NaCN中毒对缺氧大鼠心脏损伤作用及干预措施研究
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摘要
氢氰酸(HCN)和氯化氰(CNCl)是外军重点装备的全身中毒性毒剂,因分子中都含有氰根,故又称之为氰类毒剂。它们的毒性大、吸收迅速、防护困难,是典型的速杀性战剂。除HCN和CNCl外,属于氰类的还有无机的氰化钠、氰化钾等,都是剧毒的化工原料,平时的工农业生产中也可能由于泄露而引起人畜中毒。有机氰和植物中存在的氰类化合物也能对机体产生毒害作用。因此,氰类毒物中毒的防治是军事预防医学的重要任务。
氰化物中毒后迅速出现缺氧、窒息、惊厥等中毒症状,来势凶猛、发展迅速,可在数分钟至数十分钟内死于呼吸、循环衰竭。氰化物进入机体后能迅速离解出氰根离子(CN-),能阻断细胞呼吸和氧化磷酸化,对细胞线粒体呼吸链末端氧化酶产生抑制作用,引起组织中毒性缺氧,进而细胞内生物氧化发生一系列变化,造成“内呼吸”障碍。
一般将海拔高度大于3 000m的地区成为高原。低压低氧(习惯上称作高原缺氧)是高原地区的主要环境特征。部分人在这种环境下会出现明显的症状和体征。而超过这个高度时,其生理、生化和解剖等方面的改变就会变得越来越明显[1]。我国是一个高原地区面积广阔的国家,仅青藏高原的面积就达到250万平方公里。高原地区多与他国相邻,边境漫长,有重要的军事战略地位。
当高原缺氧复合NaCN中毒时,缺氧和NaCN中毒这“内”、“外”两种缺氧因素同时作用机体,将对机体产生双重缺氧的联合打击。因此,对缺氧复合氰化物(NaCN)中毒的研究对我国的国防保障和未来的战争胜负都会产生极其重要的影响。
急性缺氧对机体的血压(SP)、心率(HR)的影响已有相关报道。有多篇文献提出,急性低压缺氧大鼠心室收缩功能指标如左室收缩压(LVSP),左室压力最大上升速率(+dP/dtmax)等均显著降低。心肌细胞缺氧不仅是严重创伤后心功能衰竭的重要原因,也是启动和诱发其它脏器损伤形成多脏器功能不全/衰竭的重要始动因素[2]。有研究发现,在高原缺氧条件下化学战剂毒性增强,对心功能损害效应加重,引起心率失常、心肌收缩力减弱等心脏功能异常变化[3]。目前,国内外对缺氧复合NaCN中毒所产生的联合效应对大鼠心脏结构和功能影响研究甚少,对药物预防救治效果亦未见有关评价方面的报道。
心脏是对缺氧极为敏感的器官。有研究表明,单纯缺氧和单纯NaCN中毒均可明显导致心肌细胞凋亡(Apoptosis),缺氧导致细胞死亡的主要方式是通过诱导细胞凋亡而产生的。在采用大鼠离体心肌细胞培养后、给予缺氧/复氧处理的实验研究中,发现了心肌细胞在持续较长时间的缺氧而再复氧时,细胞损伤现象严重,与曾经研究过的在体动物心肌细胞缺血/再灌注损伤情况一致。在缺氧/复氧损伤组中,流式细胞仪PI染色法的DNA分析直方图上,低于G1期的细胞数增多,明显高于其他各组,说明凋亡的细胞存在较多。透射电镜可见缺氧/复氧损伤组心肌细胞损伤严重,可有明显的凋亡前期、凋亡阶段或凋亡后期的改变,同时心肌细胞凋亡数目也明显增加,支持复氧(再灌注)损伤是心肌细胞凋亡的重要诱发因素。认为缺氧/复氧可加重心肌细胞损伤,伴着心肌细胞凋亡的增加;细胞内钙离子过多可能是触发心肌细胞凋亡的因素;超氧化物歧化酶(superoxide dismutase, SOD)可减少自由基的生成、降低心肌细胞凋亡的发生和缺氧预处理具有抗心肌细胞损伤和减轻心肌细胞凋亡的作用,均有可能是通过降低细胞内钙离子浓度而起作用的[4-6]。Fliss[7]等在在体动物模型研究中也发现了细胞凋亡是心肌再灌注损伤的特征之一,再灌注损伤可加速不可逆转的细胞凋亡。Musat-Marcu等[8]在体外灌注大鼠心脏发现其早期即可发生心肌细胞凋亡,且在抑制凋亡后伴随着心功能的恢复,提示心肌细胞凋亡参与了心功能的衰减。
细胞色素C(Cytochrome C, Cyt C)及细胞凋亡诱导因子(apoptosis-inducing factor, AIF)正常时分别位于线粒体内膜和线粒体膜间隙。缺血/再灌注(MI/R)时,PT孔受损而开放,Cyt C及AIF被释放入胞浆。Cyt C与胞浆中的凋亡激活因子-1(apoptosis- activating factor-1, Apaf-1)结合,依次激活半胱酸蛋白酶家族中的Caspase-9、Caspase-3而启动凋亡通路,活化的Caspase-3可以作用于胞质中的细胞骨架蛋白,或作用于细胞核中的DNA,引发细胞凋亡,这是线粒体caspase依赖性凋亡途径。而AIF经PT孔被释放入胞浆,其将易位入核,激活内源性核酸内切酶将染色体切割为180-200bp整数倍的DNA片断,同时加速Cyt C的释放,进一步促进细胞凋亡,这是线粒体caspase非依赖性凋亡途径。可见,线粒体caspase依赖性和caspase非依赖性这两条凋亡途径在心肌细胞凋亡中发挥重要作用。
缺氧预适应( hypoxic preconditioning, HPC),并可将其界定为“预先短时间非致死性重复缺血/缺氧后,机体组织细胞获得对随后长时间致死性缺血/缺氧损伤的高度耐受性”。缺氧预处理对心肌细胞可以产生预适应现象,缺氧预适应迄今为止被认为是最强有力的一种心肌内源性保护措施[9,10]。
人参皂苷(saponins of panax ginseng, SPG)能对抗氧自由基对心脏的损伤,保持心肌细胞膜的完整性,改善急性心肌缺血时心肌舒张功能,还能对抗心肌缺血所致心肌不可逆坏死,使SOD活性升高,心肌释放磷酸烯醇式丙酮酸羧激酶(phosphoenolpy- ruvate carboxykinase, PCK)减少。人参总皂苷(total saponins of Panax Ginseng, TSPG)对缺血再灌注损伤中的细胞坏死和细胞凋亡均有显著的保护作用[11-14]。人参皂苷尤其是Re可能具有阻滞K+通道的作用,可使离体豚鼠乳头状肌细胞动作电位时程和有效不应期均延长,人参皂苷抗心律失常的作用[15]。人参皂苷抗心肌缺血等作用研究表明,人参皂苷能对抗氧自由基对心脏的损伤,保持心肌细胞膜的完整性,改善急性心肌缺血时心肌舒张功能,还能对抗心肌缺血所致心肌不可逆坏死,使SOD活性升高,心肌释放PCK减少。侯明晓等利用体外培养的心肌细胞缺血再灌注损伤的模型的结果表明:TSPG对缺血再灌注损伤中的细胞坏死和细胞凋亡均有显著的保护作用[16]。Scott等研究发现,人参皂苷Rb1可抑制心肌细胞的收缩,有助于减少心肌的耗氧量[17]。自由基亦可损伤血管内皮细胞,引起动脉粥样硬化、高血压等心脑血管疾病。有实验表明,人参皂苷可降低过氧化物丙二醛的含量,从而减轻血管内皮细胞损伤。
而牛磺酸(Taurine, Tau)能清除氧自由基、保护细胞膜、使心肌酶和肝酶释放减少、具有极强抗氧化能力。它也能调节细胞内钙的稳态,减少肢体缺血再灌注时钙离子内流,而且中性粒细胞呼吸爆发产生大量过氧化氢(hydrogen peroxide, H2O2),与CL结合产生难以清除的强氧化剂-次氯酸,牛磺酸可与次氯酸结合,形成稳定的弱氧化剂-氯胺牛磺酸,从而除次氯酸对细胞的破坏作用。因此显示牛磺酸可纠正肢体缺血再灌注后远隔器官损伤[18]。长疗程应用牛磺酸在降低血压和逆转心肌肥厚的同时,也使高血压大鼠心肌细胞凋亡减少,并能降低大鼠心肌AngⅡ含量和ACE活性。因此,我们认为,长疗程牛磺酸治疗可抑制高血压大鼠心肌细胞凋亡和逆转心肌肥厚,上述作用可能与药物拮抗组织局部AngⅡ的生成有关。牛磺酸还具有抑制心肌细胞凋亡的作用,可能与其调控凋亡相关基因Fas、Bax和Bcl-2的蛋白表达有关。牛磺酸可以有效保护心肌细胞而避免发生心脏畸形,同时有降低血压[19,20]。
血液供应和血氧含量是心脏功能正常的先决条件,因此,任何引起血液中氧含量降低或心肌供血不足的有害因素都可能导致心脏功能异常。
