index and Caspase-3 activity in ischemia-reperfusion hearts of rat, associating with application of estrogen receptor antagonist ICI 182,780 and tyrosine phosphatase inhibitor vanadate on ischemia-reperfusion heart of rat in vitro showed that the protective effect of Genistein pharmacological postconditioning related to the enhancement of myocardial NOS activity,the inhibition of cardiomyocyte apoptosis and the excitement of estrogen receptor.Its protein tyrosine kinase inhibitor property was detrimental to myocardial protection,and this may be the major reasons for a dose-dependent decrease of the protective effect in the higher dose range.
尽管在冠状动脉阻塞后及早恢复缺血心肌的血液灌注对挽救心肌死亡是至关重要的,但大量临床和实验研究发现,在一定条件下,当组织、器官重新获得血液供应时,不但未使组织细胞原有的缺血性损害减轻或恢复,反而进一步产生了新的有害损伤,这种损伤被称为缺血再灌注损伤。它是高等动物机体组织缺血再灌注后发生的普遍现象。如心脏手术、冠脉搭桥、脏器血供梗塞后再通、器官移植以及休克脏器低灌流纠正后都可能发生再灌注损伤。缺血再灌注损伤发生的机制包括氧自由基生成增多、细胞内钙超载、急性炎症反应和血管内皮细胞自稳态破坏等等。Murry等首先观察到狗心肌在经历几次反复短暂的缺血再灌注后,可以提高心肌组织对随后持续严重的缺血再灌注损伤的耐受性,此现象被称为缺血预适应。缺血预适应可以显著减轻心肌缺血再灌注损伤,但其临床应用价值有很大的局限性。Zhao等在对狗的缺血再灌注研究中首次发现,在再灌注的早期对心脏进行数次极为短暂的冠脉再灌注/再缺血可以显著减少心梗面积,减轻缺血再灌注损伤,并将此现象称为缺血后适应。缺血后适应的保护效应不亚于缺血预适应,并为临床应用提供了可操作的空间。缺血后适应现象的发现及对其产生保护效应机制的研究,给我们一个启示,如果在再灌注早期,及时给予某种药物干预,该药物在体内发挥的作用与缺血后适应类似,那么,这种药物干预对缺血再灌注损伤的保护和治疗作用将不仅具有深远的理论意义,也将具有广泛的临床应用前景。在缺血预适应产生机制的研究过程中,曾经证明在缺血前给予某些药物,可以模拟缺血预适应的心肌保护效应,这种药物干预过程称为药物预适应。因此,再灌注前的药物干预过程相应地被称为药物后适应。
Genistein(GST)是1931年第一个从大豆中分离出的异黄酮类物质,它可以通过多种机制作用于靶组织,发挥多种生物学效应。它通过抗氧化和清除活性氧、抑制心肌钙超载、降血脂、影响血管反应性、抑制平滑肌细胞增殖、抑制心肌细胞凋亡、抗血栓形成等机制,对心血管疾病的预防和治疗产生积极的作用。心肌再灌注后迅速生成的活性氧类物质和细胞内Ca~(2+)超载是启动心肌缺血再灌注损伤病理过程的两个最重要的触发因素。Genistein具有抗氧化、清除活性氧和抑制心肌钙超载的作用,因此将其确定为减轻心肌缺血再灌注损伤的候选药物。我们将利用在体、离体大鼠心肌缺血再灌注模型检验Genistein可能具有的药物后适应心肌保护作用及其机制。
研究目的:
1、利用在体大鼠心脏局部缺血再灌注模型,通过测量大鼠心肌梗塞面积、心肌酶和心功能指标的变化,以及观测心肌组织病理形态学和超微结构的变化,明确Genistein对缺血再灌注损伤后的大鼠心肌是否存在药物后适应的保护效应。
2、通过观察Genistein对缺血再灌注损伤后的大鼠心肌组织、血清中NOS活力和NO含量变化的影响,以及对缺血再灌注损伤后心肌细胞凋亡的影响,结合在离体心脏局部缺血再灌注下,观测雌激素受体拮抗剂ICI 182780和酪氨酸磷酸酶抑制剂正钒酸钠对Genistein心肌保护效应的干预效果,分析Genistein产生药物后适应心肌保护效应的可能机制。
研究方法:
1、在体大鼠心脏局部缺血再灌注模型和Genistein药物后适应给药方式的建立
实验采用240～260g健康Wistar大鼠,按照丁延风等报道的方法建立在体大鼠心脏局部缺血再灌注模型。可逆性结扎左冠状动脉前降支,阻断左冠状动脉前降支血流,造成心肌缺血,心电图显示ST段抬高≥0.15 mV时为模型缺血成功,缺血30分钟。松开结扎线实现再灌注,再灌注3小时。实验分为假手术组、缺血再灌注组、Genistein 0.1 mg/kg组、Genistein 0.3 mg/kg组、Genistein 1.0 mg/kg组、Genistein 2.0 mg/kg组和Genistein 5.0mg/kg组等七组。各不同剂量Genistein组,从再灌注开始前20秒至再灌注第1分钟结束,经股静脉微量推注方式给药。
2、心脏梗死面积的测定
实验结束时,从测压管侧孔,经颈动脉迅速注入2%Even's blue 2ml,待大鼠口唇及足部蓝染后快速取出心脏,剔除左心室以外部分,将左心室垂直于心脏纵轴方向自心底向心尖平均切成六片环形的薄片,在37℃于1%TTC中避光孵育15 min后,用10%甲醛溶液固定24～48h。用扫描仪对心脏切面进行扫描,利用image-pro plus图像分析软件测定心肌危险区与梗塞区面积,对数据进行多组间单因素方差分析(ANOVA)统计分析。
3、心肌组织病理形态学和超微结构变化的观察
