酸枣仁皂苷A对大鼠心肌缺血再灌注的保护作用及机制研究
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摘要
一、研究背景
冠心病为内科常见病及多发病,是导致人类死亡的主要原因之一,全国每年约有110万人死于冠心病。溶栓治疗、经皮冠状动脉介入术(percutaneous coronary intervention, PCI)或冠状动脉旁路移植术(coronary artery bypass graft, CABG)是缩小急性心肌梗死梗塞范围和改善临床预后的最有效方法。但部分患者或动物心肌缺血后再灌注,使缺血所致的功能和代谢障碍及结构破坏进一步加重,这种现象称为心肌缺血再灌注损伤(myocardial ischemia/reperfusion injury, MIRI),临床表现为在闭塞的冠状动脉再通、梗死区血液灌流重建后一段时间内,有的病例发生血压骤降、心功能不全、心律失常甚至猝死等病情恶化的现象。因此,MIRI的发生机制与防治越来越引起人们的关注,并一直试图寻找能对MIRI产生确切保护作用的药物。
心肌缺血再灌注损伤的发生机制十分复杂,而细胞调亡是心肌缺血再灌注损伤发生的重要机制之一。与细胞坏死过程不同,细胞凋亡又称程序性细胞死亡是指有核细胞在一定条件下通过启动其内部机制,主要通过内源性DNA内切酶的激活而发生的细胞自然死亡过程。细胞凋亡在心肌缺血再灌注损伤中的发生可能与以下因素有关：①活性氧大量生成：再灌注导致大量氧自由基释放,造成生物膜脂质过氧化、各种酶蛋白失活及DNA损伤；②细胞内钙超载：心肌缺血再灌注过程中,线粒体内Ca2+大量聚集,引起线粒体内膜上的通透性转换孔开放,释放细胞色素C入胞质内,进一步激活半胱氨酸天冬氨酸蛋白酶诱致细胞凋亡。③线粒体损伤：在细胞凋亡期间,尽管线粒体能维持其超微结构的正常,但事实上其功能已经发生显著改变,如线粒体内膜通透性增大,线粒体内膜的跨膜电位下降,能量合成水平显著降低。上述因素并不能直接引起细胞凋亡,而是通过一定的信号传递方式激活生存或死亡相关基因,然后将信号传递到核内切酶,而执行死亡的功能。因此阻断心肌缺血再灌注损伤中心肌细胞凋亡的信号转导能够阻止凋亡程序的执行,减少心肌细胞凋亡,防治心肌缺血再灌注损伤。
酸枣仁是鼠李科植物酸枣(Ziziphus psinosa Hu)的干燥成熟种子,具有敛汗,养肝,宁心安神等多种功效。酸枣仁主要含有多种酸枣仁皂苷类等化合物。酸枣仁皂苷是酸枣仁的水溶性成分,酸枣仁皂苷A是其中的主要有效成份之一。研究发现,酸枣仁提取物具有调脂、降压、抗动脉粥样硬化、抗心律失常、抗心肌缺血、抗心肌细胞缺氧-复氧损伤作用。酸枣仁皂甙A具有抑制血管平滑肌细胞过度增殖而抗动脉粥样硬化,同时对脑缺血再灌注损伤导致的细胞凋亡有明显的抑制作用,但是目前对于其对心肌缺血再灌注损伤的保护作用及机制尚未报道。
二、研究方法
目的：
在整体水平,我们采用建立大鼠在体心肌缺血再灌注模型模拟急性心肌梗死后再灌注损伤的病理生理变化,酸枣仁皂苷A于造模前腹腔注射给药干预,旨在观察其抗细胞凋亡、保护心肌等作用,并进一步探明其作用机制(主要是其对凋亡相关蛋白Bax、Bcl-、Caspase-3和CytC的影响)；在细胞水平,采用过氧化氢损伤原代大鼠心肌细胞建立氧化损伤模型,酸枣仁皂苷A预处理后观察其对心肌细胞形态及细胞凋亡的影响,探明其抑制细胞凋亡的作用机制(主要是与PKCε信号转导通路的关系),为临床上有效防治心肌缺血再灌注损伤提供新思路。本课题分三个部分进行：第一部分酸枣仁皂苷A预处理对大鼠缺血再灌注是否有保护作用,重点观察其抗氧化作用及其对心肌组织结构、心肌损伤标志物、再灌注心律失常及心功能的影响；第二部分酸枣仁皂苷A预处理对缺血再灌注心肌细胞凋亡的影响,从蛋白质水平观察酸枣仁皂苷A的抗凋亡作用,并探求其作用机制；第三部分观察酸枣仁皂苷A对过氧化氢诱导新生大鼠心肌细胞凋亡及PKCε表达的影响,进一步明确其作用机制。
研究方法：
第一部分：采用结扎(15 min)和松开(30 min)左冠状动脉前降支(LAD)的方法制备心肌缺血再灌注损伤模型。44只雄性wistar大鼠随机分为4组,分别为假手术组(n=8)、模型组(n=12)、酸枣仁皂甙A组(n=12)及表没食子儿茶素没食子酸酯组(n=12)。酸枣仁皂苷A组及EGCG组的给药剂量均为20mg·kg-1·d-1,假手术组和模型组给予等体积的生理盐水,均为腹腔注射,连续用药3d,末次给药30min后进行造模。Ⅱ导联心电图观察再灌注30min期间心律失常的发生情况,根据Roringerova T方法进行室性心律失常(ventricular arrhythmias, VA)评分,Medlab6.0生物信号采集处理系统监测左心室收缩压(LVSP)、左心室舒张末压(LVEDP)、收缩期左心室内压上升的最大变化速率(+dp/dtmax)、舒张期左心室内压下降的最大变化速率(-dp/dtmax)分别在缺血前、缺血15min、再灌注15、30min时的变化。标本收集：实验结束时腹主动脉采血,同时在心底部将心脏与大血管及组织离断,取出心脏,去除左右心房及右心室,心肌置入冰生理盐水中冲洗3次,将左心室分离为三部分,一部分用精密电子天平称取记录质量,剪碎匀浆,作为SOD、MDA检测用,一部分放入4%多聚甲醛中固定,作为组织切片备用,以观察心肌组织病理结构,另一部分放入-80℃冰箱中保存备；血液室温静放30min,在4℃离心后,取其上清置-80℃冰箱保存,检验肌酸磷酸激酶(CK)及乳酸脱氢酶(LDH)水平。
第二部分：40只雄性wistar大鼠随机分为4组,每组10只,分别为假手术组、模型组、酸枣仁皂甙A组及表没食子儿茶素没食子酸酯组,其余同第(?)部分。以末端脱氧核昔酸转移酶(TdT酶)介导的脱氧三磷酸尿昔(dUTP)缺口末端标记法(TdT-mediated du Tpniekend labeling, TUNEL)检测凋亡心肌细胞,计算细胞凋亡指数(apoptotic index, AI);取出-80℃冰箱中保存的部分心肌,提取蛋白及定量后,采用蛋白印迹法检测Bax、CytC及Bcl-2表达,采用分光光度法检测caspase-3的活性。
第三部分：采用差速贴壁法分离培养Wistar新生乳鼠心肌细胞,以过氧化氢作用心肌细胞建立氧化损伤模型,将培养72 h的原代心肌细胞,选择搏动良好、生长密度无明显差异的细胞按孔随机分为以下4组：①对照组：细胞培养过程中不给予任何处理；②模型组：心肌细胞培养基中加终浓度为500μmol/L过氧化氢作用4h；③酸枣仁皂苷A治疗组：操作同模型组,但在给予H202前加入酸枣仁皂苷A至终浓度为20mg/l干预24小时；④PKC抑制剂组(JA+Chelerythrine):操作同治疗组,但在给予H202前5min加入Chelerythrine至终浓度5umol/L。倒置显微镜观察心肌细胞形态学和搏动频率的变化；四唑盐(MTT)比色法检测心肌细胞活力；流式细胞仪检测细胞凋亡率；采用分光光度法检测caspase-3的活性；蛋白印迹检测CtyC及PKCε表达。统计分析：实验数据采用SPSS13.0统计软件进行分析。计量资料数据以x±s表示。对数据进行方差齐性检验。多组数据间比较采用单因素方差分析处理,方差齐性时两两比较采用LSD法；方差不齐时,多组数据间比较采用Welch法,两两比较采用Games-Howell法。各实验组缺血前及缺血再灌注不同时间点心功能指标采用重复测量方差分析。检验显著性水准α=0.05。
结果：
第一部分：
1、大鼠心肌组织病理形态学HE染色光镜下观察显示,假手术组大鼠心肌细胞形态规则、排列整齐,肌纤维完整,未见心肌细胞水肿、变性、坏死,未见中性粒细胞浸润。模型组心肌细胞排列紊乱,局部肌纤维横纹消失,肿胀明显,较多的中性粒细胞浸润。酸枣仁皂苷A治疗组可见心肌细胞水肿及中性粒细胞浸润,但都较模型组轻,心肌受损程度有所下降；EGCG组心肌组织病理形态学变化与酸枣仁皂苷A治疗组相似。
2、缺血再灌注对大鼠LDH、CK、SOD及MDA的影响再灌注结束后检测各组LDH、CK、SOD及MDA值。结果显示,I/R组、JA组、EGCG组的LDH及CK值均较Sham组明显增高(P<0.01)；各组SOD值均显著降低,与Sham组比较有显著性差异(P<0.01)；各组MDA值均显著增高,与Sham组相比有显著性差异(P<0.01)。表明缺血再灌注可造成心肌严重损伤,证实心肌缺血再灌注模型构建成功。
3、酸枣仁皂甙A对缺血再灌注大鼠LDH、CK、SOD及MDA的影响经统计学检验,各组间LDH、CK、SOD及MDA的水平均有显著差异(LDH:F=69.104, P=0.000; CK:F=143.320, P=0.000; SOD:F=101.844, P=0:000; MDA:F=212.780,P=0.000)。JA和EGCG均可明显抑制再灌注所导致的LDH、CK、MDA的升高,同时显著增加SOD的活性,与I/R组相比有显著性差异(P<0.01)；上述指标在JA组与EGCG组间无统计学差异(P>0.05),表明JA和EGCG均具有抗氧化作用而减轻心肌缺血再灌注损伤。