那么,缺氧环境氰化物中毒对心脏功能将会产生何种影响?由此带来的心脏损伤其机制如何?线粒体caspase依赖性和caspase非依赖性这两条凋亡途径在缺氧环境氰化物中毒诱导大鼠心肌细胞凋亡中有何种作用?缺氧预适应、人参皂苷和牛磺酸等干预措施对大鼠缺氧环境氰化物中毒有无效果?这些问题迄今未见研究报道。本课题基于上述两种情况,在前期工作的基础上,采用实验动物研究和体外细胞培养研究相结合的方法,建立缺氧复合NaCN中毒的动物模型和NaCN染毒的细胞模型,通过观察模拟高原环境缺氧复合NaCN中毒对大鼠心脏的影响、检测SPG等干预措施对缺氧复合NaCN中毒大鼠心脏保护作用、观测心肌细胞NaCN染毒后细胞凋亡情况,以及定量分析上述情况下线粒体caspase依赖性和caspase非依赖性这两条凋亡途径中Cyt C、caspase-3和AIF等因子的表达,来探讨模拟缺氧条件下NaCN中毒后心功能改变的特点和心肌细胞凋亡的特性,为今后缺氧性NaCN中毒的预防救治措施的研究提供重要的理论基4础。最终为制定缺氧条件下复合NaCN中毒的预防和救治方案提供实验和理论依据。
方法:
1.检测有无干预措施情况下缺氧复合NaCN中毒后对大鼠心脏结构和功能影响,包括血流动力学、心电图、心肌酶谱、心肌病理切片光镜电镜观测等;
2.建立体外SD乳鼠心肌细胞NaCN染毒模型,检测急性NaCN染毒后心肌细胞是否存在凋亡情况;
3.定量分析有无干预措施情况下心肌细胞缺氧复合NaCN中毒后Cyt C、caspase-3和AIF表达,分析线粒体caspase依赖性和caspase非依赖性这两条凋亡途径在缺氧复合NaCN中毒引起心肌细胞凋亡中的特点;
结果:
一、人参皂苷等干预措施对缺氧和NaCN中毒大鼠心脏的保护作用
1.平原实验组大鼠NaCN中毒后,血流动力学发生改变:HR、mLVSP、+dP/dtmax等指标均较中毒前出现显著下降(P<0.01),T波振幅则出现显著上升(P<0.01),30min后逐渐向正常值恢复。
2.高原实验组大鼠NaCN中毒后,血流动力学改变比平原实验组更为明显:HR、mLVSP、+dP/dtmax等指标均较中毒前出现显著下降(P<0.01),T波振幅则出现显著上升(P<0.01),40min后逐渐向正常值恢复,但恢复程度和效果较平原实验组差。
3.平原实验组大鼠NaCN中毒后,Cyt C活性显著降低(P<0.01)。中毒2h后,Cyt C活性有明显的恢复(P<0.01)。
4.高原实验组大鼠在NaCN中毒后,Cyt C的活性显著低于平原实验组(P<0.01);中毒2h后也有一定程度恢复,但与平原实验组比较恢复更慢(P<0.01)。
5.平原和高原实验组大鼠NaCN中毒后均出现明显的心肌酶谱(AST、LDH、CK、CK-MB)变化,高原实验组大鼠的心肌酶谱改变比平原实验组大鼠更显著(P<0.01),并且恢复缓慢。其中,LDH变化最为显著。
6.平原实验组大鼠中毒后心肌组织损伤逐渐加重,至2h后最为严重,间质有腔隙形成,出现纤维退变,横纹不清,弥漫性充血、水肿变性,少数心肌有断裂,横纹消失;中毒6h时有明显的恢复。
7.高原实验组大鼠心肌组织中毒前即见充血、水肿变性、局灶性炎症细胞等变化。NaCN中毒后心肌损伤加重,出现严重水肿变性,弥漫性细胞肿胀,胞质模糊,并有水肿囊出现,细胞变性,间质血管充血。中毒6h时,高原实验组大鼠心肌组织损伤并无明显的恢复。
8.平原实验组大鼠中毒2h后出现线粒体轻微水肿,灶性溶解,内质网扩张,线粒体部分嵴和膜融合或消失等现象。
9.高原对照组大鼠超微结构也出现轻微损伤,心肌间质有轻微水肿,线粒体略有肿胀;高原NaCN中毒组大鼠线粒体则肿胀明显,有小量的嵴和膜融合或消失,心肌间质水肿,肌节排列稍紊乱,核旁水肿处线粒体大部分嵴和膜融合或消失,粗面内质网有脱颗粒现象;损伤程度明显重于平原实验组大鼠。
10. HPC和SPG、Tau灌胃干预后,对NaCN中毒和高原缺氧复合NaCN中毒引起的大鼠血流动力学改变有明显的干预作用,HR、mLVSP、+dP/dtmax、T波振幅等指标均较未干预组变化程度小,恢复效果更明显,且有统计学意义。
11. HPC干预后的大鼠NaCN中毒后Cyt C活性也出现显著降低(P<0.01),至2h时有所恢复,效果好于高原缺氧复合NaCN中毒大鼠(P<0.05)。
二、NaCN对SD乳鼠原代心肌细胞的凋亡作用
1.建立了NaCN染毒SD乳鼠心肌细胞模型;
2.测定出SD乳鼠心肌细胞NaCN染毒的IC50值:正常条件下IC50值为87.85μmol·L-1。
3.以IC50剂量的NaCN染毒后,SD乳鼠心肌细胞出现凋亡。凋亡细胞的细胞核由于染色质浓集而呈现亮蓝色,呈分叶、碎片状等。
三、caspase依赖/非依赖性途径在大鼠心脏损伤中的作用研究
1.大鼠缺氧复合/或NaCN中毒30min后,心肌组织胞浆Cyt C、caspase-3蛋白表达和mRNA表达均上调;
2.大鼠缺氧复合/或NaCN中毒30min后,心肌组织胞浆AIF蛋白表达上调,而AIF mRNA表达对NaCN染毒诱导却并不敏感;
3. Tau、SPG干预对抑制大鼠缺氧复合/或NaCN中毒30min后心肌组织胞浆Cyt C、caspase-3和AIF蛋白表达以及Cyt C、caspase-3 mRNA表达上调有明显作用,对AIF mRNA表达无明显作用。
结论:
1.缺氧复合NaCN中毒大鼠血流动力学HR、mLVSP、+dP/dtmax等指标均显著下降,T波振幅显著增高。相对于单纯的缺氧和单纯的NaCN中毒,两种因素同时作用所引起的心脏功能损伤,特别是心脏的收缩能力的损伤更加明显;
2.缺氧复合NaCN中毒大鼠的血液生化指标,如心肌酶谱(AST、LDH、CK、CK-MB)的变化也更为明显,Cyt C活性下降也更为显著。表明两种因素作用下大鼠的心脏生化代谢变化很大,加重心脏功能的紊乱;
3.病理检测结果表明,缺氧复合NaCN中毒后大鼠心肌组织的结构损伤更为严重,超微结构的病理改变更加明显,特别是NaCN中毒的靶细胞器-线粒体的损伤,这可能是引起心肌细胞凋亡的最主要原因;
4. HPC、SPG和Tau等干预措施,对缺氧复合NaCN中毒大鼠心脏功能的恢复和维持Cyt C的活性均有一定的效果。可考虑将HPC、SPG和Tau等列入缺氧复合NaCN中毒防护措施的筛选;
5.通过建立的体外SD乳鼠心肌细胞NaCN染毒模型,发现经IC50剂量NaCN染毒SD乳鼠原代心肌细胞凋亡。提示细胞凋亡可能在缺氧复合NaCN中毒心脏损伤中发挥作用;
6.大鼠缺氧复合/或NaCN中毒30min后心肌组织胞浆Cyt C、caspase-3蛋白表达和mRNA表达上调,线粒体caspase依赖途径导致的心肌细胞凋亡十分明显,寻找阻止线粒体caspase依赖途径心肌细胞凋亡的措施可能会对缺氧复合NaCN中毒的预防和治疗带来一定的效果;
7. Tau、SPG干预,对抑制大鼠缺氧和/或NaCN中毒心肌组织胞浆Cyt C、caspase-3和AIF蛋白表达以及Cyt C、caspase-3 mRNA表达上调有明显作用,提示Tau、SPG可作为预防线粒体途径引起心肌细胞凋亡的筛选措施;
8.大鼠NaCN中毒30min后,心肌组织胞浆AIF蛋白表达虽然出现上调,但AIF mRNA表达变化却并不明显;AIF mRNA表达对缺氧的敏感程度强于对NaCN中毒,提示NaCN中毒后线粒体非caspase依赖途径引起心肌细胞凋亡的机制并不同于caspase依赖途径,缺氧、NaCN中毒引起心肌细胞凋亡可能存在不同的机制,值得深入研究。
Exemplified by the HCN, Cyanogen agents (blood agents) are highly toxic. Teir intocication effects are potent, quick and hard to defend. As a result, it has captured the attention of foreign military forces. Attachment three of the UN’s ban on chemical weapons, cyanogens chloride and hydrogen cyanide ranked 2 and 3 on the list. Both of which are cynogen agents. They are referred to as“the king of speed kills”. They are considered as the top chemical weapons of U.S. and Russia. After intoxication with the agent, the subject experiences hypoxia and difficulty breathing.
