丹酚酸B对脑缺血小鼠脑组织线粒体作用及其机制的研究

【作者】 张颖；

【导师】 蒋玉凤；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2007， 硕士

【摘要】 脑血管病中医称为中风,也叫卒中,因其发病急,变化快,类似风善行数变的特点而命名。中风的主要症状为突然昏仆,半身不遂,口舌歪斜,语言謇涩或不语,偏身或全身麻木等。中风病发病率高,起病急,后果严重,常迅速进入昏迷,并伴有抽搐、高热,高热程度越严重,时间越长,越容易引起继发性脑组织再损伤而加重症状,同时也容易引起其它重要脏器严重的并发症,形成恶性循环。脑血管病分为出血性脑血管病和缺血性脑血管病两种,出血性脑血管病约占所有脑血管病的10%～15%,脑出血病人的预后不好,病死率高。缺血性脑血管病是导致人类死亡的三大主要疾病之一,是病残的最主要原因,其主要包括脑血管痉挛、脑血栓形成和脑栓塞,缺血性脑血管病的病理基础是脑动脉粥样硬化,而脂质代谢障碍又是形成动脉粥样硬化的重要因素。随着科学技术的发展,脑血管病通过CT和MRI等技术在早期即可以进行诊断并进行相应治疗。在脑血管病的治疗上,国际上有可能在基因治疗及细胞治疗上取得突破性进展并成为脑血管病治疗最有效的方法。缺血性脑中风的病因病机是以虚为本:清代医家王清任认为,气虚是缺血性脑中风的根本,血瘀是缺血性脑中风的核心。基本病因为风、火、痰、瘀、气、虚六种,主要病机为瘀血痰浊互阻,蒙蔽清窍,而致神机失统,发展成中风。西医学中的毒性氧自由基、兴奋性氨基酸、代谢性酸中毒、钙离子超载、炎性介质、血管活性物质过度释放等,均可看作是中医的毒邪。丹参为唇形科植物的干燥根及根茎,是我国传统中药常用的理血类中药材,性微寒、味苦,归心、肝经,具有祛淤止痛、活血通经、清心除烦之功效。丹参主要含有两类有效成分:一类为脂溶性的菲醌类成份,另一类为水溶性的多聚酚酸类成份,近年来,随着对丹参的深入研究,发现水溶性丹酚酸类是丹参的主要有效成份,具有多方面的药理活性,在药理作用方面甚至超过脂溶性成份。丹酚酸B(又称丹酚酸乙,Salvianolic acid B,SalB)是丹参中重要的水溶性有效成份之一,在临床上治疗缺血性的心脑血管疾病发挥了极好的疗效,受到了医学界的首肯。有资料显示,SalB能抑制脂质过氧化、清除氧自由基的活性、保护相关内皮细胞及降低细胞内钙浓度及增强学习记忆等功能,是丹参发挥药效的主要物质基础之一。目前SalB对缺血性脑血管疾病的防治已有大量的研究报道,本实验复制小鼠脑缺血模型,探讨SalB对急性脑缺血脑组织损伤的保护作用。本论文分为文献综述和实验研究两部分。1综述综述一主要介绍现代医学对缺血性脑血管病的发病机制及治疗概况。综述二主要介绍了传统医学对缺血性脑中风的研究概况,包括对病因病机及发病部位的论述和中医治疗脑中风的常用方法和方药。2实验研究2.1实验内容实验一目的:观察SalB对脑缺血小鼠脑组织能量代谢的影响。方法:复制急性脑缺血模型,将NIH小鼠随机分为4组:假手术组、模型组、SalB治疗组和尼莫地平对照组。检测脑缺血6min、12min、18min、24min和30min的ATP、ADP、AMP、磷酸肌酸及乳酸的含量。实验二目的:观察SalB对脑缺血小鼠线粒体的影响。动物分组及模型复制同实验一,检测线粒体膜电位(MMP)、细胞内钙浓度及细胞凋亡率的变化。实验三目的:观察SalB对脑缺血小鼠脑组织Na+-K+-ATP酶和脑含水量的影响。动物分组及模型制作同实验一,检测Na+-K+-ATP酶活性,计算脑含水量和脑指数。2.2实验结果2.2.1 SalB对脑缺血小鼠脑组织能量代谢的影响急性脑缺血6min,ATP、ADP、AMP和磷酸肌酸含量开始下降,缺血12min-30min与假手术组比较差异显著(P<0.05-0.01);用SalB和Nim治疗,缺血24min-30min与模型组比较有显著差异(P<0.05-0.01)。脑缺血后,乳酸含量增加,缺血12min-30min,模型组与假手术组比差异显著(P<0.05-0.01),SalB和Nim治疗后乳酸含量降低,24min-30min,与模型组比较差异极显著(P<0.05-0.01)。2.2.2 SalB对脑缺血小鼠脑组织线粒体的影响脑缺血6min时,线粒体膜电位下降,缺血24min、30min与假手术组比较有显著差异(P<0.05-0.01),SalB和Nim治疗后,24min-30ming与模型组相比差异显著(P <0.05 -0.01)。缺血6min时,细胞内钙离子浓度和细胞凋亡率高于假手术组,钙离子缺血12min-30min模型组与假手术组比较差异显著(P<0.05-0.01),凋亡率在缺血18min-30min与假手术组比较有统计学意义(P<0.05-0.01),用SalB和Nim治疗后,细胞内钙离子浓度和凋亡率下降,钙离子浓度于缺血18min-30min与模型组比较差异极显著(P<0.05-0.01),凋亡率于缺血24min-30min与模型组比较差异明显(P<0.05-0.01)。2.2.3 SalB对脑缺血小鼠脑组织Na+-K+-ATP酶和脑含水量的影响脑缺血6min,Na+-K+-ATP酶活性开始下降,18min-30min与假手术比较差异显著(P<0.05-0.01),用SalB和Nim治疗,在缺血30min与模型组比较有显著差异(P<0.05)。脑指数和脑含水量在脑缺血6min时上升,脑指数于脑缺血24min-30min与假手术组比较差异显著(P<0.05-0.01),用SalB和Nim治疗后于脑缺血30min时与模型组比较差异显著(P<0.01)。脑含水量于脑缺血18min-30min与假手术组比较差异显著(P<0.05-0.01),而脑缺血30min时与模型组比较有差异(P<0.05)。2.3结论2.3.1 SalB通过升高ATP、ADP和PCr含量、EC值,降低乳酸含量起到改善脑缺血后脑组织能量代谢障碍的作用。2.3.2 SalB通过升高线粒体膜电位、降低细胞内钙离子浓度,从而达到保护线粒体,减少细胞凋亡的作用。2.3.3 SalB通过增强Na+-K+-ATP酶的活性,降低脑指数和脑含水量,达到减轻脑水肿的作用。更多还原