4、再灌注心律失常44只大鼠中12只被排除,剩余32只中26只发生心律失常,心律失常多出现于再灌注早期,包括室性早博、室性心动过速和心室纤颤等；假手术组仅有偶发室性早搏,未见其他类型心律失常。模型组均出现严重的心律失常,以频繁室速、室颤为主,伴有少量的房室传导阻滞,其中2只大鼠因为室颤而死亡。经统计学检验,各组间心律失常评分差异显著(F=44.699,P=0.000)。与假手术组比较,模型组心律失常评分显著上升,差异具有统计学意义(P=0.000)。酸枣仁皂苷A治疗组也出现了心律失常,以室早及室速为主,与模型组比较心律失常评分显著下降(P=0.005)。EGCG组的心律失常以室早及阵发室速为主,与酸枣仁皂苷A组比较,心律失常评分无显著性差异(P=0.080)。5、心功能指标各组缺血前的LVSP、LVEDP和±dp/dtmax差异无统计学意义(P>0.05)。缺血再灌注时各组间大鼠左室收缩末压(LVSP)由高到低依次为Sham组、EGCG组、酸枣仁皂苷A组及I/R组；等容收缩期左心室最大上升速率(+dp/dtmax)及等容舒张期左心室下降最大速率(-dp/dtmax)由高到低依次为Sham组、酸枣仁皂苷A组、EGCG组及I/R组；左室舒张末压(LVEDP)由低到高依次为Sham组、EGCG组、酸枣仁皂苷A组及I/R组。经统计学分析,缺血再灌注时个组间LVSP. +dp/dtmax、-dp/dtmax、LVEDP差异均有统计学意义(P<0.01)。不同缺血再灌注时间点检测结果表明,LVSP、+dp/dtmax、-dp/dtmax随着时间延长而降低,LVEDP随着时间延长而升高,差异有统计学意义(P<0.01)。各时间点与组别间有交互作用(P<0.01)。以上结果表明缺血再灌注可导致心脏收缩及舒张功能受损,酸枣仁皂苷A可改善大鼠收缩及舒张功能。
第二部分：
1、各组心肌组织TUNEL结果按试剂盒提供的脱氧核苷酸转移酶介导的缺口末端标记法原位检测凋亡细胞。正常心肌细胞核呈蓝色；凋亡心肌细胞核呈深浅不一的棕褐色。经统计学检验,各组间AI差异显著(F=198.001,P=0.000). I/R组AI为(18.14±2.22)%,JA和EGCG预处理后AI显著下降(P<0.01)；与Sham组比较,JA组和EGCG组中AI显著升高,差异具有统计学意义(P<0.01)。
2.Western bloting检测各组大鼠心肌Bcl-2、Bax及细胞色素C的表达通过分析目的条带的光密度值(IOD)发现,各组间Bcl-2、Bax、Bcl-2/Bax及细胞色素C的表达均有显著差异(Bcl-2:F=222.463,P=0.000；Bax:F=82.507,P=0.000；Bcl-2/Bax:F=231.350,P=0.000；细胞色素C：F=96.916,P=0.000)。在Sham组中心肌组织有一定量的Bcl-2及Bax表达,胞浆中细胞色素C的含量极少,I/R组心肌Bcl-2蛋白表达显著减少(P=0.000),而Bax及胞浆中细胞色素C蛋白表达显著增加,Bcl-2/Bax比值显著降低(P<0.01)；JA组和EGCG组中Bax及胞浆中细胞色素C表达较I/R组显著下降(P<0.01),但Bcl-2的表达较I/R组显著升高(P<0.01), Bcl-2/Bax比值显著高于I/R组(P<0.01)。JA组与EGCG组中的Bcl-2、Bax、胞浆中CytC表达及Bcl-2/Bax比值无显著性差异(P>0.05)；此两组中Bax表达与Sham组比较无显著性差异(P>0.05),Bcl-2、胞浆中CytC表达及Bcl-2/Bax比值与Sham组比较有显著性差异(P<0.01)。3、各组caspase-3活性检测结果caspase-3活性测定显示,各组间caspase-3活性差异显著(F=39.663,P=0.000)。正常心肌细胞的caspase-3活性水平很低,在体缺血15分钟,再灌注30分钟后心肌caspase-3的活性显著升高(P=0.000)；经过JA及EGCG预处理后心肌caspase-3的活性显著减少(P<0.01),但JA组与EGCG组的caspase-3活性间无显著性差异(P=0.817)。
第三部分：
1、各组心肌细胞形态学观察倒置显微镜下观察,正常心肌细胞从72h起细胞成单层成簇生长,心肌细胞核折光性好,伪足饱满并相互连接,生长呈半融合状态,搏动明显,节律一致,60～80次/min；模型组细胞核暗淡,伪足显著变细,细胞间连接明显减少,搏动节律减慢,20～30次/min；与模型组比较,酸枣仁皂苷A治疗组心肌细胞形态明显改善,细胞核折光性较好,伪足略变细,搏动有力,节律一致,50～60次/min；与酸枣仁皂苷A治疗组比较,P.KC抑制剂组心肌细胞细胞核折光性较差,伪足变细,细胞间连接减少,搏动节律减慢,30～40次/min。
2、各组心肌细胞活力与细胞凋亡的结果经统计学检验,各组间心肌细胞活力、细胞凋亡差异显著(心肌细胞活力：F=87.752,P=0.000；细胞凋亡：F=657.061,P=0.000)。与对照组比较,模型组心肌细胞活力显著降低(P=0.000),细胞凋亡率则显著增加(P=0.000)；与模型组比较,JA组心肌细胞活力显著增加(P=0.000),细胞凋亡率则显著降低(P=0.000)；与JA组比较,PKC抑制剂组心肌细胞活力显著降低(P=0.008),细胞凋亡率则显著增加(P=0.000)。
3、Western blot检测各组心肌细胞CtyC及PKCε的表达情况经统计学检验,各组间CtyC及PKCε的表达差异显著(CtyC:F=210.511,P=0.000; PKCε: F=42.589, P=0.000)。与对照组比较,模型组胞浆中细胞色素C显著增多(P=0.000), PKCε表达无显著性差异(P=0.235)；JA预处理后,细胞色素C表达显著减少(P=0.000), PKCε表达显著升高(P=0.000)；与JA组比较,PKC抑制剂组胞浆中细胞色素C显表达显著增多(P=0.000), PKCε表达显著降低(P=0.000)。
4、各组caspase-3活性检测经统计学检验,各组间caspase-3活性差异显著(F=663.629,P=0.000)。与对照组比较,模型组Caspase-3的活性显著增加(P=0.000)；与模型组比较,JA组的Caspase-3活性显著降低(P=0.000); Chelerythrin干预后,Caspase-3活性显著升高(P=0.000)。
结论：
1、本实验通过在体大鼠心肌缺血/再灌注模型,证实了心肌缺血/再灌注可导致心肌损伤,酸枣仁皂苷A预处理可以减轻心肌的损伤,降低LDH、CK、MDA值,升高SOD值,减少再灌注心律失常,改善心功能。
2.心肌缺血/再灌注和过氧化氢损伤可以引起心肌细胞凋亡,酸枣仁皂苷A预处理可以减少心肌细胞凋亡。因此酸枣仁皂苷A可减轻心肌缺血再灌注损伤,与其抑制心肌细胞凋亡有关。
3.酸枣仁皂苷A预处理可以上调Bcl-2、下调Bax蛋白表达,升高Bcl-2/Bax比值,抑制细胞色素C从线粒体中的释放,减少caspase-3在心肌组织中活性从而减少心肌细胞凋亡。
4.PKC是预适应机制的重要环节,酸枣仁皂苷A预处理可增强心肌细胞膜PKCε的蛋白表达,经Chelerythrine处理后,酸枣仁皂苷A预处理的保护作用显著减弱,因此PKCε的信号转导途径是酸枣仁皂苷A抑制心肌细胞凋亡的关键环节。
1、Background
Coronary heart disease(CHD)is a kind of common and frequently-occurring disease. CHD is the single-most important cause of death worldwhile, and about 1.1 million people die of the disease each year in china. After an acute myocardial infarction,with the use of thrombolytic therapy or primary percutaneous coronary intervention(PCI) or coronary artery bypass graft(CABG) is the most effective strategy reducing the size of myocardial infarction and improving clinical outcome.But in some animals or patients,we can find that the myocardial cell dysbolism and structural damage became more serious after ischemia-reperfusion.The phenomenon was called myocardial ischemia- reperfusion injury(MIRI). In clinical situations, reperfusion following ischemia result in a sharp drop in blood pressure,heart failure,arrhythmia and even sudden death which worsening pathogenetic condition. Therefore, people show greater concern for mechanisms and control of myocardial ischemia-reperfusion injury, and always try to find out the drugs which have exactly protective effects to myocardial ischemia-reperfusion injury.