各组实验完成后,迅速取出心脏置于0℃生理盐水中,洗净心腔内残血,剪取冠状动脉前降支结扎线下约2mm处左室前壁组织(约0.2×0.2cm~2大小),经10%甲醛溶液固定后24～48h,常规石蜡包埋,4μm切片,常规HE染色,光镜(400×)下观察心肌组织病理形态学改变。留取的心肌左室前壁组织浸于2.5%戊二醛中,固定2～4小时,再用四氧锇酸固定2小时,丙酮脱水,环氧树脂618包埋,超薄切片,铀-铅双重染色,透射电镜(6000～20000×)下观察心肌组织超微结构改变。
4、血清、心肌组织中酶学指标的测定
实验结束时,经颈动脉测压管的侧孔,取动脉血1.5 ml,2000 r/min离心10 min,取上清,进行血清酶学指标测定;实验结束后,快速取出心脏,置于0℃生理盐水中,洗净心腔内残血,剪取冠状动脉前降支结扎线下约2mm处左室前壁组织(约0.2×0.2cm~2大小),制备10%的大鼠心肌组织匀浆,取心肌组织匀浆,10000 r/min离心10 min,取上清,进行心肌酶学指标测定。
采用Bionewtrans Pharmaciutical Biotechnology Co.,Ltd.的goat anti-rat cardiac TroponinⅠ试剂盒,用夹心酶联免疫吸附测试法(定量)检测血清中心肌肌钙蛋白Ⅰ(Tn-Ⅰ)含量;采用南京建成生物工程研究所的丙二醛(MDA)试剂盒,用TAB法测定心肌组织中的MDA含量;采用南京建成生物工程研究所的超氧化物歧化酶(SOD)试剂盒,用黄嘌呤氧化酶法测定心肌组织中的总超氧化物歧化酶(T-SOD)活力,对数据进行多组间单因素方差分析(ANOVA)统计分析。
5、心功能指标的监测
在实验过程中用RM6240B生物信号采集处理系统2.0e全程记录从缺血前到再灌注3小时结束后的心电图和左心室内压力波形。在缺血后30 min、再灌注10 min、再灌注30 min、再灌注60 min、再灌注120 min和再灌注180 min等时间点,测定HR、LVSP、LVDP、+dp/dtmax和-dp/dtmax等心功能指标与缺血前相比,计算得到HR、LVSP-LVDP、+dp/dtmax和-dp/dtmax的回复率数据,应用重复测量的方差分析模型进行对比分析。
6、血清NO含量和心肌组织NOS活力(分型)的测定
血清和心肌组织匀浆检测标本的制备同方法4。采用南京建成生物工程研究所的一氧化氮(NO)测定试剂盒,用化学法测定血清中NO含量;采用南京建成生物工程研究所的一氧化氮合酶(nitric oxide synthase,NOS)测定试剂盒(分型),测定心肌组织中的总NOS活力和iNOS活力,计算cNOS活力,对数据进行多组间单因素方差分析(ANOVA)统计分析。
7、心肌细胞凋亡的观测
采用德国Roche Diagnostics GmbH Mannheim公司的TUNEL凋亡检测试剂盒测定各实验组心肌组织中的凋亡细胞。留取的心肌左室前壁组织,经10%甲醛溶液固定后24～48h,常规石蜡包埋,4μm切片,用经多聚赖氨酸包被的玻片制作石蜡切片,严格按试剂盒说明书操作,制作TUNEL法凋亡检测石蜡切片,用荧光显微镜对切片上的凋亡细胞进行计数,计算凋亡指数,对数据进行多组间单因素方差分析(ANOVA)统计分析。
心肌组织匀浆检测标本的制备同方法4,利用美国Biological公司的Caspase-3凋亡检测试剂盒测定各实验组心肌组织中的Caspase-3活性,计算各组Caspase-3的比活性,对数据进行多组间单因素方差分析(ANOVA)统计分析。
8、离体大鼠心脏局部缺血再灌注模型和药物干预方式的建立
实验采用240-260g健康Wistar大鼠,利用Langendorff离体心脏灌流技术,按照Hausenloy等报道的方法建立离体大鼠心脏局部缺血再灌注模型。离体心脏从主动脉插管,悬挂于Langendorff装置上,行主动脉逆行灌注,恒定流速9 ml/min。可逆性结扎左冠状动脉前降支,阻断左冠状动脉前降支灌流,造成心肌缺血,心电图显示ST段抬高≥0.15 mV时为模型缺血成功,缺血30分钟。松开结扎线实现再灌注,再灌注3小时。应用工具药物雌激素受体拮抗剂ICI 182780和酪氨酸磷酸酶抑制剂正钒酸钠,观测离体大鼠心肌梗塞面积、心肌酶和心功能等指标变化。实验分为假结扎组、缺血再灌注组、Genistein组、ICI182780组、正钒酸钠组、Genistein+ICI 182780组和Genistein+正钒酸钠组等七组。在再灌注前1 min,分别用含有药物的灌注液给予干预,直到再灌注后9 min,共干预10 min,建立不同药物干预组。
研究结果:
1、局部缺血再灌注对大鼠在体心脏的损伤作用
缺血再灌注组心肌梗死区面积占危险区面积百分比、血清样品中心肌肌钙蛋白Ⅰ含量、心肌组织样品中MDA含量和SOD活力的测定值分别为47.25±1.89%、0.72±0.06 ng/ml、2.42±0.15 nmol/mgprot和23.38±1.67 U/mgprot,而假手术组各测定值分别为4.36±0.77%、0.22±0.01 ng/ml、1.27±0.10 nmol/mgprot和44.21±1.38 U/mgprot。与假手术组相比,缺血再灌注组显著增高了心肌梗死区面积、心肌肌钙蛋白Ⅰ和MDA含量,降低了心肌组织的SOD活力。缺血再灌注组的心率、LVSP-LVDP和-dp/dtmax回复率均显著低于假手术组。同时,缺血再灌注组的心肌组织表现出广泛坏死,肌纤维排列紊乱、断裂,部分心肌细胞核肿胀、固缩、破裂、溶解,线粒体形态异常,大量炎性细胞渗出等严重损伤的病理形态学和超微结构变化。以上结果显示局部缺血再灌注对在体心脏具有明显的损伤作用,同时也证实我们比较成功地建立了在体心脏缺血再灌注实验模型。
2、Genistein药物后适应对心肌梗死区面积的影响