The toxicant acts quickly and disseminates through the body with incredible speed. It could kill the subject within 10 minutes. After the cynogen agents enters the body, the CN ion stops cells from performing respiratory functions and oxidative phosphorylation. It inhibits oxidases the end of the Mitochondrial respiratory chain. This causes the tissues to die due to lack of toxic lack of oxygen. Also known as damage to“internal respiratory”. Medical definition for plateau is 3 000m above sea level. Hypoxia is a characteristic of plateaus. Some people demonstrate clear symptoms of hypoxia. Any level higher than this could have substantial effects on Physiology, biochemistry and anatomy. Our country is full of plateaus. Just the Tibetan plateau alone covers 250 square kilometers of land. The plateau borders many other countries. The military values of these plateaus are of great importance. When NaCN is mixed in with the harsh environment of the plateaus, hypoxia kicks in from both external and internal. When this occurs, the rate at which a subject dies doubles. To understand and analyze the use and nature of NaCN intoxication is of great importance for national defense of our country.
The heart functions under the condition of ample blood supply and oxygen. Therefore, any lack of blood supply or the level of oxygen in the blood will cause the heart to function abnormally. Sudden decrease in oxygen level could lead to fluctuation in blood pressure and heart rate. Many studies have shown that when hypoxia is induced in lab rat, the left ventricle pressure and maximum pressure decreases dramatically. Myocardial hypoxia is not only a serious post-traumatic heart failure, but also other start-induced organ damage and the formation of multiple organ dysfunction and failure of the move before an important factor. Studies have found that oxygen under the conditions of soman toxicity increased.
Heart is a very sensitive organ to hypoxia; studies have shown that simple and pure hypoxia induced by NaCN poisoning can result in significant cardiomyocyte apoptosis, hypoxia leading to the main form of cell death through induction of apoptosis arising. In the use of isolated rat cardiac cells to give H / R in the way of experimental studies have found that the myocardial cells in continued lack of oxygen condition for a longer period of time have achieved reoxygenation. The animal myocardial cells in vivo ischemia / reperfusion injury is in the same situation. In hypoxia and reoxygenation, there is injury in the group. PI staining by flow of cytometry analysis of DNA histogram showed that it’s lower than the number of cells in G1 phase of the increase and it was significantly higher than other groups. There are more cell apoptosis. Transmission of electron microscopy can see hypoxia / reoxygenation group of serious cardiac cell injury is a clear pre-apoptosis. Apoptosis in the late stages changes at the same time the number of cardiomyocyte apoptosis is also a noticeable increase in support for the resumption of oxygen (again Reperfusion). Injury in myocardial apoptosis is an important predisposing factor. The H / R can increase the myocardial cell damage, accompanied by an increase in myocardial apoptosis; intracellular calcium may be triggered by excessive apoptosis of myocardial factors; SOD (superoxide dismutase, SOD) can be reduced. The generation of free radicals and reduced the incidence of myocardial apoptosis and hypoxic preconditioning with anti-myocardial cells to reduce myocardial injury and apoptosis. It’s likely to have lower intracellular calcium ion concentration. Fliss, and so on in the body in an animal subject study also found that the apoptosis of myocardial reperfusion injury is one of the characteristics. Reperfusion injury can accelerate irreversible apoptosis. Musat-Marcu and so on at the heart of rat in vitro perfusion can be found in its early occurrence of myocardial apoptosis, and inhibit apoptosis in the post along with the restoration of heart functions. This suggests that cardiomyocyte apoptosis isinvolved in the attenuation of cardiac function.
Cytochrome C (Cyt C) and apoptosis-inducing factor are located in normal mitochondria mitochondrial membrane and space. Ischemia / reperfusion time, PT and the opening hole damage, Cyt C and AIF were released into the cytoplasm. Cyt C with the cytoplasm of apoptosis-activating factor-1(apoptosis-activating factor-1, Apaf-1), followed by activation of acid cysteine protease family of Caspase-9, Caspase-3 and start the apoptosis pathway, activation of Caspase-3 can be in the cytoplasm of the role of the cytoskeleton protein, or acting on the nuclear DNA, lead to apoptosis. The AIF was released by PT hole into the cytoplasm, the translocation into the nucleus, activated endogenous endonuclease chromosome will be cut for several times the entire 180-200bp fragment of DNA, at the same time to speed up the release of cytochrome C and further promote cell Apoptosis. Cyt C, AIF can be seen as apoptosis of the two channels in cardiac cells. They play an important role in apoptosis.
Hypoxic preconditioning, and can be defined as "a short period of time in advance to repeat non-fatal ischemic / hypoxic, the body of the cell was followed by a long-fatal ischemic / hypoxic injury A high degree of tolerance.”Hypoxic preconditioning on myocardial cells can generate Preconditioning. Hypoxic preconditioning so far is considered to be one of the most powerful endogenous myocardial protective measures. Ginsenosides can act as antioxidants to free radical damages to the heart, to maintain the integrity of myocardial cell membrane and improve acute myocardial ischemia diastolic function. But also against myocardial ischemia-induced myocardial necrosis is irreversible, so that the increased activity of SOD can release myocardial PCK Reduction. Ginsenosides have significant protective effects on ischemia-reperfusion injury in cell necrosis and apoptosis.
In addition, taurine removes oxygen free radicals and protects the cell membrane, so that the enzyme reduces the release of liver enzymes, which are very anti-oxidized. It also can regulate the intracellular calcium homeostasis, reduces limb ischemia reperfusion. During calcium influx, neutrophil respiratory burst a large amount of H2O2 and CL, having a combination of difficulty to get rid of the strong oxidizer - hypochlorous acid and Taurine With hypochlorous acid, to form a stable low-oxidants-taurine chloramine, and thus clear the hypochlorite on the role of cell damage. Therefore taurine corrects limb ischemia-reperfusion injury in distant organs. Application of taurine in the long course of treatment to lower blood pressure and reversal of cardiac hypertrophy are highly possible. High blood pressure also reduces myocardial apoptosis, and can reduce myocardial AngⅡcontent and ACE activity. As a result, we believe that the long course of taurine treatment of high blood pressure can inhibit myocardial apoptosis and reverse cardiac hypertrophy. The role of antagonist drugs may be related to local organizations related to the generation of AngⅡ. Taurine inhibition of myocardial apoptosis also may be related to apoptosis-related gene regulation Fas, Bax and Bcl-2 protein expressions. Taurine can protect myocardial cells and avoid the occurrence of cardiac malformation, at the same time have lower blood pressure.
The normal supply of blood and oxygen to heart bring into full play. Accordingly, some adverse factor that result lower oxygen content of blood and insufficiency of blood for the cardiac muscle will lead to abnormal cardiac function.