The causing mechanisms of myocardial ischemia-reperfusion injury are very complicated, and cell apoptosis is one of the important mechanisms of myocardial ischemia-reperfusion injury. Apoptosis is distinguished from necrosis,apoptosis is the process of programmed cell death in which nucleated cells occur the process of natural cell death on condition that through activation of an internally suicide program,mainly through activation of endogeneity DNA endonuclease.Apoptosis occurs in ischemia-reperfusion injury, which may be concerned with the following factors:①lots of reactive oxygen species generation:a burst of Oxygen free radicals generation occur during myocardial reperfusion, Oxygen free radicals generation results in membrane lipid peroxidation、inactivation to kinds of apoenzymes and damage to DNA.②intracellular calcium overload:lots of calcium concentrates in mitochondrial membrane during the process of myocardial ischemia-reperfusion injury, mitochondrial calcium overload results in the opening of mitochondrial permeability transition pore, releases of cytochrome c, and caspase activation leading to myocardial apoptosis.③mitochondria damage:mitochondria still can maintain normal ultrastructure during the process of apoptosis, but its function has been changed significantly, such as increase of inner mitochondrial membrane permeability, decrease of mitochondrial transmembrane potential and the levels of energy production decreased significantly. But these factors can not induce cell apoptosis dircetly,they activate survival or death gene by signal transmission,then the signal transmit to the incision enzyme intranuclear to perform the function of death.To block the signal transduction of cardiomyocyte apoptosis induced by myocardial ischemia-reperfusion may inhibit cardiomyocyte apoptosis, Prevent or cure myocardial ischemia-reperfusion injury.
Semen Ziziphi Spinosae is the dried ripe seed of Ziziphu spsinosa Hu, It has the efficacies of constraining sweat, protecting liver, quieting the heart, inducing sleep,and so on.Semen Ziziphi Spinosae mainly contains kinds of jujubosides.Jujubosides are water-soluble constituents of Semen Ziziphi Spinosae,and jujuboside A is one of main effective components. Studies had found that Spine Date Seed PE could regulate blood lipid, lower blood pressure,anti-atherosclerosis, anti-arrhythmia,anti-myocardial ischemia,and,relieve cardiomyocyte following hypoxia/reoxy-genation injury.The others studies had confirmed jujuboside A could anti-atherosclerosis by means of inhibiting overproliferation of vascular smooth muscle cells, and it also could inhibit cell apotosis significantly during the processof cerebral ischemia-reperfusion injury, but now there is no Study on protective effects and mechanisms of jujuboside A on myocardial ischemia reperfusion injury.
2、Methods:
Objective:
In vivo we set up myocardial ischemia-reperfusion injury models in rats simulate the pathophysiologic changes in myocardial ischemia-reperfusion injury, and jujuboside A was administrated by intraperitonea injection before myocardial ischemia. The aim of this study is to demonstrate if jujuboside A can inhibit apoptosis and protect myocardium from ischemia-reperfusion injury, discuss the mechanism involved,chiefly find out the effects of preconditioning with jujuboside A on apoptosis related protein Bax,Bcl-2,Caspase-3 and CytC; In cellular level we set up oxidative injury models by damage to neonate rat myocardial cell with hydrogen peroxide, The aim of this study is to observe the effects of preconditioning with jujuboside A on cardiomyocytes morphology and apoptosis, discuss the mechanism involved, chiefly find out the relation between the protection and PKCs pathway, and find new evidence to protect myocardium from ischemia-reperfusion injury. Our study includes three parts:The first part is to find out if jujuboside A can protect myocardium from ischemia-reperfusion injury when it was administrated by intraperitonea injection before myocardial ischemia,especially to explore the effect of jujuboside A on the change of myocardial tissue、the marker of myocardial damage、the use of antioxidatio、the occurrence of arrhythmia after reperfusion and heart function. The second part is to demonstrate the effects of preconditioning with jujuboside A on cardiomyocytes apoptosis in protein,discuss the mechanism involved;The third part is to demonstrate the effects of preconditioning with jujuboside A on neonate rat myocardial cell apoptosis induced by hydrogen peroxide and the experssion of PKCs,identify the mechanism involved.
Methods:
The first part of this study:The left anterior descending (LAD) coronary artery was occluded for 15min followed by reperfusion for 30min to establish myocardial ischemia-reperfusion injury model.44 androgenic wistar rats were randomly divided into four groups:the Sham group (n=8), the I/R control group(n= 12), the jujuboside A group (n=12)and the EGCG group (n=12). Rats of the jujuboside A group and the EGCG group were given drug at a dose of 20mg/kg for three consecutive days(intraperitoneal injection), rats of the Sham group and the I/R control group were given physiological saline of the same volume for three consecutive days (intraperitoneal injection),the operation was conducted 30 min after the last intraperitoneal injection in rats of each group. We observed the occurrence of arrhythmias in electrocardiogram of lead II during myocardial reperfusion for 30min, and scored the ventricular arrhythmias (VA)acceording to the method of Ravingerova T. Through the medlab 6.0 biological signal collecting and processing system, the left ventricle systolic pressure(LVSP)、end-diastolic (LVEDP) pressure and the max rate of rise and decline(±dp/dtmax) were measured at the time of before ischemia, ischemia 15min, reperfusion 15min and reperfusion 30min respectivly. Specimen collection:After the experiment had been done, we drew blood from the abdominal aortic vascular of rats, mutilated the great vessels and tissues from the cardiac base, took out of the heart, removed the atriums and the right ventricles, washed the hearts by cold physiological saline for three times, cut the heart to take the damaged cardiae muscles according to the need of detection, divided the left ventricle in three parts.the first part was weighed by electronic precision balance,enoughly cut and then homogenized about8 stroke with a glass Dounce homogenizer, measured the contents of MDA and SOD in heart;the second part was fixed with 4% polysorbate; the third part was keptin icebox at 80℃below zero. The blood was stood for 30min at room temperature, then centrifugated at 4℃.the serum was kept in icebox at 80℃below zero, measured the levels of CK and LDH.
The second part of this study:40 androgenic wistar rats were randomly divided into four groups:the Sham group (n=10), the I/R control group(n=10),the jujuboside A group (n=10)and the EGCG group (n=10), The rest is the same as the first part. The apoptotic cardiomyocytes were detected by TUNEL staining, counted apoptotic index.The expression of Bcl-2 and Bax protein were measured by western blotting,cytochrome C in the cytoplasm were detected by western blotting, the activity of caspase-3 was measured by spectrophotometry in each group.The results were compared among the groups.
The third part of this study:Primary cultures of cardiac myocytes were prepared from ventricles of 1～3 day new born witar rats and cultured for 72 hours. we set up oxidative injury models by damage to neonate rat myocardial cell with hydrogen peroxide.the neonate rat myocardial cells were divided as follows:①Control group:There was not any treatment in the cultural course of neonate rat myocardial cells.②Model group:The model group was treatment with the final concentration of 500μmol/L hydrogen peroxide in the cell culture fluid in 4 hours. ③Jujuboside A treatment group:The treatment was similar to that of the model group,but dealed with the final concentration of 20mg/L jujuboside A in the cell culture fluid in 24 hours before adding hydrogen peroxide.④PKC inhibitor group
(JA+Chelerythrine), The treatment was similar to that of the jujuboside A treatment group,but dealed with the final concentration of 5umol/L chelerythrine in the cell culture fluid in 5min before adding hydrogen peroxide. Morphology and the beating rate change of primary cultured neonatal rat myocardial cell were observed under the inverted microscope; viability of myocardial cell was assayed by MTT; Apoptosis rate was determined by flow cytometric analysis. The activity of caspase-3 in myocardial cell was measured by spectrophotometry,and Western blotting was used to analyze expression of cytochrome C of the cytoplasm and expression of PKCs of the cytomembrane in each group.The results were compared among the groups.