Genistein 0.1 mg/kg组、0.3 mg/kg组、1.0 mg/kg组、2.0 mg/kg组和5.0 mg/kg组的心肌梗死区面积占危险区面积百分比分别为20.37±2.05%、16.93±2.26%、8.76±2.17%、28.46±2.34%和40.75±1.90%。与缺血再灌注组47.25±1.89%相比,分别显著减少了心肌梗死面积56.89%、64.17%、81.46%、39.77%和13.76%。但0.1～1.0 mg/kg组表现出剂量依赖性减少心梗面积,1.0～5.0 mg/kg组表现出剂量依赖性增加心梗面积。
3、Genistein药物后适应对心肌组织病理形态学和超微结构变化的影响
用光镜和电镜观察各组心肌组织病理形态学和超微结构的变化,经历30分钟缺血、3小时再灌注以后,心肌组织光镜下表现为广泛坏死,肌纤维排列紊乱、断裂,部分心肌细胞核溶解、消失,中性粒细胞大量渗出等变化;电镜下表现为心肌肌原纤维排列紊乱、大面积断裂、融合、消失,肌节结构不清,心肌细胞核肿胀、空亮,核膜不完整,核染色欠均匀,核染色质固缩、边聚,线粒体形态异常,肿胀,嵴排列紊乱,嵴断裂、消失形成大空泡,线粒体间糖原颗粒消失,双层细胞膜内包裹部分线粒体,与母细胞完全脱离,形成凋亡小体等变化。0.1～5.0 mg/kg各不同剂量Genistein组中,心肌组织损伤的病理形态学和超微结构变化随给药剂量不同而变化,呈现出0.1～1.0 mg/kg时损伤逐渐变轻,而1.0-5.0mg/kg时又逐渐加重。
4、Genistein药物后适应对心肌组织和血清中酶学指标的影响
0.1～5.0 mg/kg各不同剂量Genistein组和缺血再灌注组相比,血清样品中心肌肌钙蛋白Ⅰ含量、心肌组织样品中MDA含量均显著少于缺血再灌注组,心肌组织样品中SOD活力显著高于缺血再灌注组。0.1～1.0 mg/kg组表现出剂量依赖性减少心肌肌钙蛋白Ⅰ和MDA含量,增高SOD活力,1.0～5.0 mg/kg组表现出剂量依赖性增加心肌肌钙蛋白Ⅰ和MDA含量,降低SOD活力。
5、Genistein药物后适应对心功指标回复率的影响
Genistein 1.0 mg/kg组的心率、LVSP-LVDP、+dp/dtmax和-dp/dtmax等指标的回复率(%)分别为100.40%、109.69%、133.28%和116.03%,均显著高于缺血再灌注组,缺血再灌注组各指标的回复率分别为78.51%、77.96%、80.66%和79.32%。
6、Genistein药物后适应对心肌组织各型NOS活力和血清NO含量的影响
各不同剂量Genistein组和缺血再灌注组相比,血清样品中NO含量显著高于缺血再灌注组,其中,1.0 mg/kg组的NO含量最高;心肌组织样品中总NOS活力和cNOS活力显著高于缺血再灌注组,其中,1.0 mg/kg组的总NOS活力和cNOS活力最高;iNOS活力在各组之间没有差异,且iNOS活力比cNOS活力低得多。
7、Genistein药物后适应对心肌细胞凋亡的影响
缺血再灌注组的心肌损伤严重,凋亡指数高达28.58±2.37%,在0.1、1.0和5.0 mg/kg三个Genistein组中,凋亡指数分别为19.48±2.13%、10.55±1.93%和21.49±2.34%,其中,1.0 mg/kg组最低,各不同剂量Genistein组的凋亡指数与缺血再灌注组相比均有显著下降。设定假手术组心肌细胞Caspase-3比活性为1.00,缺血再灌注组与之相比,Caspase-3比活性增高至2.75±0.25,在0.1、1.0和5.0 mg/kg三个Genistein组中,Caspase-3比活性分别为1.51±0.15、1.28±0.10和1.71±0.13,其中,1.0 mg/kg组最低,各不同剂量Genistein组的Caspase-3比活性与缺血再灌注组相比均有显著下降。
8、雌激素受体拮抗剂ICI 182780和酪氨酸磷酸酶抑制剂正钒酸钠对Genistein药物后适应作用的干预
离体大鼠心脏局部缺血再灌注模型经不同药物干预后,ICI 182780组、正钒酸钠组、Genistein+ICI 182780组的心肌梗死区面积占危险区面积的百分比分别为48.92±1.63%、46.07±1.81%和29.02±1.65%,显著高于Genistein组10.35±1.74%,而Genistein+正钒酸钠组仅为7.45±1.40%,显著低于Genistein组。各药物干预组心脏灌流漏出液中T-SOD活力的变化趋势与心肌梗死区面积的变化趋势相反。离体心脏经过缺血30分钟、再灌注3小时后,心率回复率在各组之间不具有显著性差异;ICI 182780组、正钒酸钠组、Genistein+ICI 182780组的LVSP-LVDP、+dp/dtmax和-dp/dtmax各指标回复率显著劣于Genistein组;Genistein+ICI 182780组的各项心功能指标回复率数值,比较接近缺血再灌注组;Genistein+正钒酸钠组的各项心功能指标回复率,均与Genistein组非常接近,且数值上略好于Genistein组;ICI 182780组、正钒酸钠组的大部分心功能指标回复率与缺血再灌注组相比没有显著性差异。
研究结论:
1、利用在体大鼠心脏局部缺血再灌注模型证实,在再灌注早期静脉给予Genistein能有效减轻心肌缺血再灌注损伤。表现为心肌梗死面积减小,心肌肌钙蛋白Ⅰ和MDA含量下降,SOD活力升高,心功能指标回复率提高,心肌超微结构的破坏明显减轻,多方面地显示了Genistein药物后适应的心肌保护作用。
2、在0.1～5.0 mg/kg剂量范围内,Genistein都具有明显的心肌保护作用,但表现出双向的剂量依赖性。在0.1～1.0 mg/kg剂量范围内其保护效应呈剂量依赖性增强,而在1.0～5.0mg/kg剂量范围内呈剂量依赖性减弱。
3、通过心肌NOS活性与血清NO含量的测定,凋亡指数和Caspase-3比活性的测定,结合对雌激素受体拮抗剂和酪氨酸磷酸酶抑制剂对Genistein药物后适应作用干预的分析,提示Genistein药物后适应的心肌保护作用与增强心肌NOS活性、抑制细胞凋亡、和对雌激素受体的激动作用有关;它本身的酪氨酸蛋白激酶抑制作用,对心肌保护是不利的,这可能是大剂量范围内保护效应呈剂量依赖性减弱的重要原因。