Hypoxic environment of cyanide poisoning changes how the heart functions? How the mechanism of the heart resulting in injury? What are the characteristics of myocardial apoptosis? How to search for possible interventions? Research on these issues has not been reported so far. To be the subject of the preliminary research on the basis of the establishment of hypoxia and cyanide poisoning of animal models and cell models to study hypoxia on the compound sodium cyanide poisoning Cyt C, AIF apoptosis two way interference with the role of hypoxia to find Cyanide poisoning and two different factors at the same time under myocardial apoptosis. The key to explore the hypoxic preconditioning, as well as ginsenosides is taurine and anti-hypoxia medicines interference with the effects of hypoxia environment for the prevention and treatment of cyanide poisoning research Theory. Based on this subject in both cases, the use of laboratory animals in general studies and cell culture in vitro study of the combination will implement both methods. By observing the simulation of composite NaCN high altitude hypoxia in rat poisoning on heart structure and function as well as myocardial hypoxia and NaCN poisoning After Cyt C, caspase-3 and AIF expression of quantitative analysis, as well as Cyt C and AIF caused by apoptosis analytical effect, to explore simulated hypoxic conditions of NaCN poisoning after cardiac function changes in the characteristics of myocardial apoptosis. The future of hypoxic NaCN poisoning prevention measures the medical treatment studies and it provides important theoretical basis. For the ultimate development of composites hypoxia NaCN poisoning prevention and treatment programs, it will implement the following methods.
Method:
1. Testing whether interventions of hypoxia and NaCN intoxication after rat cardiac structures will function. Utilizing methods include hemodynamics, ECG, myocardial enzymes, myocardial biopsy light microscope, such as electron microscopy observation;
2. the establishment of in vitro NaCN poisoning induced by hypoxia and myocardial cell damage model to detect hypoxia and NaCN poisoning under the conditions of myocardial apoptosis and its interventions on the impact of cardiac apoptosis;
3. any intervention under the circumstances NaCN complex cardiomyocytes hypoxia poisoning after caspase-3, Cyt C and AIF expression detection, analysis Cyt C and AIF caused by the two channels the characteristics of apoptosis;
Results:
Part one: Change of hypoxia and NaCN intoxication on structure and fuction in rat.
1. Hemodynamics of rat in the plain experimental group takes place change after NaCN intoxication. These markers, such as HR, mLVSP, +dP/dtmax show up significance descent(P<0.01) and the amplitude of T wave show up significance elevation(P<0.01) after NaCN intoxication than no NaCN intoxication. Then, they get a recover to normal value after 30 minute.
2. Hemodynamics of rat in plateau experimental group take place more evident change after NaCN intoxication than the plain experimental group. These markers, such as HR, mLVSP, +dP/dtmax show up significance descent(P<0.01) and the amplitude of T wave show up significance elevation(P<0.01) after NaCN intoxication than no NaCN intoxication. Then, they get a recover to normal value after 40 minute. Nevertheless, the recover degree and effect is bad.
3. Change of the cardiac muscle enzymogram in the plateau experimental group is more obviously than in plain experimental group, and it shows up significant difference (P<0.01). This change difficultly put back. Variation of LDH value is the most significant, and it is with one accord that anaerobic respiration corresponding reinforce in the hypoxia environment.
4. The activity of Cyt C show up significant decrease after NaCN intoxication in plain experimental group. Behind tow hours, the activity occur evident recovery.
5. Compare to in plain experimental group, the activity of Cyt C in plateau experimental group show up significance decrease after NaCN intoxication. Behind tow hours, the activity occur recovery to normal value, but this recovery is the more slowing.
6. Cardiac muscle tissue damage of rat gradually aggravate in plain experimental group after NaCN intoxication. It is the most severity after two hour. There is lacune formation in interstitial substance, and showing up fibrilla cataplasia, transverse striation no clear, diffuse hyperemia, hydropic degeneration, and so on. There is apparent recovery after six hour.
7. Cardiac muscle tissue of normal rat occur some symptom in plateau experimental group, including hyperemia, hydropic degeneration, colour shallowness, focal inflammation cell, etc. Myocardial damage rapidly aggravate after NaCN intoxication. There are severely hydropic degeneration, diffuse cellular swelling, cytoplasm ambiguity, hydrops capsule, cell degeneration and interstitial substance vasocongestion. These symptoms don’t emerge recovery after six hour.
8. Mitochondrion of rat occur light hydrops and swelling and distension, deliquescence, cristae and membrane fusion or delitescence in plain experimental group after NaCN intoxication two hour.
9. Mitochondrion of rat occur light hydrops and swelling and distension in plateau experimental group. It shows up obviously Swelling, cristae and membrane fusion or delitescence, etc. after NaCN intoxication. Degree of injury is the more severity.
10. HPC, SPG and Tau bring out comparative effect to Hemodynamics of rat after hypoxia and NaCN intoxication. These indexes, such as HR, mLVSP, +dP/dtmax and amplitude of T wave show up less change degree. Rat gets obviously recovery.
11. Activity of Cyt C of HPC rat shows up significance depress after NaCN intoxication, and gets recovery after two hour. The recovery effectiveness is better in HPC rat group than in plateau rat group, and worse in HPC rat group than in plain rat group.
Part two: Apoptosis detection of origin generation cardiac muscle cell of SD neonate rat
1. Model constructing of cardiac muscle cell of SD neonate rat used NaCN narcotics.
2. Assay of IC50 value of origin generation cardiac muscle cell of SD neonate rat. IC50 value is 87.85mmol/L.
3. Origin generation cardiac muscle cell of SD neonate rat show up apoptosis by IC50 dosage NaCN used narcotics, and Cell Nucleus appear sapphirine owing to chromatin enrich, and so on.
Part three: Expression detection of Cyt C, caspase-3, AIF in cardiac muscle cell of SPG and Tau intervention rat after NaCN intoxication
1. There are Expression up-regulation of Cyt C, caspase-3 and their mRNA of hypoxia and NaCN intoxication rat after 30 minute in cardiac muscle tissue endochylema.
2. There is Expression up-regulation of AIF of hypoxia and NaCN intoxication rat after 30 minute in cardiac muscle tissue endochylema. But, Expression of AIF mRNA show up insensitivity to NaCN used narcotics.
3. HPC, SPG and Tau interventions bring out comparative effect to Expression depress of Cyt C, caspase-3 and their mRNA, AIF after hypoxia and NaCN intoxication.
Conclusion:
1. Hemodynamics markers, such as HR, mLVSP, +dP/dtmax of rat in plateau experimental group show up significance descent after NaCN intoxication, and amplitude of T wave show up significance heightening. These direct that cardiac contractile function of rat occur the more severity injury.
2. Biochemical indicator, such as cardiac muscle enzymogram and Cyt C activity of rat in plateau experimental group shows up significance change after NaCN intoxication. These direct that heart biochemistry metabolism of rat occur large change, and it results to cardiac function disorder.
3. Ultrastructural pathology variation and injury of cardiac muscle tissue of rat in plateau experimental group get all the more severity after NaCN intoxication. Mitochondrial injury especially is possible to cause cardiac muscle cell apoptosis.
4. These intervention study including HPC, SPG and Tau are effect for recover of cardiac function and Cyt C activity. They act as bolting measures for prevention and treatment of hypoxia and NaCN intoxication.
5. Origin generation cardiac muscle cell of SD neonate rat show up apoptosis by IC50 dosage NaCN used narcotics. Otherwise, cardiac muscle cell apoptosis has induced by purely hypoxia. These hint cardiac muscle cell apoptosis all the more severity under hypoxia and NaCN intoxication koinonia, and it may be important reason of heart injury.
6. There are Expression up-regulation of Cyt C, caspase-3 and their mRNA of hypoxia and NaCN intoxication rat after 30 minute in cardiac muscle tissue endochylema. Action of mitochondria caspase dependency way is obviously to cardiac muscle cell apoptosis, and it may bring out good effect for prevention and treatment of hypoxia and NaCN intoxication.
7. SPG and Tau interventions bring out comparative effect to Expression depress of Cyt C, caspase-3 and their mRNA, AIF after hypoxia and NaCN intoxication. It hint SPG and Tau maybe act as bolting measures for prevention and treatment of hypoxia and NaCN intoxication.
