知母有效成分对脑缺血的保护作用及其机制研究

【作者】 邓云；

【导师】 徐秋萍；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2004， 博士

【摘要】 随着人口老龄化的加剧,脑血管病,尤其是缺血性脑血管病越来越成为威胁人类健康的主要疾病之一,它不仅严重损害了患者的健康和生命质量,也给社会和家庭带来了沉重的负担。因此,明确脑缺血损伤的病理生理机制,研制和开发出新型、有效、副作用小的防治药物势在必行。 脑缺血的病理生理机制复杂,研究认为其与缺血再灌注后能量代谢障碍、兴奋性氨基酸毒性、自由基大量产生、细胞内钙超载、血小板激活、炎症损伤等诸多因素相关。现今血小板活化、炎症免疫损伤、核转录因子的调控下细胞因子的释放以及粘附分子的表达等机制越来越受到研究者的重视。 知母有效成分(化合物 9714)是军事医学科学院放射医学研究所提供的一种来源于中药知母的含有两个糖链的水溶性甾体皂苷化合物,结构类似糖皮质激素。本课题根据知母有效成分(化合物 9714)的结构特征,并结合脑缺血的研究现况,从整体、细胞、分子等不同水平,体内外研究相结合,利用生化测定、免疫组织化学、酶联免疫、分子生物学、细胞培养等多种方法系统分析探讨了化合物 9714 防治脑缺血损伤的药理作用及可能机制,为研究开发抗脑缺血新药提供一定的理论和实验依据。研究共分为以下五个部分:1 化合物 9714 对实验动物脑功能衰退的改善作用 学习记忆能力下降是脑缺血致脑功能障碍中的一种典型表现。本实验采用双侧颈总动脉结扎这一经典脑缺血模型,用 Morris 水迷宫测定各组动物空间学习记忆能力的差异,用 HE 染色及 SABC 免疫组织化学染色分析化合物 9714 对脑缺血模型大鼠病理形态学及神经生长因子、粘附分子表达的影响;同时鉴于胆碱系统的功能紊乱、胆碱受体密度降低与学习记忆、认知障碍、脑功能衰退之间的重要关系,实验中采用 M 受体阻断剂东莨菪碱 ip 0.5mg/kg 致获得性记忆障碍小鼠模型,观察化合物 9714 对模型小鼠学习记忆能力的影响,及对其海马中胆碱能受体的影响。结果表明,化合物 9714 10mg/kg、20mg/kg、40mg/kg 造模后连续给药 40 天,可以明显改善脑缺血大鼠的学习记忆障碍;改善神经细胞的变性坏死;增强脑皮质中脑源性神经生长因子(BDNF)的表达,降低细胞间粘附分子-1(ICAM-1)的表达;化合物 9714 15mg/kg、30mg/kg、60mg/kg 连续预防给药 30天,能明显改善获得性记忆障碍模型小鼠的学习记忆能力,并明显上调其海马中胆碱能M1 受体,同时具有明显上调 N 受体亚型 α7(Nα7)的作用趋势,与空白组相比有显著性差异,对 N受体亚型 α4无明显作用。2 化合物 9714 对局灶性脑缺血大鼠的保护作用 常用的脑缺血模型包括全脑缺血模型和局灶性脑缺血模型两大类。本实验采用 FeCl3化学敷贴法致大脑中动脉血栓形成(MCAT)模型及栓线法致大脑中动脉(MCA)缺血再灌注模型,观察化合物 9714 对脑缺血大鼠神经症状、脑梗塞范围、脑水含量、脑血流<WP=5>2 知母有效成分对脑缺血的保护作用及其机制研究量及多项生化指标的影响。结果显示,化合物 9714 20mg/kg、40mg/kg能明显缩小 MCAT模型大鼠的脑梗塞范围,改善其神经症状,并显著降低患侧脑水含量,改善脑水肿;化合物 9714 10mg/kg、20mg/kg、40mg/kg 能显著减低脑组织表面滴加 FeCl3 致血栓形成模型大鼠造模后 50min 时的脑血流量下降幅度,并延长脑血流量下降 50% 所需时间;化合物9714 20mg/kg、40mg/kg 能明显降低栓线法致 MCAO 再灌模型大鼠脑组织中 MPO 活性、提高 T-SOD活性、降低 MDA含量,以及降低 T-NOS、i-NOS 活性、减少 NO含量。3 化合物 9714 对血栓形成、血小板聚集和血液流变学的影响 血栓形成是引起缺血性脑血管病及血栓栓塞性疾病的重要病理过程之一,与血管壁损伤、血液成分异常及血流动力学改变密切相关。研究表明,脑缺血后血小板激活,血液流变学异常,血栓易于形成。血小板活化是动脉血栓的起始因素,花生四烯酸代谢通路是其活化的重要途径之一。本实验采用动静脉旁路血栓形成模型,胶原、ADP 诱导血小板聚集模型,以及皮下注射肾上腺素加冰水浸泡致急性血瘀模型,观察了化合物 9714 对动静脉旁路血栓、血小板聚集、血液流变学以及花生四烯酸代谢通路的影响。结果表明,化合物 9714 10mg/kg、20mg/kg、40mg/kg 能明显抑制动-静脉旁路血栓形成;明显抑制胶原、ADP 诱导的大鼠血小板聚集;明显降低急性血瘀模型大鼠全血粘度及血浆粘度,提高其红细胞变形性,降低红细胞聚集性;以及能明显降低 MCAO 再灌注模型大鼠血浆中TXB2/6-Keto-PGF1α 比值。同时体外实验中我们首次发现,化合物 9714 发挥血小板聚集作用的有效成分为其代谢产物(10-4, 10-5, 10-6 mol/L),而 9714 原药无此作用。这可能正是药效学实验中该药起效较慢,给药时间较长的原因之一。4 化合物 9714 对脑缺血再灌大鼠细胞因子、粘附分子、核因子 NF-κB 表达的影响 缺血性脑损伤与缺血再灌注后的炎症免疫反应密切相关。研究表明,细胞因子的释放、粘附分子的表达以及炎症免疫靶细胞的激活在脑缺血损伤过程中意义重大。本实验采用栓线法致 MCA 缺血再灌大鼠模型,通过双抗体夹心 ABC-ELISA 法以及免疫组化染色法研究化合物 971更多还原