Background:
Although the early restoration of blood flow to the ischemiac myocardium after coronary artery occlusion is critical to salvage myocytes from eventual death,accumulating clinical and experimental studies have demonstrated that reperfusion has additional deleterious injury on the ischemic myocardium that are not expressed during ischemia,which was referred to as ischemic reperfusion injury.It is the universal phenomenon in the higher animals when tissue and organ are subjected to ischemia-reperfusion.It often occurred in cardiac surgery,coronary bypass. infarct recanalization,organ transplantation and so on.The mechanisms responsible for ischemia reperfusion injury include increasing of oxygen free radicals,overloading of intracellular calcium,acute inflammatory response and damage of vascular endothelial cell auto-homeostasis, etc.Murry,et al.first discovered that repetitions of brief ischemia-reperfusion led to a pronounced protection against subsequent lethal ischemia.This phenomenon was known as ischemia preconditioning.Ischemic preconditioning can significantly reduce myocardial ischemia-reperfusion injury,but the clinical application is limited.In 2003,Zhao,et al.witnessed for the first time that repetitions short-term coronary ischemia and reperfusion at early period of reperfusion significantly reduced the infarct size and alleviated ischemia reperfusion injury.This phenomenon was called ischemic postconditioning.Postconditioning possesses a comparable protective effect as preconditioning and provides operation spaces for clinical application.After ischemic postconditioning phenomenon was recognized,the possible mechanisms of its protective effect were explored gradually.We were encouraged to testify the hypothesis that the administration of drug intervention at the early period of reperfusion can simulate the protective effect of postconditioning.The protective and remedial effect of this drug intervention will have not only profound significance of theoretical study,but also broad prospects of clinical application.The process of drug intervention before ischemia was known as pharmacological preconditioning.Therefore,the process of drug intervention before reperfusion was accordingly called pharmacological postconditioning.