8. There is expression up-regulation of AIF of anoxia and NaCN intoxication rat after 30 minute in cardiac muscle tissue endochylema. But, it is not obviously to expression change of AIF mRNA. These results direct that sensitive degree of AIF mRNA expression is better in hypoxia rat than in NaCN intoxication rat, and cardiac muscle cell apoptosis mechanism of mitochondria caspase dependency way is different with mitochondria non-caspase dependency way after NaCN intoxication, and cardiac muscle cell apoptosis mechanism of hypoxia is different with NaCN intoxication. These results are worth for lucubrate.
氰化物中毒后迅速出现缺氧、窒息、惊厥等中毒症状,来势凶猛、发展迅速,可在数分钟至数十分钟内死于呼吸、循环衰竭。氰化物进入机体后能迅速离解出氰根离子(CN-),能阻断细胞呼吸和氧化磷酸化,对细胞线粒体呼吸链末端氧化酶产生抑制作用,引起组织中毒性缺氧,进而细胞内生物氧化发生一系列变化,造成“内呼吸”障碍。
一般将海拔高度大于3 000m的地区成为高原。低压低氧(习惯上称作高原缺氧)是高原地区的主要环境特征。部分人在这种环境下会出现明显的症状和体征。而超过这个高度时,其生理、生化和解剖等方面的改变就会变得越来越明显[1]。我国是一个高原地区面积广阔的国家,仅青藏高原的面积就达到250万平方公里。高原地区多与他国相邻,边境漫长,有重要的军事战略地位。
当高原缺氧复合NaCN中毒时,缺氧和NaCN中毒这“内”、“外”两种缺氧因素同时作用机体,将对机体产生双重缺氧的联合打击。因此,对缺氧复合氰化物(NaCN)中毒的研究对我国的国防保障和未来的战争胜负都会产生极其重要的影响。
急性缺氧对机体的血压(SP)、心率(HR)的影响已有相关报道。有多篇文献提出,急性低压缺氧大鼠心室收缩功能指标如左室收缩压(LVSP),左室压力最大上升速率(+dP/dtmax)等均显著降低。心肌细胞缺氧不仅是严重创伤后心功能衰竭的重要原因,也是启动和诱发其它脏器损伤形成多脏器功能不全/衰竭的重要始动因素[2]。有研究发现,在高原缺氧条件下化学战剂毒性增强,对心功能损害效应加重,引起心率失常、心肌收缩力减弱等心脏功能异常变化[3]。目前,国内外对缺氧复合NaCN中毒所产生的联合效应对大鼠心脏结构和功能影响研究甚少,对药物预防救治效果亦未见有关评价方面的报道。
心脏是对缺氧极为敏感的器官。有研究表明,单纯缺氧和单纯NaCN中毒均可明显导致心肌细胞凋亡(Apoptosis),缺氧导致细胞死亡的主要方式是通过诱导细胞凋亡而产生的。在采用大鼠离体心肌细胞培养后、给予缺氧/复氧处理的实验研究中,发现了心肌细胞在持续较长时间的缺氧而再复氧时,细胞损伤现象严重,与曾经研究过的在体动物心肌细胞缺血/再灌注损伤情况一致。在缺氧/复氧损伤组中,流式细胞仪PI染色法的DNA分析直方图上,低于G1期的细胞数增多,明显高于其他各组,说明凋亡的细胞存在较多。透射电镜可见缺氧/复氧损伤组心肌细胞损伤严重,可有明显的凋亡前期、凋亡阶段或凋亡后期的改变,同时心肌细胞凋亡数目也明显增加,支持复氧(再灌注)损伤是心肌细胞凋亡的重要诱发因素。认为缺氧/复氧可加重心肌细胞损伤,伴着心肌细胞凋亡的增加;细胞内钙离子过多可能是触发心肌细胞凋亡的因素;超氧化物歧化酶(superoxide dismutase, SOD)可减少自由基的生成、降低心肌细胞凋亡的发生和缺氧预处理具有抗心肌细胞损伤和减轻心肌细胞凋亡的作用,均有可能是通过降低细胞内钙离子浓度而起作用的[4-6]。Fliss[7]等在在体动物模型研究中也发现了细胞凋亡是心肌再灌注损伤的特征之一,再灌注损伤可加速不可逆转的细胞凋亡。Musat-Marcu等[8]在体外灌注大鼠心脏发现其早期即可发生心肌细胞凋亡,且在抑制凋亡后伴随着心功能的恢复,提示心肌细胞凋亡参与了心功能的衰减。
细胞色素C(Cytochrome C, Cyt C)及细胞凋亡诱导因子(apoptosis-inducing factor, AIF)正常时分别位于线粒体内膜和线粒体膜间隙。缺血/再灌注(MI/R)时,PT孔受损而开放,Cyt C及AIF被释放入胞浆。Cyt C与胞浆中的凋亡激活因子-1(apoptosis- activating factor-1, Apaf-1)结合,依次激活半胱酸蛋白酶家族中的Caspase-9、Caspase-3而启动凋亡通路,活化的Caspase-3可以作用于胞质中的细胞骨架蛋白,或作用于细胞核中的DNA,引发细胞凋亡,这是线粒体caspase依赖性凋亡途径。而AIF经PT孔被释放入胞浆,其将易位入核,激活内源性核酸内切酶将染色体切割为180-200bp整数倍的DNA片断,同时加速Cyt C的释放,进一步促进细胞凋亡,这是线粒体caspase非依赖性凋亡途径。可见,线粒体caspase依赖性和caspase非依赖性这两条凋亡途径在心肌细胞凋亡中发挥重要作用。
缺氧预适应( hypoxic preconditioning, HPC),并可将其界定为“预先短时间非致死性重复缺血/缺氧后,机体组织细胞获得对随后长时间致死性缺血/缺氧损伤的高度耐受性”。缺氧预处理对心肌细胞可以产生预适应现象,缺氧预适应迄今为止被认为是最强有力的一种心肌内源性保护措施[9,10]。
人参皂苷(saponins of panax ginseng, SPG)能对抗氧自由基对心脏的损伤,保持心肌细胞膜的完整性,改善急性心肌缺血时心肌舒张功能,还能对抗心肌缺血所致心肌不可逆坏死,使SOD活性升高,心肌释放磷酸烯醇式丙酮酸羧激酶(phosphoenolpy- ruvate carboxykinase, PCK)减少。人参总皂苷(total saponins of Panax Ginseng, TSPG)对缺血再灌注损伤中的细胞坏死和细胞凋亡均有显著的保护作用[11-14]。人参皂苷尤其是Re可能具有阻滞K+通道的作用,可使离体豚鼠乳头状肌细胞动作电位时程和有效不应期均延长,人参皂苷抗心律失常的作用[15]。人参皂苷抗心肌缺血等作用研究表明,人参皂苷能对抗氧自由基对心脏的损伤,保持心肌细胞膜的完整性,改善急性心肌缺血时心肌舒张功能,还能对抗心肌缺血所致心肌不可逆坏死,使SOD活性升高,心肌释放PCK减少。侯明晓等利用体外培养的心肌细胞缺血再灌注损伤的模型的结果表明:TSPG对缺血再灌注损伤中的细胞坏死和细胞凋亡均有显著的保护作用[16]。Scott等研究发现,人参皂苷Rb1可抑制心肌细胞的收缩,有助于减少心肌的耗氧量[17]。自由基亦可损伤血管内皮细胞,引起动脉粥样硬化、高血压等心脑血管疾病。有实验表明,人参皂苷可降低过氧化物丙二醛的含量,从而减轻血管内皮细胞损伤。
而牛磺酸(Taurine, Tau)能清除氧自由基、保护细胞膜、使心肌酶和肝酶释放减少、具有极强抗氧化能力。它也能调节细胞内钙的稳态,减少肢体缺血再灌注时钙离子内流,而且中性粒细胞呼吸爆发产生大量过氧化氢(hydrogen peroxide, H2O2),与CL结合产生难以清除的强氧化剂-次氯酸,牛磺酸可与次氯酸结合,形成稳定的弱氧化剂-氯胺牛磺酸,从而除次氯酸对细胞的破坏作用。因此显示牛磺酸可纠正肢体缺血再灌注后远隔器官损伤[18]。长疗程应用牛磺酸在降低血压和逆转心肌肥厚的同时,也使高血压大鼠心肌细胞凋亡减少,并能降低大鼠心肌AngⅡ含量和ACE活性。因此,我们认为,长疗程牛磺酸治疗可抑制高血压大鼠心肌细胞凋亡和逆转心肌肥厚,上述作用可能与药物拮抗组织局部AngⅡ的生成有关。牛磺酸还具有抑制心肌细胞凋亡的作用,可能与其调控凋亡相关基因Fas、Bax和Bcl-2的蛋白表达有关。牛磺酸可以有效保护心肌细胞而避免发生心脏畸形,同时有降低血压[19,20]。
血液供应和血氧含量是心脏功能正常的先决条件,因此,任何引起血液中氧含量降低或心肌供血不足的有害因素都可能导致心脏功能异常。