Statistiealanalysis:The data was analyzed by SPSS13.0.measurement data was presented as the means±SEM.Test of homogeneity of variance was done in each group.If datas accorded with homogeneity of variance statistical analysis was made by one-way ANOVA followed by LSD test, otherwise statistical analysis was made by rank sum test, and multiple comparison was made by Games-Howell. Heart function parameters in different time points was analyzed by repetitive measurement and analysis of varianee.Level of signifieance a=0.05.
Results:
The first part of this study:
1、Histopathologic observation of the myocardium in rats:Pathologic changes of myocardial tissue were observed under hematoxylin-eosine staining(HE).In sham group there was no detectable histopathological change. Rats in the I/R control group displayed a serious degree of myocardial neutrophilic infiltrate, necrosis,hemorrhage, and spindle-shaped interstitial cells as compared with rats in the sham group. pathologic changes of cardiac tissue in the JA and EGCG group were significantly milder than that of the I/R group.
2、The effects of myocardial ischemia-reperfusion on the levels of LDH、CK、SOD and MDA in rats:Compared with Sham group,I/R group、JA group and EGCG group markedly increased the activity of LDH and CK in serum,the content of MDA in myocardium,but significantly decreased the activity of SOD in myocardium (P< 0.01). The results indicated that ischemia-reperfusion could result in serious injury of myocardium, and confirmed that myocardial ischemia-reperfusion injury model was established successfuly.
3、The effects of JA on the levels of LDH、CK、SOD and MDA of myocardial ischemia-reperfusion in rats:Compared with I/R group, JA group and EGCG group markedly decreased the activity of LDH、CK in serum and the content of MDA in myocardium,but significantly increased the activity of SOD in myocardium (P<0.01).The values of MDA,CK,LDH and SOD in JA and EGCG groups had no significant difference(P>0.05).The results indicated that effects of JA and EGCG antioxidation decreased the injury of ischemia/reperfusion of myocardium.
4、Reperfusion arrhythmia:Twelve rats were excluded from entry to this study,arrhythmia was found in 26 rats,arrhythmia occurred mainly at the early period of reperfusion. The arrhythmia appeared in rats inclding ventricular premature beat、ventricular tachycardia(VT)、ventricular fibrillation(VF),and so on.Only accidental ventricular premature beat appeared in rats of Sham group. The serious arrhythmia appeared in rats of I/R group,remained frequent ventricular tachycardia(VT)、frequent ventricular fibrillation(VF),and accompanied a small quantity of atrioventricular block,there were 2 dead rats in I/R group,most of arrhythmia occurred in reperfusion. Arrhythmia score in I/R group was more than that in sham group (P=0.000).The arrhythmia also appeared in rats of JA group mainly inclding ventricular premature beat and ventricular tachycardia(VT), and Arrhythmia score in the group was lower than that in I/R group (P=0.005).The arrhythmia appeared in rats of EGCG group mainly inclding ventricular premature beat and paroxysmal ventricular tachycardia(VT),Compared with JA group,There was no difference between them (P=0.080).
5、Index of cardiac function:No significant difference of LVSP、LVEDP and±dp/dtmax were found in all groups of preischemic (P>0.05). LVSP was significantly decreased in the sequence of Sham group, EGCG group, JA group and I/R group,±dp/dtmax was significantly decreased in the sequence of Sham group, JA group, EGCG group and I/R group, while LVEDP was significantly increased in the sequence of Sham group, EGCG group, JA group and I/R group.The results indicated that ischemia-reperfusion could lead toheart malfunction,and pretreatmenting with jujuboside A can improve the heart function.
The second part of this study:
1、Myocardial TUNEL staining:The apoptotic cardiomyocytes were detected by Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) assay.The normal cardiomyocytic nucleus is blue, The nucleus of apoptotic cardiomyocyte is brown.AI of I/R group was (18.14±2.22)%,after pretreatmenting with jujuboside A and EGCG, AI was significantly lower than that of I/R group (P=0.000).Compared with sham group, AI of JA group and EGCG group were significantly raised (P<0.01)
2、Bcl-2、Bax and CytC protein expression:According to the densitometry of bands under the CCD imagine system, a certain amount of Bcl-2 and Bax were expressed in myocardium of sham group, the cytochrome C expression in cytoplasm was little. Compared with the sham group,the expression of Bcl-2 and the rate of Bcl-2/Bax decreased in the I/R group, the expression of Bax and the cytochrome C expression in cytoplasm increased with significant difference (P<0.01). Compared with the I/R group,the expression of Bcl-2 and the rate of Bcl-2/Bax increased in the JA group and EGCG, the expression of Bax and the cytochrome C expression in cytoplasm decreased with significant difference (P<0.01).The expression of Bcl-2、Bax、cytochrome C and the rate of Bcl-2/Bax were no significant difference between JA group and EGCG group(P>0.05), and there was no a significant difference of the Bax expression between the two groups and sham group (P>0.05), but The expression of Bcl-2、cytochrome C and the rate of Bcl-2/Bax were significant difference between the two groups and sham group(P<0.01).
3、The results of Caspase-3 Activity:The levels ofcaspase-3 activity in normal cardiomyocyte were very low, the levels of caspase-3 activity in I/R group were increased significantly, which was decreased significantly in JA and EGCG groups(P <0.01). The caspase-3 activity was no significant difference between JA group and EGCG group (P=0.817).
The third part of this study:
1、Morphological characters of cellular:Cell morphology was observed under reverse microscope.From 72 hours on,the normal cardiomyocytes grew in clusters,the reflected light of nucleus was good,and the pseudopodia was plump, connected reciprocally. In addition, cell beating intension of cardiac cells was apparent,the rhythm was 60-80 beats per minute.Most of cells in H2O2 group were crenulated, the nucleus were dim, the pseudopodia were became thin significantly, the rhythm of cell beating was decreased significantly, the.frequency was 20～30 beats per minute. Compared with the I/R group, the morphological characters of JA group was improved significantly.the morphological characters of PKC inhibitor group was worse that of JA group.
2、The results of viability and apoptosis rate in myocardial cell:Compared with the control group, the viability in myocardial cell of model group was decreased significantly,and the apoptosis rate was increased significantly (P<0.01).Compared with the model group, the viability in myocardial cell of JA group was increased significantly, and the apoptosis rate was decreased significantly (P<0.01). Compared with the JA group, the viability in myocardial cell of PKC inhibitor group was decreased significantly, and the apoptosis rate was increased significantly (P<0.01,).
3、Protein expression of CytC and PKCs were measured by Western blot analysis: Compared with the control group, the expression of cytochrome C in the cytosol of model group was increased significantly (P=0.000), and the expression of PKCεin the membrane was no significant difference between them (P=0.235).Treatments with jujuboside A (20 mg/L) significantly decreased the expression of cytochrome C in the cytosol, and significantly increased the expression of PKCεin the membrane(P <0.01). Compared with the jujuboside A group, the expression of cytochrome c in the cytosol of PKC inhibitor group was increased significantly, and the expression of PKCεin the membrane was decreased significantly (P< 0.01).
4、The results of Caspase-3 Activity in cardiomyocyte:Compared with the control group,the Caspase-3 Activity of model group was increased significantly (P=0.000). Compared with the model group,the caspase-3 activity of jujuboside A group was decreased significantly (P=0.000).Treatments with Chelerythrin significantly increased the caspase-3 activity (P=0.000).
Conclusion:
1、The experiment has proved that myocardial ischemia/reperfusion could lead to myocardial injury by an in vivo myocardial ischemic reperfusion model in rats. Pretreatment with jujuboside A could decrease the injury of ischemia/reperfusion of myocardium,decrease the levels of LDH、CK and MDA, increase the activity of SOD, decrease the reperfusion arrhythmia and improve cardiac function.
2、Myocardial ischemia/reperfusion and hydrogen peroxide injury could result in cardiomyocyte apoptosis,pretreatment with jujuboside A could decrease cardiomyocyte apoptosis.Pretreatment with jujuboside A can relieve the ischemic reperfusion injury,the mechanism of which may be related with the inhibited myocardial apoptosis.
3、The protective effect of preconditioning with jujuboside A was probably achieved through decreasing myocardium apoptosis,and modulating expression of Bcl-2 and Bax,increasing the ratios of Bcl-2/Bax,inhibiting the release of cytochrome C and decreasing the activity of caspase-3.
4、Protein kinase C is the important link of preconditioning, pretreatment with jujuboside A can strengthen the expression of PKCεin the membrane,but the PKC inhibitor can significantly attenuate the effects of jujuboside A, and so the PKCεsignaling pathway may be a key point in the anti-apoptosis of jujuboside A.