Genistein,one of the isoflavones was first isolated from soybeans in 1931.It has diverse bioactivity on target tissue via different mechanisms.Genistein is beneficial to the prevention and treatment of cardiovascular diseases by anti-oxidation,scavenging reactive oxygen species, inhibiting myocardial calcium overload,hypolipidemic,affecting vascular reactivity,inhibiting smooth muscle cell proliferation,prohibiting cardiomyocyte apoptosis,anti-thrombosis formation,and so on.Reactive oxygen species(ROS)and calcium overloading generated right after reperfusion are two most important triggers to ischemia reperfusion injury.Genistein can resist oxidant,scavenge reactive oxygen species and inhibit myocardial calcium overload. Therefore,we choose Genistein as our drug candidate.We will utilize rat myocardial ischemia reperfusion in vivo and in vitro model to testify the potential pharmacological postconditioning protective effect of genistein to myocardium and its mechanisms.
Aim:
1.To establish the rat heart ischemia-reperfusion in vivo model,compare the differences in myocardial infarction,myocardial enzyme,heart function indexes,heart pathomorphology and ultrastructure between Genistein given and Genistein not given in early stage of reperfusion,and identify the pharmacological postconditioning protective effect of Genistein on rat's myocardial ischemia-reperfusion injury.
2.To observe the effect of Genistein on myocardial NOS activity,serumal NO content and cardiomyocyte apoptosis in rats' ischemia-reperfusion in vivo hearts,and study the interventional effects of estrogen receptor antagonist ICI 182,780 and tyrosine phosphatase inhibitor vanadate on cardioprotection of Genistein against heart ischemia-reperfusion injury in an in vitro model, we analyze the potential mechanisms of Genistein pharmacological postconditioning.
Materials and methods:
1.The establishment of the rat heart ischemia-reperfusion in vivo model and the definement of the drug injection methods in Genistein pharmacological postconditioning
Healthy male adult Wistar rats weighed 240-260 g were selected to establish the ischemia reperfusion model according to the method published in literature.After blocking the left anterior descending coronary artery reversibly,when electrocardiogram showed ST-segment elevation＞0.15 mV,we thought ischemia model was finished.The experiment was divided into such seven groups as the sham group,ischemia-reperfusion group,Genistein 0.1 mg/kg group, Genistein 0.3 mg/kg group,Genistein 1.0 mg/kg group,Genistein 2.0 mg/kg group and Genistein 5.0 mg/kg group.In the early stage of reperfusion,different doses of Genistein were given by an intravenous microinjection.