那么,缺氧环境氰化物中毒对心脏功能将会产生何种影响?由此带来的心脏损伤其机制如何?线粒体caspase依赖性和caspase非依赖性这两条凋亡途径在缺氧环境氰化物中毒诱导大鼠心肌细胞凋亡中有何种作用?缺氧预适应、人参皂苷和牛磺酸等干预措施对大鼠缺氧环境氰化物中毒有无效果?这些问题迄今未见研究报道。本课题基于上述两种情况,在前期工作的基础上,采用实验动物研究和体外细胞培养研究相结合的方法,建立缺氧复合NaCN中毒的动物模型和NaCN染毒的细胞模型,通过观察模拟高原环境缺氧复合NaCN中毒对大鼠心脏的影响、检测SPG等干预措施对缺氧复合NaCN中毒大鼠心脏保护作用、观测心肌细胞NaCN染毒后细胞凋亡情况,以及定量分析上述情况下线粒体caspase依赖性和caspase非依赖性这两条凋亡途径中Cyt C、caspase-3和AIF等因子的表达,来探讨模拟缺氧条件下NaCN中毒后心功能改变的特点和心肌细胞凋亡的特性,为今后缺氧性NaCN中毒的预防救治措施的研究提供重要的理论基4础。最终为制定缺氧条件下复合NaCN中毒的预防和救治方案提供实验和理论依据。
方法:
1.检测有无干预措施情况下缺氧复合NaCN中毒后对大鼠心脏结构和功能影响,包括血流动力学、心电图、心肌酶谱、心肌病理切片光镜电镜观测等;
2.建立体外SD乳鼠心肌细胞NaCN染毒模型,检测急性NaCN染毒后心肌细胞是否存在凋亡情况;
3.定量分析有无干预措施情况下心肌细胞缺氧复合NaCN中毒后Cyt C、caspase-3和AIF表达,分析线粒体caspase依赖性和caspase非依赖性这两条凋亡途径在缺氧复合NaCN中毒引起心肌细胞凋亡中的特点;
结果:
一、人参皂苷等干预措施对缺氧和NaCN中毒大鼠心脏的保护作用
1.平原实验组大鼠NaCN中毒后,血流动力学发生改变:HR、mLVSP、+dP/dtmax等指标均较中毒前出现显著下降(P<0.01),T波振幅则出现显著上升(P<0.01),30min后逐渐向正常值恢复。
2.高原实验组大鼠NaCN中毒后,血流动力学改变比平原实验组更为明显:HR、mLVSP、+dP/dtmax等指标均较中毒前出现显著下降(P<0.01),T波振幅则出现显著上升(P<0.01),40min后逐渐向正常值恢复,但恢复程度和效果较平原实验组差。
3.平原实验组大鼠NaCN中毒后,Cyt C活性显著降低(P<0.01)。中毒2h后,Cyt C活性有明显的恢复(P<0.01)。
4.高原实验组大鼠在NaCN中毒后,Cyt C的活性显著低于平原实验组(P<0.01);中毒2h后也有一定程度恢复,但与平原实验组比较恢复更慢(P<0.01)。
5.平原和高原实验组大鼠NaCN中毒后均出现明显的心肌酶谱(AST、LDH、CK、CK-MB)变化,高原实验组大鼠的心肌酶谱改变比平原实验组大鼠更显著(P<0.01),并且恢复缓慢。其中,LDH变化最为显著。
6.平原实验组大鼠中毒后心肌组织损伤逐渐加重,至2h后最为严重,间质有腔隙形成,出现纤维退变,横纹不清,弥漫性充血、水肿变性,少数心肌有断裂,横纹消失;中毒6h时有明显的恢复。
7.高原实验组大鼠心肌组织中毒前即见充血、水肿变性、局灶性炎症细胞等变化。NaCN中毒后心肌损伤加重,出现严重水肿变性,弥漫性细胞肿胀,胞质模糊,并有水肿囊出现,细胞变性,间质血管充血。中毒6h时,高原实验组大鼠心肌组织损伤并无明显的恢复。
8.平原实验组大鼠中毒2h后出现线粒体轻微水肿,灶性溶解,内质网扩张,线粒体部分嵴和膜融合或消失等现象。
9.高原对照组大鼠超微结构也出现轻微损伤,心肌间质有轻微水肿,线粒体略有肿胀;高原NaCN中毒组大鼠线粒体则肿胀明显,有小量的嵴和膜融合或消失,心肌间质水肿,肌节排列稍紊乱,核旁水肿处线粒体大部分嵴和膜融合或消失,粗面内质网有脱颗粒现象;损伤程度明显重于平原实验组大鼠。
10. HPC和SPG、Tau灌胃干预后,对NaCN中毒和高原缺氧复合NaCN中毒引起的大鼠血流动力学改变有明显的干预作用,HR、mLVSP、+dP/dtmax、T波振幅等指标均较未干预组变化程度小,恢复效果更明显,且有统计学意义。
11. HPC干预后的大鼠NaCN中毒后Cyt C活性也出现显著降低(P<0.01),至2h时有所恢复,效果好于高原缺氧复合NaCN中毒大鼠(P<0.05)。
二、NaCN对SD乳鼠原代心肌细胞的凋亡作用
1.建立了NaCN染毒SD乳鼠心肌细胞模型;
2.测定出SD乳鼠心肌细胞NaCN染毒的IC50值:正常条件下IC50值为87.85μmol·L-1。
3.以IC50剂量的NaCN染毒后,SD乳鼠心肌细胞出现凋亡。凋亡细胞的细胞核由于染色质浓集而呈现亮蓝色,呈分叶、碎片状等。
三、caspase依赖/非依赖性途径在大鼠心脏损伤中的作用研究
1.大鼠缺氧复合/或NaCN中毒30min后,心肌组织胞浆Cyt C、caspase-3蛋白表达和mRNA表达均上调;
2.大鼠缺氧复合/或NaCN中毒30min后,心肌组织胞浆AIF蛋白表达上调,而AIF mRNA表达对NaCN染毒诱导却并不敏感;
3. Tau、SPG干预对抑制大鼠缺氧复合/或NaCN中毒30min后心肌组织胞浆Cyt C、caspase-3和AIF蛋白表达以及Cyt C、caspase-3 mRNA表达上调有明显作用,对AIF mRNA表达无明显作用。
结论:
1.缺氧复合NaCN中毒大鼠血流动力学HR、mLVSP、+dP/dtmax等指标均显著下降,T波振幅显著增高。相对于单纯的缺氧和单纯的NaCN中毒,两种因素同时作用所引起的心脏功能损伤,特别是心脏的收缩能力的损伤更加明显;
2.缺氧复合NaCN中毒大鼠的血液生化指标,如心肌酶谱(AST、LDH、CK、CK-MB)的变化也更为明显,Cyt C活性下降也更为显著。表明两种因素作用下大鼠的心脏生化代谢变化很大,加重心脏功能的紊乱;
3.病理检测结果表明,缺氧复合NaCN中毒后大鼠心肌组织的结构损伤更为严重,超微结构的病理改变更加明显,特别是NaCN中毒的靶细胞器-线粒体的损伤,这可能是引起心肌细胞凋亡的最主要原因;
4. HPC、SPG和Tau等干预措施,对缺氧复合NaCN中毒大鼠心脏功能的恢复和维持Cyt C的活性均有一定的效果。可考虑将HPC、SPG和Tau等列入缺氧复合NaCN中毒防护措施的筛选;
5.通过建立的体外SD乳鼠心肌细胞NaCN染毒模型,发现经IC50剂量NaCN染毒SD乳鼠原代心肌细胞凋亡。提示细胞凋亡可能在缺氧复合NaCN中毒心脏损伤中发挥作用;
6.大鼠缺氧复合/或NaCN中毒30min后心肌组织胞浆Cyt C、caspase-3蛋白表达和mRNA表达上调,线粒体caspase依赖途径导致的心肌细胞凋亡十分明显,寻找阻止线粒体caspase依赖途径心肌细胞凋亡的措施可能会对缺氧复合NaCN中毒的预防和治疗带来一定的效果;
7. Tau、SPG干预,对抑制大鼠缺氧和/或NaCN中毒心肌组织胞浆Cyt C、caspase-3和AIF蛋白表达以及Cyt C、caspase-3 mRNA表达上调有明显作用,提示Tau、SPG可作为预防线粒体途径引起心肌细胞凋亡的筛选措施;
8.大鼠NaCN中毒30min后,心肌组织胞浆AIF蛋白表达虽然出现上调,但AIF mRNA表达变化却并不明显;AIF mRNA表达对缺氧的敏感程度强于对NaCN中毒,提示NaCN中毒后线粒体非caspase依赖途径引起心肌细胞凋亡的机制并不同于caspase依赖途径,缺氧、NaCN中毒引起心肌细胞凋亡可能存在不同的机制,值得深入研究。
Exemplified by the HCN, Cyanogen agents (blood agents) are highly toxic. Teir intocication effects are potent, quick and hard to defend. As a result, it has captured the attention of foreign military forces. Attachment three of the UN’s ban on chemical weapons, cyanogens chloride and hydrogen cyanide ranked 2 and 3 on the list. Both of which are cynogen agents. They are referred to as“the king of speed kills”. They are considered as the top chemical weapons of U.S. and Russia. After intoxication with the agent, the subject experiences hypoxia and difficulty breathing.