冠心病为内科常见病及多发病,是导致人类死亡的主要原因之一,全国每年约有110万人死于冠心病。溶栓治疗、经皮冠状动脉介入术(percutaneous coronary intervention, PCI)或冠状动脉旁路移植术(coronary artery bypass graft, CABG)是缩小急性心肌梗死梗塞范围和改善临床预后的最有效方法。但部分患者或动物心肌缺血后再灌注,使缺血所致的功能和代谢障碍及结构破坏进一步加重,这种现象称为心肌缺血再灌注损伤(myocardial ischemia/reperfusion injury, MIRI),临床表现为在闭塞的冠状动脉再通、梗死区血液灌流重建后一段时间内,有的病例发生血压骤降、心功能不全、心律失常甚至猝死等病情恶化的现象。因此,MIRI的发生机制与防治越来越引起人们的关注,并一直试图寻找能对MIRI产生确切保护作用的药物。
心肌缺血再灌注损伤的发生机制十分复杂,而细胞调亡是心肌缺血再灌注损伤发生的重要机制之一。与细胞坏死过程不同,细胞凋亡又称程序性细胞死亡是指有核细胞在一定条件下通过启动其内部机制,主要通过内源性DNA内切酶的激活而发生的细胞自然死亡过程。细胞凋亡在心肌缺血再灌注损伤中的发生可能与以下因素有关：①活性氧大量生成：再灌注导致大量氧自由基释放,造成生物膜脂质过氧化、各种酶蛋白失活及DNA损伤；②细胞内钙超载：心肌缺血再灌注过程中,线粒体内Ca2+大量聚集,引起线粒体内膜上的通透性转换孔开放,释放细胞色素C入胞质内,进一步激活半胱氨酸天冬氨酸蛋白酶诱致细胞凋亡。③线粒体损伤：在细胞凋亡期间,尽管线粒体能维持其超微结构的正常,但事实上其功能已经发生显著改变,如线粒体内膜通透性增大,线粒体内膜的跨膜电位下降,能量合成水平显著降低。上述因素并不能直接引起细胞凋亡,而是通过一定的信号传递方式激活生存或死亡相关基因,然后将信号传递到核内切酶,而执行死亡的功能。因此阻断心肌缺血再灌注损伤中心肌细胞凋亡的信号转导能够阻止凋亡程序的执行,减少心肌细胞凋亡,防治心肌缺血再灌注损伤。
酸枣仁是鼠李科植物酸枣(Ziziphus psinosa Hu)的干燥成熟种子,具有敛汗,养肝,宁心安神等多种功效。酸枣仁主要含有多种酸枣仁皂苷类等化合物。酸枣仁皂苷是酸枣仁的水溶性成分,酸枣仁皂苷A是其中的主要有效成份之一。研究发现,酸枣仁提取物具有调脂、降压、抗动脉粥样硬化、抗心律失常、抗心肌缺血、抗心肌细胞缺氧-复氧损伤作用。酸枣仁皂甙A具有抑制血管平滑肌细胞过度增殖而抗动脉粥样硬化,同时对脑缺血再灌注损伤导致的细胞凋亡有明显的抑制作用,但是目前对于其对心肌缺血再灌注损伤的保护作用及机制尚未报道。
二、研究方法
目的：
在整体水平,我们采用建立大鼠在体心肌缺血再灌注模型模拟急性心肌梗死后再灌注损伤的病理生理变化,酸枣仁皂苷A于造模前腹腔注射给药干预,旨在观察其抗细胞凋亡、保护心肌等作用,并进一步探明其作用机制(主要是其对凋亡相关蛋白Bax、Bcl-、Caspase-3和CytC的影响)；在细胞水平,采用过氧化氢损伤原代大鼠心肌细胞建立氧化损伤模型,酸枣仁皂苷A预处理后观察其对心肌细胞形态及细胞凋亡的影响,探明其抑制细胞凋亡的作用机制(主要是与PKCε信号转导通路的关系),为临床上有效防治心肌缺血再灌注损伤提供新思路。本课题分三个部分进行：第一部分酸枣仁皂苷A预处理对大鼠缺血再灌注是否有保护作用,重点观察其抗氧化作用及其对心肌组织结构、心肌损伤标志物、再灌注心律失常及心功能的影响；第二部分酸枣仁皂苷A预处理对缺血再灌注心肌细胞凋亡的影响,从蛋白质水平观察酸枣仁皂苷A的抗凋亡作用,并探求其作用机制；第三部分观察酸枣仁皂苷A对过氧化氢诱导新生大鼠心肌细胞凋亡及PKCε表达的影响,进一步明确其作用机制。
研究方法：
第一部分：采用结扎(15 min)和松开(30 min)左冠状动脉前降支(LAD)的方法制备心肌缺血再灌注损伤模型。44只雄性wistar大鼠随机分为4组,分别为假手术组(n=8)、模型组(n=12)、酸枣仁皂甙A组(n=12)及表没食子儿茶素没食子酸酯组(n=12)。酸枣仁皂苷A组及EGCG组的给药剂量均为20mg·kg-1·d-1,假手术组和模型组给予等体积的生理盐水,均为腹腔注射,连续用药3d,末次给药30min后进行造模。Ⅱ导联心电图观察再灌注30min期间心律失常的发生情况,根据Roringerova T方法进行室性心律失常(ventricular arrhythmias, VA)评分,Medlab6.0生物信号采集处理系统监测左心室收缩压(LVSP)、左心室舒张末压(LVEDP)、收缩期左心室内压上升的最大变化速率(+dp/dtmax)、舒张期左心室内压下降的最大变化速率(-dp/dtmax)分别在缺血前、缺血15min、再灌注15、30min时的变化。标本收集：实验结束时腹主动脉采血,同时在心底部将心脏与大血管及组织离断,取出心脏,去除左右心房及右心室,心肌置入冰生理盐水中冲洗3次,将左心室分离为三部分,一部分用精密电子天平称取记录质量,剪碎匀浆,作为SOD、MDA检测用,一部分放入4%多聚甲醛中固定,作为组织切片备用,以观察心肌组织病理结构,另一部分放入-80℃冰箱中保存备；血液室温静放30min,在4℃离心后,取其上清置-80℃冰箱保存,检验肌酸磷酸激酶(CK)及乳酸脱氢酶(LDH)水平。
第二部分：40只雄性wistar大鼠随机分为4组,每组10只,分别为假手术组、模型组、酸枣仁皂甙A组及表没食子儿茶素没食子酸酯组,其余同第(?)部分。以末端脱氧核昔酸转移酶(TdT酶)介导的脱氧三磷酸尿昔(dUTP)缺口末端标记法(TdT-mediated du Tpniekend labeling, TUNEL)检测凋亡心肌细胞,计算细胞凋亡指数(apoptotic index, AI);取出-80℃冰箱中保存的部分心肌,提取蛋白及定量后,采用蛋白印迹法检测Bax、CytC及Bcl-2表达,采用分光光度法检测caspase-3的活性。
第三部分：采用差速贴壁法分离培养Wistar新生乳鼠心肌细胞,以过氧化氢作用心肌细胞建立氧化损伤模型,将培养72 h的原代心肌细胞,选择搏动良好、生长密度无明显差异的细胞按孔随机分为以下4组：①对照组：细胞培养过程中不给予任何处理；②模型组：心肌细胞培养基中加终浓度为500μmol/L过氧化氢作用4h；③酸枣仁皂苷A治疗组：操作同模型组,但在给予H202前加入酸枣仁皂苷A至终浓度为20mg/l干预24小时；④PKC抑制剂组(JA+Chelerythrine):操作同治疗组,但在给予H202前5min加入Chelerythrine至终浓度5umol/L。倒置显微镜观察心肌细胞形态学和搏动频率的变化；四唑盐(MTT)比色法检测心肌细胞活力；流式细胞仪检测细胞凋亡率；采用分光光度法检测caspase-3的活性；蛋白印迹检测CtyC及PKCε表达。统计分析：实验数据采用SPSS13.0统计软件进行分析。计量资料数据以x±s表示。对数据进行方差齐性检验。多组数据间比较采用单因素方差分析处理,方差齐性时两两比较采用LSD法；方差不齐时,多组数据间比较采用Welch法,两两比较采用Games-Howell法。各实验组缺血前及缺血再灌注不同时间点心功能指标采用重复测量方差分析。检验显著性水准α=0.05。
结果：
第一部分：
1、大鼠心肌组织病理形态学HE染色光镜下观察显示,假手术组大鼠心肌细胞形态规则、排列整齐,肌纤维完整,未见心肌细胞水肿、变性、坏死,未见中性粒细胞浸润。模型组心肌细胞排列紊乱,局部肌纤维横纹消失,肿胀明显,较多的中性粒细胞浸润。酸枣仁皂苷A治疗组可见心肌细胞水肿及中性粒细胞浸润,但都较模型组轻,心肌受损程度有所下降；EGCG组心肌组织病理形态学变化与酸枣仁皂苷A治疗组相似。
2、缺血再灌注对大鼠LDH、CK、SOD及MDA的影响再灌注结束后检测各组LDH、CK、SOD及MDA值。结果显示,I/R组、JA组、EGCG组的LDH及CK值均较Sham组明显增高(P<0.01)；各组SOD值均显著降低,与Sham组比较有显著性差异(P<0.01)；各组MDA值均显著增高,与Sham组相比有显著性差异(P<0.01)。表明缺血再灌注可造成心肌严重损伤,证实心肌缺血再灌注模型构建成功。
3、酸枣仁皂甙A对缺血再灌注大鼠LDH、CK、SOD及MDA的影响经统计学检验,各组间LDH、CK、SOD及MDA的水平均有显著差异(LDH:F=69.104, P=0.000; CK:F=143.320, P=0.000; SOD:F=101.844, P=0:000; MDA:F=212.780,P=0.000)。JA和EGCG均可明显抑制再灌注所导致的LDH、CK、MDA的升高,同时显著增加SOD的活性,与I/R组相比有显著性差异(P<0.01)；上述指标在JA组与EGCG组间无统计学差异(P>0.05),表明JA和EGCG均具有抗氧化作用而减轻心肌缺血再灌注损伤。