2.The determination of myocardial infarct size
When the experiment was over,through the side hole of the pressure pipe,2%Even's blue was injected rapidly into left ventricular by carotid.After rats' lips and foot became blue,the heart was taken out quickly from thorax.The atrium and right ventricular was removed.The left ventricular was averagely cut into six annular slices perpendicular to the longitudinal axis direction of heart from the bottom to the cusp,and the slices were incubated at 37℃in 1% TTC for 15 min,and then were fixed 24 to 48 h with 10%formaldehyde solution.The images of the heart were scanned by scanner,and then the dangerous area and infarct size were determined by image-pro plus image analysis software.The date was analyzed by the one-way ANOVA of the multi-groups.
3.The observation of the pathomorphological and ultrastructural changes of myocardial tissue
When the experiment was over,the heart was taken out quickly from thorax,and was washed with the saline solution at 0℃.The myocardial tissue of left ventricular anterior wall under the ligation thread of anterior descending coronary artery about 2 mm was scissored to the size of about 0.2×0.2 cm~2.After being fixed for 24 to 48 h in 10%formaldehyde solution, followed conventional paraffin-embedded,it was sectioned to 4μm sections.Followed conventional HE staining,the sections were observed by the optical microscope(400×)for pathomorphology.The myocardial tissue of left ventricular anterior wall was fixed in 2.5% glutaraldehyde for 2 to 4 h and in osmium tetroxide acid for 2 h one by one.After acetone dehydration,618 epoxy-embedded,it was sectioned to ultrathin sections.Followed uranium-lead double staining,the ultrathin sections were observed by the TEM(6000～20000×)for ultrastructure.
4.The determination of myocardial and serumal enzyme indexes
The experiment was over,through the side hole of the pressure pipe,1.5 ml artery blood was drawn out.After the blood was centrifugated for 10 min at 2000 rpm,the supernatant was collected,and the serumal enzyme indexes were measured.10%myocardial tissue homogenate was prepared with the myocardial tissue of left ventricular anterior wall.The homogenate was centrifugated for 10 min at 10000 rpm.The supernatant was collected,and then the myocardial enzyme indexes were measured.
By sandwich enzyme-linked immunosorbent assay test(quantitative),the serumal cardiac troponin I(Tn-I)content was detected with the goat anti-rat cardiac troponin I kit,which was purchased from Bionewtrans Pharmaciutical Biotechnology Co.,Ltd.The myocardial MDA content was detected by TAB test with the maleic dialdehyde detection kit,which was purchased from Nanjing Jiancheng Bioengineering Institute.The myocardial SOD activity was detected by xanthine oxidase method with the superoxide dismutase detection kit,which was purchased from Nanjing Jiancheng Bioengineering Institute.The date was analyzed by the one-way ANOVA of the multi-groups.
5.The monitoring of the heart function indexes
During the experiment,the ECG and left ventricular pressure waveform were entirely recorded using RM6240B biological signal acquisition and processing system 2.0e from before ischemia beginning to after ischemia-reperfusion being finished for 3 h.The data of HR,LVSP, LVDP,+dp/dtmax and -dp/dtmax in 30 min after the ischemia and in 10 minutes,30 min,60 min,120 min,180 min after the reperfusion were compared with before ischemia,then the recovery rate of the HR,LVSP-LVDP,+dp/dtmax and -dp/dtmax at each time point were calculated.The date of recovery rate was analyzed by a variance analysis model of repeated measures.
6.The determination of serumal NO content and myocardial NOS activity
The preparation of serum and myocardial specimens for NO and NOS detections were defined as method 4.The serumal NO content was detected by chemic method with the nitric oxide detection kit,which was purchased from Nanjing Jiancheng Bioengineering Institute.The activities of each type NOS in myocardial tissue were detected with the nitric oxide synthase detection kit(typing),which was purchased from Nanjing Jiancheng Bioengineering Institute. The data was analyzed by the one-way ANOVA of the multi-groups.
7.The observation of the cardiomyocyte apoptosis
The cardiomyocyte apoptosis was detected with in situ cell death detection kit,which was purchased from Germany Roche Diagnostics Mannheim GmbH Company.Myocardial tissue specimens were prepared to TUNEL paraffin sections strictly in accordance with the manual method.Apoptotic cells in TUNEL paraffin sections were counted by a fluorescence microscopy, and apoptosis index was calculated.The data was analyzed by the one-way ANOVA of the multi-groups.
The preparation of myocardial detection specimens was defined as method 4.The myocardial Caspase-3 activity was detected with Caspase-3 activity detection kit,which was purchased from American Biological Company.The ratios of Caspase-3 activity in each group were calculated.The data was analyzed by the one-way ANOVA of the multi-groups.