The toxicant acts quickly and disseminates through the body with incredible speed. It could kill the subject within 10 minutes. After the cynogen agents enters the body, the CN ion stops cells from performing respiratory functions and oxidative phosphorylation. It inhibits oxidases the end of the Mitochondrial respiratory chain. This causes the tissues to die due to lack of toxic lack of oxygen. Also known as damage to“internal respiratory”. Medical definition for plateau is 3 000m above sea level. Hypoxia is a characteristic of plateaus. Some people demonstrate clear symptoms of hypoxia. Any level higher than this could have substantial effects on Physiology, biochemistry and anatomy. Our country is full of plateaus. Just the Tibetan plateau alone covers 250 square kilometers of land. The plateau borders many other countries. The military values of these plateaus are of great importance. When NaCN is mixed in with the harsh environment of the plateaus, hypoxia kicks in from both external and internal. When this occurs, the rate at which a subject dies doubles. To understand and analyze the use and nature of NaCN intoxication is of great importance for national defense of our country.
The heart functions under the condition of ample blood supply and oxygen. Therefore, any lack of blood supply or the level of oxygen in the blood will cause the heart to function abnormally. Sudden decrease in oxygen level could lead to fluctuation in blood pressure and heart rate. Many studies have shown that when hypoxia is induced in lab rat, the left ventricle pressure and maximum pressure decreases dramatically. Myocardial hypoxia is not only a serious post-traumatic heart failure, but also other start-induced organ damage and the formation of multiple organ dysfunction and failure of the move before an important factor. Studies have found that oxygen under the conditions of soman toxicity increased.
Heart is a very sensitive organ to hypoxia; studies have shown that simple and pure hypoxia induced by NaCN poisoning can result in significant cardiomyocyte apoptosis, hypoxia leading to the main form of cell death through induction of apoptosis arising. In the use of isolated rat cardiac cells to give H / R in the way of experimental studies have found that the myocardial cells in continued lack of oxygen condition for a longer period of time have achieved reoxygenation. The animal myocardial cells in vivo ischemia / reperfusion injury is in the same situation. In hypoxia and reoxygenation, there is injury in the group. PI staining by flow of cytometry analysis of DNA histogram showed that it’s lower than the number of cells in G1 phase of the increase and it was significantly higher than other groups. There are more cell apoptosis. Transmission of electron microscopy can see hypoxia / reoxygenation group of serious cardiac cell injury is a clear pre-apoptosis. Apoptosis in the late stages changes at the same time the number of cardiomyocyte apoptosis is also a noticeable increase in support for the resumption of oxygen (again Reperfusion). Injury in myocardial apoptosis is an important predisposing factor. The H / R can increase the myocardial cell damage, accompanied by an increase in myocardial apoptosis; intracellular calcium may be triggered by excessive apoptosis of myocardial factors; SOD (superoxide dismutase, SOD) can be reduced. The generation of free radicals and reduced the incidence of myocardial apoptosis and hypoxic preconditioning with anti-myocardial cells to reduce myocardial injury and apoptosis. It’s likely to have lower intracellular calcium ion concentration. Fliss, and so on in the body in an animal subject study also found that the apoptosis of myocardial reperfusion injury is one of the characteristics. Reperfusion injury can accelerate irreversible apoptosis. Musat-Marcu and so on at the heart of rat in vitro perfusion can be found in its early occurrence of myocardial apoptosis, and inhibit apoptosis in the post along with the restoration of heart functions. This suggests that cardiomyocyte apoptosis isinvolved in the attenuation of cardiac function.
Cytochrome C (Cyt C) and apoptosis-inducing factor are located in normal mitochondria mitochondrial membrane and space. Ischemia / reperfusion time, PT and the opening hole damage, Cyt C and AIF were released into the cytoplasm. Cyt C with the cytoplasm of apoptosis-activating factor-1(apoptosis-activating factor-1, Apaf-1), followed by activation of acid cysteine protease family of Caspase-9, Caspase-3 and start the apoptosis pathway, activation of Caspase-3 can be in the cytoplasm of the role of the cytoskeleton protein, or acting on the nuclear DNA, lead to apoptosis. The AIF was released by PT hole into the cytoplasm, the translocation into the nucleus, activated endogenous endonuclease chromosome will be cut for several times the entire 180-200bp fragment of DNA, at the same time to speed up the release of cytochrome C and further promote cell Apoptosis. Cyt C, AIF can be seen as apoptosis of the two channels in cardiac cells. They play an important role in apoptosis.
Hypoxic preconditioning, and can be defined as "a short period of time in advance to repeat non-fatal ischemic / hypoxic, the body of the cell was followed by a long-fatal ischemic / hypoxic injury A high degree of tolerance.”Hypoxic preconditioning on myocardial cells can generate Preconditioning. Hypoxic preconditioning so far is considered to be one of the most powerful endogenous myocardial protective measures. Ginsenosides can act as antioxidants to free radical damages to the heart, to maintain the integrity of myocardial cell membrane and improve acute myocardial ischemia diastolic function. But also against myocardial ischemia-induced myocardial necrosis is irreversible, so that the increased activity of SOD can release myocardial PCK Reduction. Ginsenosides have significant protective effects on ischemia-reperfusion injury in cell necrosis and apoptosis.
In addition, taurine removes oxygen free radicals and protects the cell membrane, so that the enzyme reduces the release of liver enzymes, which are very anti-oxidized. It also can regulate the intracellular calcium homeostasis, reduces limb ischemia reperfusion. During calcium influx, neutrophil respiratory burst a large amount of H2O2 and CL, having a combination of difficulty to get rid of the strong oxidizer - hypochlorous acid and Taurine With hypochlorous acid, to form a stable low-oxidants-taurine chloramine, and thus clear the hypochlorite on the role of cell damage. Therefore taurine corrects limb ischemia-reperfusion injury in distant organs. Application of taurine in the long course of treatment to lower blood pressure and reversal of cardiac hypertrophy are highly possible. High blood pressure also reduces myocardial apoptosis, and can reduce myocardial AngⅡcontent and ACE activity. As a result, we believe that the long course of taurine treatment of high blood pressure can inhibit myocardial apoptosis and reverse cardiac hypertrophy. The role of antagonist drugs may be related to local organizations related to the generation of AngⅡ. Taurine inhibition of myocardial apoptosis also may be related to apoptosis-related gene regulation Fas, Bax and Bcl-2 protein expressions. Taurine can protect myocardial cells and avoid the occurrence of cardiac malformation, at the same time have lower blood pressure.
The normal supply of blood and oxygen to heart bring into full play. Accordingly, some adverse factor that result lower oxygen content of blood and insufficiency of blood for the cardiac muscle will lead to abnormal cardiac function.