4、再灌注心律失常44只大鼠中12只被排除,剩余32只中26只发生心律失常,心律失常多出现于再灌注早期,包括室性早博、室性心动过速和心室纤颤等；假手术组仅有偶发室性早搏,未见其他类型心律失常。模型组均出现严重的心律失常,以频繁室速、室颤为主,伴有少量的房室传导阻滞,其中2只大鼠因为室颤而死亡。经统计学检验,各组间心律失常评分差异显著(F=44.699,P=0.000)。与假手术组比较,模型组心律失常评分显著上升,差异具有统计学意义(P=0.000)。酸枣仁皂苷A治疗组也出现了心律失常,以室早及室速为主,与模型组比较心律失常评分显著下降(P=0.005)。EGCG组的心律失常以室早及阵发室速为主,与酸枣仁皂苷A组比较,心律失常评分无显著性差异(P=0.080)。5、心功能指标各组缺血前的LVSP、LVEDP和±dp/dtmax差异无统计学意义(P>0.05)。缺血再灌注时各组间大鼠左室收缩末压(LVSP)由高到低依次为Sham组、EGCG组、酸枣仁皂苷A组及I/R组；等容收缩期左心室最大上升速率(+dp/dtmax)及等容舒张期左心室下降最大速率(-dp/dtmax)由高到低依次为Sham组、酸枣仁皂苷A组、EGCG组及I/R组；左室舒张末压(LVEDP)由低到高依次为Sham组、EGCG组、酸枣仁皂苷A组及I/R组。经统计学分析,缺血再灌注时个组间LVSP. +dp/dtmax、-dp/dtmax、LVEDP差异均有统计学意义(P<0.01)。不同缺血再灌注时间点检测结果表明,LVSP、+dp/dtmax、-dp/dtmax随着时间延长而降低,LVEDP随着时间延长而升高,差异有统计学意义(P<0.01)。各时间点与组别间有交互作用(P<0.01)。以上结果表明缺血再灌注可导致心脏收缩及舒张功能受损,酸枣仁皂苷A可改善大鼠收缩及舒张功能。
第二部分：
1、各组心肌组织TUNEL结果按试剂盒提供的脱氧核苷酸转移酶介导的缺口末端标记法原位检测凋亡细胞。正常心肌细胞核呈蓝色；凋亡心肌细胞核呈深浅不一的棕褐色。经统计学检验,各组间AI差异显著(F=198.001,P=0.000). I/R组AI为(18.14±2.22)%,JA和EGCG预处理后AI显著下降(P<0.01)；与Sham组比较,JA组和EGCG组中AI显著升高,差异具有统计学意义(P<0.01)。
2.Western bloting检测各组大鼠心肌Bcl-2、Bax及细胞色素C的表达通过分析目的条带的光密度值(IOD)发现,各组间Bcl-2、Bax、Bcl-2/Bax及细胞色素C的表达均有显著差异(Bcl-2:F=222.463,P=0.000；Bax:F=82.507,P=0.000；Bcl-2/Bax:F=231.350,P=0.000；细胞色素C：F=96.916,P=0.000)。在Sham组中心肌组织有一定量的Bcl-2及Bax表达,胞浆中细胞色素C的含量极少,I/R组心肌Bcl-2蛋白表达显著减少(P=0.000),而Bax及胞浆中细胞色素C蛋白表达显著增加,Bcl-2/Bax比值显著降低(P<0.01)；JA组和EGCG组中Bax及胞浆中细胞色素C表达较I/R组显著下降(P<0.01),但Bcl-2的表达较I/R组显著升高(P<0.01), Bcl-2/Bax比值显著高于I/R组(P<0.01)。JA组与EGCG组中的Bcl-2、Bax、胞浆中CytC表达及Bcl-2/Bax比值无显著性差异(P>0.05)；此两组中Bax表达与Sham组比较无显著性差异(P>0.05),Bcl-2、胞浆中CytC表达及Bcl-2/Bax比值与Sham组比较有显著性差异(P<0.01)。3、各组caspase-3活性检测结果caspase-3活性测定显示,各组间caspase-3活性差异显著(F=39.663,P=0.000)。正常心肌细胞的caspase-3活性水平很低,在体缺血15分钟,再灌注30分钟后心肌caspase-3的活性显著升高(P=0.000)；经过JA及EGCG预处理后心肌caspase-3的活性显著减少(P<0.01),但JA组与EGCG组的caspase-3活性间无显著性差异(P=0.817)。
第三部分：
1、各组心肌细胞形态学观察倒置显微镜下观察,正常心肌细胞从72h起细胞成单层成簇生长,心肌细胞核折光性好,伪足饱满并相互连接,生长呈半融合状态,搏动明显,节律一致,60～80次/min；模型组细胞核暗淡,伪足显著变细,细胞间连接明显减少,搏动节律减慢,20～30次/min；与模型组比较,酸枣仁皂苷A治疗组心肌细胞形态明显改善,细胞核折光性较好,伪足略变细,搏动有力,节律一致,50～60次/min；与酸枣仁皂苷A治疗组比较,P.KC抑制剂组心肌细胞细胞核折光性较差,伪足变细,细胞间连接减少,搏动节律减慢,30～40次/min。
2、各组心肌细胞活力与细胞凋亡的结果经统计学检验,各组间心肌细胞活力、细胞凋亡差异显著(心肌细胞活力：F=87.752,P=0.000；细胞凋亡：F=657.061,P=0.000)。与对照组比较,模型组心肌细胞活力显著降低(P=0.000),细胞凋亡率则显著增加(P=0.000)；与模型组比较,JA组心肌细胞活力显著增加(P=0.000),细胞凋亡率则显著降低(P=0.000)；与JA组比较,PKC抑制剂组心肌细胞活力显著降低(P=0.008),细胞凋亡率则显著增加(P=0.000)。
3、Western blot检测各组心肌细胞CtyC及PKCε的表达情况经统计学检验,各组间CtyC及PKCε的表达差异显著(CtyC:F=210.511,P=0.000; PKCε: F=42.589, P=0.000)。与对照组比较,模型组胞浆中细胞色素C显著增多(P=0.000), PKCε表达无显著性差异(P=0.235)；JA预处理后,细胞色素C表达显著减少(P=0.000), PKCε表达显著升高(P=0.000)；与JA组比较,PKC抑制剂组胞浆中细胞色素C显表达显著增多(P=0.000), PKCε表达显著降低(P=0.000)。
4、各组caspase-3活性检测经统计学检验,各组间caspase-3活性差异显著(F=663.629,P=0.000)。与对照组比较,模型组Caspase-3的活性显著增加(P=0.000)；与模型组比较,JA组的Caspase-3活性显著降低(P=0.000); Chelerythrin干预后,Caspase-3活性显著升高(P=0.000)。
结论：
1、本实验通过在体大鼠心肌缺血/再灌注模型,证实了心肌缺血/再灌注可导致心肌损伤,酸枣仁皂苷A预处理可以减轻心肌的损伤,降低LDH、CK、MDA值,升高SOD值,减少再灌注心律失常,改善心功能。
2.心肌缺血/再灌注和过氧化氢损伤可以引起心肌细胞凋亡,酸枣仁皂苷A预处理可以减少心肌细胞凋亡。因此酸枣仁皂苷A可减轻心肌缺血再灌注损伤,与其抑制心肌细胞凋亡有关。
3.酸枣仁皂苷A预处理可以上调Bcl-2、下调Bax蛋白表达,升高Bcl-2/Bax比值,抑制细胞色素C从线粒体中的释放,减少caspase-3在心肌组织中活性从而减少心肌细胞凋亡。
4.PKC是预适应机制的重要环节,酸枣仁皂苷A预处理可增强心肌细胞膜PKCε的蛋白表达,经Chelerythrine处理后,酸枣仁皂苷A预处理的保护作用显著减弱,因此PKCε的信号转导途径是酸枣仁皂苷A抑制心肌细胞凋亡的关键环节。
1、Background
Coronary heart disease(CHD)is a kind of common and frequently-occurring disease. CHD is the single-most important cause of death worldwhile, and about 1.1 million people die of the disease each year in china. After an acute myocardial infarction,with the use of thrombolytic therapy or primary percutaneous coronary intervention(PCI) or coronary artery bypass graft(CABG) is the most effective strategy reducing the size of myocardial infarction and improving clinical outcome.But in some animals or patients,we can find that the myocardial cell dysbolism and structural damage became more serious after ischemia-reperfusion.The phenomenon was called myocardial ischemia- reperfusion injury(MIRI). In clinical situations, reperfusion following ischemia result in a sharp drop in blood pressure,heart failure,arrhythmia and even sudden death which worsening pathogenetic condition. Therefore, people show greater concern for mechanisms and control of myocardial ischemia-reperfusion injury, and always try to find out the drugs which have exactly protective effects to myocardial ischemia-reperfusion injury.