8.The establishment of the rat heart ischemia-reperfusion in vitro model and the definement of the drug intervention methods in Genistein pharmacological postconditioning
Healthy Wistar male rats weighed 240-260 g were selected to establish the ischemia reperfusion in vitro model by Langendorff isolated heart perfusion technology according to the method in literature.After blocking the left anterior descending coronary artery reversibly,when electrocardiogram showed ST-segment elevation≥0.15 mV,we thought ischemia model finished. Using estrogen receptor antagonist ICI 182,780 and tyrosine phosphatase inhibitor vanadate as tools drug,the area of myocardial infarction,activities of myocardial enzyme and heart function indexes in isolated rat myocardium were observed.The model rats was divided into such seven groups as sham group,ischemia-reperfusion group,Genistein group,ICI 182780 group,vanadate group,Genistein + ICI 182780 group and Genistein + vanadate groups.The fusion fluid containing drug was used to intervene at 1 min before reperfusion until 9 min after reperfusion,a total of 10 min intervention,and the different drug intervention groups were established.
Result:
1.The damage effect of regional ischemia-reperfusion on rat hearts in vivo
The percentage of myocardial infarct size to the ischemic area at risk,the cardiac troponin I levels of serumal samples,the MDA levels of myocardial tissue samples and the activity of SOD of myocardial tissue samples of the ischemia-reperfusion group were 47.25±1.89%,0.72±0.06 ng/ml,2.42±0.15 nmol/mgprot and 23.38±1.67 U/mgprot,respectively,and the dates of the sham group were 4.36±0.77%,0.22±0.01 ng/ml,1.27±0.10 nmol/mgprot and 44.21±1.38 U/mgprot.The ischemia-reperfusion group significantly increased the area of myocardial infarction,cardiac troponin I and MDA levels,and reduced SOD activity than the sham group. These results indicated that ischemia-reperfusion had obvious injury effect on rat in vivo heart, and it also proved that we were successful in establishing the myocardial ischemia-reperfusion in vivo model.
2.The effect of Genistein pharmacological postconditioning on myocardial infarction size
The percentage of myocardial infarct size to the ischemic area at risk of Genistein 0.1 mg/kg group,0.3 mg/kg group,1.0 mg/kg group,2.0 mg/kg group and 5.0 mg/kg group were respectively 20.37±2.05%,16.93±2.26%,8.76±2.17%,28.46±2.34%and 40.75±1.90%.They were significantly reduced myocardial infarction area than the ischemia-reperfusion group, which was 47.25±1.89%.In 0.1～1.0 mg/kg group,there were a dose-dependent decrease of myocardial infarction area,but in 1.0～5.0 mg/kg group,it showed a dose-dependent increase.
3.The effect of Genistein pharmacological postconditioning on the pathomorphological and ultrastructural changes of myocardial tissue
By the optical microscopy and TEM,the pathomorphology and ultrastructure of the myocardial tissue after ischemia or reperfusion for 30 minutes and 3 hours were observed.By the optical microscopy,the myocardial tissue showed extensive necrosis and severe muscle fiber fracture,myocardial cells dissolution and disappearance,neutrophils exudation,and so on.Under the TEM,it showed myofibrils disarrangement,large areas of myofibrils fracture,amalgamation, disappearance,sarcomere structure unclear,myocardial nuclear swelling,the nuclear membrane rupture,nuclear chromatin asymmetry,condensation,margination,mitochondrial morphological abnormalities and swelling,the ridge disarrangement,fracture,disappearance,forming cavity, glycogen granules between the mitochondrial vanishment,some mitochondria parceled by double membrane,and completely desquamated from the mother cells,forming apoptotic bodies. In the different dosage Genistein groups,with the difference of doses,myocardial pathomorphological and ultrastructural changes were discriminating,it was showed that the injury became gradually lighter in 0.1～1.0 mg/kg group,however,gradually more serious in 1.0～5.0 mg/kg group.
4.The effect of Genistein pharmacological postconditioning on myocardial and serumal enzyme indexes
In 0.1～5.0mg/kg dose range,the cardiac troponin I levels and MDA levels of the different dosage Genistein groups were significantly less than ischemia-reperfusion group,and the activity of SOD of myocardial tissue samples were significantly higher than ischemia-reperfusion group. In 0.1～1.0 mg/kg group,there were a dose-dependent decrease of the cardiac troponin I levels and MDA levels,and a dose-dependent increase of the SOD activity,but in 1.0-5.0 mg/kg group, it was the opposite.