Hypoxic environment of cyanide poisoning changes how the heart functions? How the mechanism of the heart resulting in injury? What are the characteristics of myocardial apoptosis? How to search for possible interventions? Research on these issues has not been reported so far. To be the subject of the preliminary research on the basis of the establishment of hypoxia and cyanide poisoning of animal models and cell models to study hypoxia on the compound sodium cyanide poisoning Cyt C, AIF apoptosis two way interference with the role of hypoxia to find Cyanide poisoning and two different factors at the same time under myocardial apoptosis. The key to explore the hypoxic preconditioning, as well as ginsenosides is taurine and anti-hypoxia medicines interference with the effects of hypoxia environment for the prevention and treatment of cyanide poisoning research Theory. Based on this subject in both cases, the use of laboratory animals in general studies and cell culture in vitro study of the combination will implement both methods. By observing the simulation of composite NaCN high altitude hypoxia in rat poisoning on heart structure and function as well as myocardial hypoxia and NaCN poisoning After Cyt C, caspase-3 and AIF expression of quantitative analysis, as well as Cyt C and AIF caused by apoptosis analytical effect, to explore simulated hypoxic conditions of NaCN poisoning after cardiac function changes in the characteristics of myocardial apoptosis. The future of hypoxic NaCN poisoning prevention measures the medical treatment studies and it provides important theoretical basis. For the ultimate development of composites hypoxia NaCN poisoning prevention and treatment programs, it will implement the following methods.
Method:
1. Testing whether interventions of hypoxia and NaCN intoxication after rat cardiac structures will function. Utilizing methods include hemodynamics, ECG, myocardial enzymes, myocardial biopsy light microscope, such as electron microscopy observation;
2. the establishment of in vitro NaCN poisoning induced by hypoxia and myocardial cell damage model to detect hypoxia and NaCN poisoning under the conditions of myocardial apoptosis and its interventions on the impact of cardiac apoptosis;
3. any intervention under the circumstances NaCN complex cardiomyocytes hypoxia poisoning after caspase-3, Cyt C and AIF expression detection, analysis Cyt C and AIF caused by the two channels the characteristics of apoptosis;
Results:
Part one: Change of hypoxia and NaCN intoxication on structure and fuction in rat.
1. Hemodynamics of rat in the plain experimental group takes place change after NaCN intoxication. These markers, such as HR, mLVSP, +dP/dtmax show up significance descent(P<0.01) and the amplitude of T wave show up significance elevation(P<0.01) after NaCN intoxication than no NaCN intoxication. Then, they get a recover to normal value after 30 minute.
2. Hemodynamics of rat in plateau experimental group take place more evident change after NaCN intoxication than the plain experimental group. These markers, such as HR, mLVSP, +dP/dtmax show up significance descent(P<0.01) and the amplitude of T wave show up significance elevation(P<0.01) after NaCN intoxication than no NaCN intoxication. Then, they get a recover to normal value after 40 minute. Nevertheless, the recover degree and effect is bad.
3. Change of the cardiac muscle enzymogram in the plateau experimental group is more obviously than in plain experimental group, and it shows up significant difference (P<0.01). This change difficultly put back. Variation of LDH value is the most significant, and it is with one accord that anaerobic respiration corresponding reinforce in the hypoxia environment.
4. The activity of Cyt C show up significant decrease after NaCN intoxication in plain experimental group. Behind tow hours, the activity occur evident recovery.
5. Compare to in plain experimental group, the activity of Cyt C in plateau experimental group show up significance decrease after NaCN intoxication. Behind tow hours, the activity occur recovery to normal value, but this recovery is the more slowing.
6. Cardiac muscle tissue damage of rat gradually aggravate in plain experimental group after NaCN intoxication. It is the most severity after two hour. There is lacune formation in interstitial substance, and showing up fibrilla cataplasia, transverse striation no clear, diffuse hyperemia, hydropic degeneration, and so on. There is apparent recovery after six hour.
7. Cardiac muscle tissue of normal rat occur some symptom in plateau experimental group, including hyperemia, hydropic degeneration, colour shallowness, focal inflammation cell, etc. Myocardial damage rapidly aggravate after NaCN intoxication. There are severely hydropic degeneration, diffuse cellular swelling, cytoplasm ambiguity, hydrops capsule, cell degeneration and interstitial substance vasocongestion. These symptoms don’t emerge recovery after six hour.
8. Mitochondrion of rat occur light hydrops and swelling and distension, deliquescence, cristae and membrane fusion or delitescence in plain experimental group after NaCN intoxication two hour.
9. Mitochondrion of rat occur light hydrops and swelling and distension in plateau experimental group. It shows up obviously Swelling, cristae and membrane fusion or delitescence, etc. after NaCN intoxication. Degree of injury is the more severity.
10. HPC, SPG and Tau bring out comparative effect to Hemodynamics of rat after hypoxia and NaCN intoxication. These indexes, such as HR, mLVSP, +dP/dtmax and amplitude of T wave show up less change degree. Rat gets obviously recovery.
11. Activity of Cyt C of HPC rat shows up significance depress after NaCN intoxication, and gets recovery after two hour. The recovery effectiveness is better in HPC rat group than in plateau rat group, and worse in HPC rat group than in plain rat group.
Part two: Apoptosis detection of origin generation cardiac muscle cell of SD neonate rat
1. Model constructing of cardiac muscle cell of SD neonate rat used NaCN narcotics.
2. Assay of IC50 value of origin generation cardiac muscle cell of SD neonate rat. IC50 value is 87.85mmol/L.
3. Origin generation cardiac muscle cell of SD neonate rat show up apoptosis by IC50 dosage NaCN used narcotics, and Cell Nucleus appear sapphirine owing to chromatin enrich, and so on.
Part three: Expression detection of Cyt C, caspase-3, AIF in cardiac muscle cell of SPG and Tau intervention rat after NaCN intoxication
1. There are Expression up-regulation of Cyt C, caspase-3 and their mRNA of hypoxia and NaCN intoxication rat after 30 minute in cardiac muscle tissue endochylema.
2. There is Expression up-regulation of AIF of hypoxia and NaCN intoxication rat after 30 minute in cardiac muscle tissue endochylema. But, Expression of AIF mRNA show up insensitivity to NaCN used narcotics.
3. HPC, SPG and Tau interventions bring out comparative effect to Expression depress of Cyt C, caspase-3 and their mRNA, AIF after hypoxia and NaCN intoxication.
Conclusion:
1. Hemodynamics markers, such as HR, mLVSP, +dP/dtmax of rat in plateau experimental group show up significance descent after NaCN intoxication, and amplitude of T wave show up significance heightening. These direct that cardiac contractile function of rat occur the more severity injury.
2. Biochemical indicator, such as cardiac muscle enzymogram and Cyt C activity of rat in plateau experimental group shows up significance change after NaCN intoxication. These direct that heart biochemistry metabolism of rat occur large change, and it results to cardiac function disorder.
3. Ultrastructural pathology variation and injury of cardiac muscle tissue of rat in plateau experimental group get all the more severity after NaCN intoxication. Mitochondrial injury especially is possible to cause cardiac muscle cell apoptosis.
4. These intervention study including HPC, SPG and Tau are effect for recover of cardiac function and Cyt C activity. They act as bolting measures for prevention and treatment of hypoxia and NaCN intoxication.
5. Origin generation cardiac muscle cell of SD neonate rat show up apoptosis by IC50 dosage NaCN used narcotics. Otherwise, cardiac muscle cell apoptosis has induced by purely hypoxia. These hint cardiac muscle cell apoptosis all the more severity under hypoxia and NaCN intoxication koinonia, and it may be important reason of heart injury.
6. There are Expression up-regulation of Cyt C, caspase-3 and their mRNA of hypoxia and NaCN intoxication rat after 30 minute in cardiac muscle tissue endochylema. Action of mitochondria caspase dependency way is obviously to cardiac muscle cell apoptosis, and it may bring out good effect for prevention and treatment of hypoxia and NaCN intoxication.
7. SPG and Tau interventions bring out comparative effect to Expression depress of Cyt C, caspase-3 and their mRNA, AIF after hypoxia and NaCN intoxication. It hint SPG and Tau maybe act as bolting measures for prevention and treatment of hypoxia and NaCN intoxication.
8. There is expression up-regulation of AIF of anoxia and NaCN intoxication rat after 30 minute in cardiac muscle tissue endochylema. But, it is not obviously to expression change of AIF mRNA. These results direct that sensitive degree of AIF mRNA expression is better in hypoxia rat than in NaCN intoxication rat, and cardiac muscle cell apoptosis mechanism of mitochondria caspase dependency way is different with mitochondria non-caspase dependency way after NaCN intoxication, and cardiac muscle cell apoptosis mechanism of hypoxia is different with NaCN intoxication. These results are worth for lucubrate.
引文
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