The causing mechanisms of myocardial ischemia-reperfusion injury are very complicated, and cell apoptosis is one of the important mechanisms of myocardial ischemia-reperfusion injury. Apoptosis is distinguished from necrosis,apoptosis is the process of programmed cell death in which nucleated cells occur the process of natural cell death on condition that through activation of an internally suicide program,mainly through activation of endogeneity DNA endonuclease.Apoptosis occurs in ischemia-reperfusion injury, which may be concerned with the following factors:①lots of reactive oxygen species generation:a burst of Oxygen free radicals generation occur during myocardial reperfusion, Oxygen free radicals generation results in membrane lipid peroxidation、inactivation to kinds of apoenzymes and damage to DNA.②intracellular calcium overload:lots of calcium concentrates in mitochondrial membrane during the process of myocardial ischemia-reperfusion injury, mitochondrial calcium overload results in the opening of mitochondrial permeability transition pore, releases of cytochrome c, and caspase activation leading to myocardial apoptosis.③mitochondria damage:mitochondria still can maintain normal ultrastructure during the process of apoptosis, but its function has been changed significantly, such as increase of inner mitochondrial membrane permeability, decrease of mitochondrial transmembrane potential and the levels of energy production decreased significantly. But these factors can not induce cell apoptosis dircetly,they activate survival or death gene by signal transmission,then the signal transmit to the incision enzyme intranuclear to perform the function of death.To block the signal transduction of cardiomyocyte apoptosis induced by myocardial ischemia-reperfusion may inhibit cardiomyocyte apoptosis, Prevent or cure myocardial ischemia-reperfusion injury.
Semen Ziziphi Spinosae is the dried ripe seed of Ziziphu spsinosa Hu, It has the efficacies of constraining sweat, protecting liver, quieting the heart, inducing sleep,and so on.Semen Ziziphi Spinosae mainly contains kinds of jujubosides.Jujubosides are water-soluble constituents of Semen Ziziphi Spinosae,and jujuboside A is one of main effective components. Studies had found that Spine Date Seed PE could regulate blood lipid, lower blood pressure,anti-atherosclerosis, anti-arrhythmia,anti-myocardial ischemia,and,relieve cardiomyocyte following hypoxia/reoxy-genation injury.The others studies had confirmed jujuboside A could anti-atherosclerosis by means of inhibiting overproliferation of vascular smooth muscle cells, and it also could inhibit cell apotosis significantly during the processof cerebral ischemia-reperfusion injury, but now there is no Study on protective effects and mechanisms of jujuboside A on myocardial ischemia reperfusion injury.
2、Methods:
Objective:
In vivo we set up myocardial ischemia-reperfusion injury models in rats simulate the pathophysiologic changes in myocardial ischemia-reperfusion injury, and jujuboside A was administrated by intraperitonea injection before myocardial ischemia. The aim of this study is to demonstrate if jujuboside A can inhibit apoptosis and protect myocardium from ischemia-reperfusion injury, discuss the mechanism involved,chiefly find out the effects of preconditioning with jujuboside A on apoptosis related protein Bax,Bcl-2,Caspase-3 and CytC; In cellular level we set up oxidative injury models by damage to neonate rat myocardial cell with hydrogen peroxide, The aim of this study is to observe the effects of preconditioning with jujuboside A on cardiomyocytes morphology and apoptosis, discuss the mechanism involved, chiefly find out the relation between the protection and PKCs pathway, and find new evidence to protect myocardium from ischemia-reperfusion injury. Our study includes three parts:The first part is to find out if jujuboside A can protect myocardium from ischemia-reperfusion injury when it was administrated by intraperitonea injection before myocardial ischemia,especially to explore the effect of jujuboside A on the change of myocardial tissue、the marker of myocardial damage、the use of antioxidatio、the occurrence of arrhythmia after reperfusion and heart function. The second part is to demonstrate the effects of preconditioning with jujuboside A on cardiomyocytes apoptosis in protein,discuss the mechanism involved;The third part is to demonstrate the effects of preconditioning with jujuboside A on neonate rat myocardial cell apoptosis induced by hydrogen peroxide and the experssion of PKCs,identify the mechanism involved.
Methods:
The first part of this study:The left anterior descending (LAD) coronary artery was occluded for 15min followed by reperfusion for 30min to establish myocardial ischemia-reperfusion injury model.44 androgenic wistar rats were randomly divided into four groups:the Sham group (n=8), the I/R control group(n= 12), the jujuboside A group (n=12)and the EGCG group (n=12). Rats of the jujuboside A group and the EGCG group were given drug at a dose of 20mg/kg for three consecutive days(intraperitoneal injection), rats of the Sham group and the I/R control group were given physiological saline of the same volume for three consecutive days (intraperitoneal injection),the operation was conducted 30 min after the last intraperitoneal injection in rats of each group. We observed the occurrence of arrhythmias in electrocardiogram of lead II during myocardial reperfusion for 30min, and scored the ventricular arrhythmias (VA)acceording to the method of Ravingerova T. Through the medlab 6.0 biological signal collecting and processing system, the left ventricle systolic pressure(LVSP)、end-diastolic (LVEDP) pressure and the max rate of rise and decline(±dp/dtmax) were measured at the time of before ischemia, ischemia 15min, reperfusion 15min and reperfusion 30min respectivly. Specimen collection:After the experiment had been done, we drew blood from the abdominal aortic vascular of rats, mutilated the great vessels and tissues from the cardiac base, took out of the heart, removed the atriums and the right ventricles, washed the hearts by cold physiological saline for three times, cut the heart to take the damaged cardiae muscles according to the need of detection, divided the left ventricle in three parts.the first part was weighed by electronic precision balance,enoughly cut and then homogenized about8 stroke with a glass Dounce homogenizer, measured the contents of MDA and SOD in heart;the second part was fixed with 4% polysorbate; the third part was keptin icebox at 80℃below zero. The blood was stood for 30min at room temperature, then centrifugated at 4℃.the serum was kept in icebox at 80℃below zero, measured the levels of CK and LDH.
The second part of this study:40 androgenic wistar rats were randomly divided into four groups:the Sham group (n=10), the I/R control group(n=10),the jujuboside A group (n=10)and the EGCG group (n=10), The rest is the same as the first part. The apoptotic cardiomyocytes were detected by TUNEL staining, counted apoptotic index.The expression of Bcl-2 and Bax protein were measured by western blotting,cytochrome C in the cytoplasm were detected by western blotting, the activity of caspase-3 was measured by spectrophotometry in each group.The results were compared among the groups.
The third part of this study:Primary cultures of cardiac myocytes were prepared from ventricles of 1～3 day new born witar rats and cultured for 72 hours. we set up oxidative injury models by damage to neonate rat myocardial cell with hydrogen peroxide.the neonate rat myocardial cells were divided as follows:①Control group:There was not any treatment in the cultural course of neonate rat myocardial cells.②Model group:The model group was treatment with the final concentration of 500μmol/L hydrogen peroxide in the cell culture fluid in 4 hours. ③Jujuboside A treatment group:The treatment was similar to that of the model group,but dealed with the final concentration of 20mg/L jujuboside A in the cell culture fluid in 24 hours before adding hydrogen peroxide.④PKC inhibitor group
(JA+Chelerythrine), The treatment was similar to that of the jujuboside A treatment group,but dealed with the final concentration of 5umol/L chelerythrine in the cell culture fluid in 5min before adding hydrogen peroxide. Morphology and the beating rate change of primary cultured neonatal rat myocardial cell were observed under the inverted microscope; viability of myocardial cell was assayed by MTT; Apoptosis rate was determined by flow cytometric analysis. The activity of caspase-3 in myocardial cell was measured by spectrophotometry,and Western blotting was used to analyze expression of cytochrome C of the cytoplasm and expression of PKCs of the cytomembrane in each group.The results were compared among the groups.