【Abstract】 Stroke, especially ischemic stroke, is a common disease which is threatening the health ofpeople. The disease not only impaired patients’ health, but also brings burden to the social.Therefore,it is an urgent task to further definite the pathophysiological mechanism of cerebralischemia and to develop effective drugs of preventing and treating ischemic stroke. The pathophysiological mechanism of cerebral ischemia is very complex. Previous studieshave demonstrated that it related with many factors, such as energy metabolism dysfunction ofbrain tissue following cerebral ischemic injury , massive free radicals producing, [Ca2+]irising,platelet activation, changes of hemorheology, inflammation damage, etc. At present thetheory about cascade of damage have been raised, it included energy failure and excitotocity,peri-infarct depolarizations, inflammation and programmed cell death. Now the relationsbetween cytokines,adhesion molecules, NF-κB and brain ischemia have been recognized. Effective component in Zhimu(Compound 9714)was a soluble steroidal saponin which wasextracted from Chinese medcine Zhimu.In this paper,the effects and mechanisms of Compound9714 on protecting cerebral ischemia were studied from different levels with the methods ofbiochemical assay, immunohistochemistry, enzyme-linked immunosorbent assay, molecularbiology, cell culture, etc. So that it can provide the theory and experiment proof for studying newmedicine of curing brain ischemia. The main results were as follows:1 Improvement effect of compound 9714 on brain function deterioration of animals The descent of learning and memory ability was a typical display of brain disfunctioninduced by ischemia. Morris water maze was used to measure the effect of compound 9714 onlearning and memory ability of brain ischemia rats induced by common carotid arteryocc1usion(CCAO), HE and SABC immunohistochemistry staining were used to study itsmechanism. Mice water maze was used to measure learning and memory ability of mice inducedby scopolamine. Western Blottin was used to measure the effect of compound 9714 onCholinergic receptor. Results showed that compound 9714 10, 20, 40mg/kg can shorten theescape latency time and increase the percent of primary flat floor quadrant swimming distance ofmodle rats in Morris water maze.Compound 9714 can improve the degeneration and necrosis ofneuron in HE staining.Compound 9714 20mg/kg, 40mg/kg can improve the expression of BDNFand decresed the expression of ICAM-1. Compound 9714 15mg/kg, 30mg/kg, 60mg/kg canimprove the learning and memory ability of acquired memory disturbance mice induced byscopolamine. Compound 9714 15mg/kg, 60mg/kg can inhance the cholinergic receptor(M1,Nalpha7) density of acquired memory disturbance mice induced by Scopolamine.<WP=8>英文摘要 52 Protective effect of compound 9714 on ischemia injury rats Brain ischemia model included focal brain ischemia and whole brain ischemia. Middlecerebral artery thrombus(MCAT) model induced by FeCl3 and middle cerebral arteryocclusion(MCAO) model induced by inserting a monofilament suture were used to study theeffect of Compound 9714 on ischemia injury rats. Results showed that Compound 9714 20,40mg/kg can obviously reduce infarct size, attenuate the neurologic symptoms, decrease thewater content of brain tissue. Compound 9714 10, 20, 40mg/kg can also improve brain bloodflow of model rats, can inhance the activity of T-SOD, decrease the activity of MPO, T-NOS, i-NOS, decrease the content of NO and MDA in brain tissue of MCAO model rats.3 The influence of compound 9714 on thrombosis, platelet aggregation and hemorheology Thrombosis formation was an important course in brain ischemia, it related to vessel, bloodcomponent and blood flow. After cerebral ischemia, platelet aggregated and blood component,hemorheology changed. Platelet aggregation was the initiation factor of thrombosi更多还原