【Abstract】 Cerebrovascular disease consisted of ischemia and Hemorrhagic which accounted for 10-15%. The rate of death that patients suffered from hemorrhagic is totally higher. Ischemic cerebrovascular disease that included transient ischemia paroxysm(10%),cerbral thromosis and cerebral embolism(50%)is one of the three principal disease causing peple to death in which seconded only to heart disease and cancer, but it was the main reason of disabled people. transient ischemia paroxysm called transient cerebral ischemia by Chinese traditional medicine is forerunner of cerbral thromosis and cerebral embolism. The pathology basis of ischemic cerebrovascular disease was cerebral atherosclerosis that causes by dyslipidosis,dyslipidoses. From the study carried on ischemic cerebrvascular disease by scholars all over the world, ischemic cerebrovascular disease will have its breakthrough in theraphy from gene therapy and cell therapy which will become the most effective methods to cure it at last.Ischemic Cerebralvascular was called stroke by Chinese traditional medicine which is characteristic of high morbidity, rapid change . The main symptoms was suddenly falling into a swoon, hemiparalysis, crooked tongue, numbness of half body and so on. Because of high, acute, serious morbidity, the stroke easily evoked muddled, convulsion, hyperpyrexia, with the severity of hyperpyrexia and length of muddled, serious complication of other organs appeared easily, which gived arise to infernal circle on entirely body.The etiology and pathgenesis of stroke was mainly on the basis of endopenia of qi and blood. Wang Qiren thought that the foundation of stroke was deficiency of vital energy and the core was stagnation of blood. Main etiology of stroke was wind, fire, sputum, stasis, qi and deficiency, and the basic pathogenesis of stroke was toxicity due to stasis and phlegm which spirt was out of control and further develop stroke..EAAs, overloading calcium, toxicity of microbe and so on in western medicine were seen the toxicity in TCM.Dashen that was general chinese medicine all purpose was the root and rhizomaof Salvia miltiorrrhiza Bge. With frigidity, bitter in taste, because it had affect on heart and liver meridian, it can activate blood to promote menstruation, ecercise slit and alleviate pain,schedul heart to vex. Dashen was thought as chinese medicine regulating blood that was generally used. Dashen was composed of two active components:fat-soluble PAQ and water-soluble poly-phenolic acid. Recently, with the further study, as for pharmaco-activity it found that the effects was more stronger in water-soluble Salvianolic compared with fat-soluble . Being one of the important active substances in water-soluble components, SalB have excenllently therapeutic effect in curing CAD and cerebrovascular disease. Some documents showed that SalB can protect endotheliocyte, depress the concentration of calcium and enhance the fundation of study and memory by inhibiting lipid peroxidation, scavenging the acvities of oxyradical. The experiment duplicate the model of acute cerebral ischemia to investigate effects of SalB on brain injury induced by cerebral ischemia.1 Overview:Overview 1: pathogenesy and method of treatment on cerebralvascular disease was mainly introduced。