Statistiealanalysis:The data was analyzed by SPSS13.0.measurement data was presented as the means±SEM.Test of homogeneity of variance was done in each group.If datas accorded with homogeneity of variance statistical analysis was made by one-way ANOVA followed by LSD test, otherwise statistical analysis was made by rank sum test, and multiple comparison was made by Games-Howell. Heart function parameters in different time points was analyzed by repetitive measurement and analysis of varianee.Level of signifieance a=0.05.
Results:
The first part of this study:
1、Histopathologic observation of the myocardium in rats:Pathologic changes of myocardial tissue were observed under hematoxylin-eosine staining(HE).In sham group there was no detectable histopathological change. Rats in the I/R control group displayed a serious degree of myocardial neutrophilic infiltrate, necrosis,hemorrhage, and spindle-shaped interstitial cells as compared with rats in the sham group. pathologic changes of cardiac tissue in the JA and EGCG group were significantly milder than that of the I/R group.
2、The effects of myocardial ischemia-reperfusion on the levels of LDH、CK、SOD and MDA in rats:Compared with Sham group,I/R group、JA group and EGCG group markedly increased the activity of LDH and CK in serum,the content of MDA in myocardium,but significantly decreased the activity of SOD in myocardium (P< 0.01). The results indicated that ischemia-reperfusion could result in serious injury of myocardium, and confirmed that myocardial ischemia-reperfusion injury model was established successfuly.
3、The effects of JA on the levels of LDH、CK、SOD and MDA of myocardial ischemia-reperfusion in rats:Compared with I/R group, JA group and EGCG group markedly decreased the activity of LDH、CK in serum and the content of MDA in myocardium,but significantly increased the activity of SOD in myocardium (P<0.01).The values of MDA,CK,LDH and SOD in JA and EGCG groups had no significant difference(P>0.05).The results indicated that effects of JA and EGCG antioxidation decreased the injury of ischemia/reperfusion of myocardium.
4、Reperfusion arrhythmia:Twelve rats were excluded from entry to this study,arrhythmia was found in 26 rats,arrhythmia occurred mainly at the early period of reperfusion. The arrhythmia appeared in rats inclding ventricular premature beat、ventricular tachycardia(VT)、ventricular fibrillation(VF),and so on.Only accidental ventricular premature beat appeared in rats of Sham group. The serious arrhythmia appeared in rats of I/R group,remained frequent ventricular tachycardia(VT)、frequent ventricular fibrillation(VF),and accompanied a small quantity of atrioventricular block,there were 2 dead rats in I/R group,most of arrhythmia occurred in reperfusion. Arrhythmia score in I/R group was more than that in sham group (P=0.000).The arrhythmia also appeared in rats of JA group mainly inclding ventricular premature beat and ventricular tachycardia(VT), and Arrhythmia score in the group was lower than that in I/R group (P=0.005).The arrhythmia appeared in rats of EGCG group mainly inclding ventricular premature beat and paroxysmal ventricular tachycardia(VT),Compared with JA group,There was no difference between them (P=0.080).
5、Index of cardiac function:No significant difference of LVSP、LVEDP and±dp/dtmax were found in all groups of preischemic (P>0.05). LVSP was significantly decreased in the sequence of Sham group, EGCG group, JA group and I/R group,±dp/dtmax was significantly decreased in the sequence of Sham group, JA group, EGCG group and I/R group, while LVEDP was significantly increased in the sequence of Sham group, EGCG group, JA group and I/R group.The results indicated that ischemia-reperfusion could lead toheart malfunction,and pretreatmenting with jujuboside A can improve the heart function.
The second part of this study:
1、Myocardial TUNEL staining:The apoptotic cardiomyocytes were detected by Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) assay.The normal cardiomyocytic nucleus is blue, The nucleus of apoptotic cardiomyocyte is brown.AI of I/R group was (18.14±2.22)%,after pretreatmenting with jujuboside A and EGCG, AI was significantly lower than that of I/R group (P=0.000).Compared with sham group, AI of JA group and EGCG group were significantly raised (P<0.01)
2、Bcl-2、Bax and CytC protein expression:According to the densitometry of bands under the CCD imagine system, a certain amount of Bcl-2 and Bax were expressed in myocardium of sham group, the cytochrome C expression in cytoplasm was little. Compared with the sham group,the expression of Bcl-2 and the rate of Bcl-2/Bax decreased in the I/R group, the expression of Bax and the cytochrome C expression in cytoplasm increased with significant difference (P<0.01). Compared with the I/R group,the expression of Bcl-2 and the rate of Bcl-2/Bax increased in the JA group and EGCG, the expression of Bax and the cytochrome C expression in cytoplasm decreased with significant difference (P<0.01).The expression of Bcl-2、Bax、cytochrome C and the rate of Bcl-2/Bax were no significant difference between JA group and EGCG group(P>0.05), and there was no a significant difference of the Bax expression between the two groups and sham group (P>0.05), but The expression of Bcl-2、cytochrome C and the rate of Bcl-2/Bax were significant difference between the two groups and sham group(P<0.01).
3、The results of Caspase-3 Activity:The levels ofcaspase-3 activity in normal cardiomyocyte were very low, the levels of caspase-3 activity in I/R group were increased significantly, which was decreased significantly in JA and EGCG groups(P <0.01). The caspase-3 activity was no significant difference between JA group and EGCG group (P=0.817).
The third part of this study:
1、Morphological characters of cellular:Cell morphology was observed under reverse microscope.From 72 hours on,the normal cardiomyocytes grew in clusters,the reflected light of nucleus was good,and the pseudopodia was plump, connected reciprocally. In addition, cell beating intension of cardiac cells was apparent,the rhythm was 60-80 beats per minute.Most of cells in H2O2 group were crenulated, the nucleus were dim, the pseudopodia were became thin significantly, the rhythm of cell beating was decreased significantly, the.frequency was 20～30 beats per minute. Compared with the I/R group, the morphological characters of JA group was improved significantly.the morphological characters of PKC inhibitor group was worse that of JA group.
2、The results of viability and apoptosis rate in myocardial cell:Compared with the control group, the viability in myocardial cell of model group was decreased significantly,and the apoptosis rate was increased significantly (P<0.01).Compared with the model group, the viability in myocardial cell of JA group was increased significantly, and the apoptosis rate was decreased significantly (P<0.01). Compared with the JA group, the viability in myocardial cell of PKC inhibitor group was decreased significantly, and the apoptosis rate was increased significantly (P<0.01,).
3、Protein expression of CytC and PKCs were measured by Western blot analysis: Compared with the control group, the expression of cytochrome C in the cytosol of model group was increased significantly (P=0.000), and the expression of PKCεin the membrane was no significant difference between them (P=0.235).Treatments with jujuboside A (20 mg/L) significantly decreased the expression of cytochrome C in the cytosol, and significantly increased the expression of PKCεin the membrane(P <0.01). Compared with the jujuboside A group, the expression of cytochrome c in the cytosol of PKC inhibitor group was increased significantly, and the expression of PKCεin the membrane was decreased significantly (P< 0.01).
4、The results of Caspase-3 Activity in cardiomyocyte:Compared with the control group,the Caspase-3 Activity of model group was increased significantly (P=0.000). Compared with the model group,the caspase-3 activity of jujuboside A group was decreased significantly (P=0.000).Treatments with Chelerythrin significantly increased the caspase-3 activity (P=0.000).
Conclusion:
1、The experiment has proved that myocardial ischemia/reperfusion could lead to myocardial injury by an in vivo myocardial ischemic reperfusion model in rats. Pretreatment with jujuboside A could decrease the injury of ischemia/reperfusion of myocardium,decrease the levels of LDH、CK and MDA, increase the activity of SOD, decrease the reperfusion arrhythmia and improve cardiac function.
2、Myocardial ischemia/reperfusion and hydrogen peroxide injury could result in cardiomyocyte apoptosis,pretreatment with jujuboside A could decrease cardiomyocyte apoptosis.Pretreatment with jujuboside A can relieve the ischemic reperfusion injury,the mechanism of which may be related with the inhibited myocardial apoptosis.
3、The protective effect of preconditioning with jujuboside A was probably achieved through decreasing myocardium apoptosis,and modulating expression of Bcl-2 and Bax,increasing the ratios of Bcl-2/Bax,inhibiting the release of cytochrome C and decreasing the activity of caspase-3.
4、Protein kinase C is the important link of preconditioning, pretreatment with jujuboside A can strengthen the expression of PKCεin the membrane,but the PKC inhibitor can significantly attenuate the effects of jujuboside A, and so the PKCεsignaling pathway may be a key point in the anti-apoptosis of jujuboside A.
引文
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