5.The effect of Genistein pharmacological postconditioning on the heart function indexes
The recovery rates of the HR,LVSP-LVDP,+dp/dtmax and -dp/dtmax in Genistein 1.0 mg/kg group were respectively 100.40%,109.69%,133.28%and 116.03%,and they were significantly higher than in ischemia-reperfusion group,which were respectively 78.51%, 77.96%,80.66%and 79.32%.
6.The effect of Genistein pharmacological postconditioning on the serumal NO content and myocardial NOS activity
The NO contents detected in different doses of Genistein groups were significantly higher than in ischemia-reperfusion group,the NO content detected in Genistein 1.0 mg/kg group was the highest.The activities of T-NOS and cNOS of myocardial tissue samples detected in the different doses of Genistein groups were significantly higher than in ischemia-reperfusion group, the activity of T-NOS and cNOS detected in Genistein 1.0 mg/kg group was the highest.There were no differences in activity of iNOS detected among the groups,and the activity of iNOS was much lower than cNOS.
7.The effect of Genistein pharmacological postconditioning on the cardiomyocyte apoptosis
Ischemia-reperfusion injury of ischemia-reperfusion group was more serious,in which the apoptosis index went up to 28.58±2.37%.Apoptosis index in 0.1,1.0 and 5.0 mg/kg genistein treated groups were respectively 19.48±2.13%,10.55±1.93%and 21.49±2.34%,and the 1.0 mg/kg genistein groups treated showed the lowest apoptosis index.Apoptosis index in the different dose of Genistein treated groups were significantly lower than in ischemia-reperfusion group.
After the ratio of Caspase-3 activity in sham group was set at 1.00,the ratios of Caspase-3 activity in other groups were calculated,which increased to 2.75±0.25 in ischemia-reperfusion group and which decreased tol.51±0.15,1.28±0.10 and 1.71±0.13 in 0.1,1.0,5.0 mg/kg groups.The ratios of Caspase-3 activity in Genistein given groups were significantly lower than in ischemia-reperfusion group.
8.The intervention effect of estrogen receptor antagonist ICI 182,780 and tyrosine phosphatase inhibitor vanadate on cardioprotection against ischemia-reperfusion injury by Genistein pharmacological postconditioning
After ischemia-reperfusion,the percentage of myocardial infarct size to the ischemic area at risk of isolated rat heart in ICI 182780,vanadate group and Genistein + ICI 182780 group were respectively 48.92±1.63%,46.07±1.81%and 29.02±1.65%,and they were significantly higher than in Genistein group,which was 10.35±1.74%.It was significantly lower in Genistein + vanadate group than in Genistein given group,which only was 7.45±1.40%.The varying tendency of SOD activity in heart perfusate in each group was completely opposite to the varying tendency of myocardial infarction area.There was no significant difference in the recovery rates of the HR between the groups after reperfusion for 180 min.The recovery rates of LVSP-LVDP,+dp/dtmax and -dp/dtmax in ICI 182780 group,vanadate group and Genistein + ICI 182780 group were significantly lower than in Genistein group.The recovery rates in Genistein+ICI 182780 group was almost as high as in ischemia-reperfusion group.The recovery rates in Genistein + vanadate group was almost as high as in Genistein group.Most of the recovery rates in ICI 182780 group and vanadate group did not differ from in ischemia-reperfusion group.
Conclusion:
1.Using the heart ischemia-reperfusion model of rat in vivo,it was identified that genistein could effectively reduce myocardial ischemia-reperfusion injury by intravenous injection in early reperfusion,which was mainly manifested in the reduction of the myocardial infarction size, lower activity of cardiac troponin I and MDA,higher activity of SOD,the improvement of the recovery of the heart function,and the marked attenuation of destruction of myocardial ultrastructure,indicating genistein has cardioprotection effect against ischemia-reperfusion injury by the pharmacological postconditioning.
2.Generally,genistein showed protective effects on myocardial ischemia-reperfusion injury in 0.1～5.0 mg/kg dose range.However,this protective effect showed two opposite trends in different dose range:a dose-dependent increasing in 0.1～1.0 mg/kg dose range,while a dose-dependent weakening in 1.0～5.0 mg/kg dose range.
3.The effects of genistein on the myocardial NOS activity,serumal NO content, cardiomyocyte apoptosis index and Caspase-3 activity in ischemia-reperfusion hearts of rat, associating with application of estrogen receptor antagonist ICI 182,780 and tyrosine phosphatase inhibitor vanadate on ischemia-reperfusion heart of rat in vitro showed that the protective effect of Genistein pharmacological postconditioning related to the enhancement of myocardial NOS activity,the inhibition of cardiomyocyte apoptosis and the excitement of estrogen receptor.Its protein tyrosine kinase inhibitor property was detrimental to myocardial protection,and this may be the major reasons for a dose-dependent decrease of the protective effect in the higher dose range.
引文
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