Overview 2: The research on stroke was mainly introduced, which included etiopathogenisis and pathogenes, position of disease, general medthod of therapy and fomula and pharmacon.2 Experiment research2.1 The contents of experimentExperiment 1: To observe the effects of SalB on energy metabolism in mice with cerebral ischemia. After acute cerebral ischemia model were duplicated and NIH mice were divided into four groups: sham group, model group, SalB group, Nim group, the experiment was operated at 6min, 12min, 18min, 24min, 30min with HPLC that ATP, ADP,AMP and PCr were detected and Lac kit that Lac was detected.Experiment 2: To observe protective effect of SalB on mitochondrial in mice with cerebral ischemia. The group and model were the same as the experiment1.MMP, content of calcium and apoptosis were detected with FCM. Experiment 3: to intestigate the effect of SalB on brain water in mice with cerebral ischemia. Group and model were the same as experiment1, the activity of Na+-K+-ATPase was detected by kit, and the brain index and the brain water were detected after the brain tissue was oven dry.2.2 The results of experiments:2.2.1 The effect of SalB on energy metobolism in mice with cerebral ischemia After acute cerebral ischemia at 6min, EC and the content of PCr were decreasing, compared with sham group, EC and content of PCr were dramatically different at 12min(P<0.05). By SalB and Nim treating, the two groups have differences compared with model group from 12min. When cerebral ischemia appeared, the conten of Lac was increasing. Compared with sham group, the difference was began at 12min. after SalB and Nim treating, the content of Lac began to decress. There was difference from 24min compared model group( P<0.05-0.01).2.2.2 Protective effect of SalB on mitochondrial in mice with cerebral ischemia The MMP began to decreased when crerbral ischemia. Compared with sham group, there was difference(P<0.05-0.01) from 24min. By treating by SalB and Nim, there was difference from 24min ( P<0.05-0.01). The content of calcium and rate of apoptosis began to increased after cerebral ischemia, and there were difference between sham group and model group at 12min and 18min. after SalB and Nim treating there were difference with the content of calcium at 18min and the rate of apoptosis at 24min( P<0.05-0.01).2.2.3 Inhibitory effect of SalB on edema in mice with cerebral ischemia. After cerebral ischemia, the brain index and brain water were increasing, compared sham group, there were difference with brain water at 18min and brain index at 24min(P<0.05-0.01). After SalB and Nim treating, difference appeared at 30min(P<0.05-0.01); The activity of Na+-K+-ATPase began to decrease, there was difference at 18min compared with sham group(P<0.05-0.01), after SalB and Nim treating there was difference at 30min(P<0.05).2.3 Conclusion2.3.1 SalB can improve energy metabolis of brain tissue with ischemia;2.3.2 SalB can protect the mitochondrial of brain tissue with ischemia2.3.3 SalB can inhibit the edema after cerebral ischemia.更多还原