【作者】 严华；

【导师】 王永炎； 黄璐琦；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2002， 博士

【摘要】 中风病是临床常见病、疑难病。相当于现代医学的脑血管疾病，以脑梗塞和脑出血为主。随着人类社会的发展，人类疾病谱的变化，中风病已成为我国三大病死原因之一。目前临床上有三个显著的特征、即年轻化，轻型化、椎-甚底动脉病变增加。主要分为三大类型：神经功能缺失、精神障碍、无症状与无偏瘫。随着影像学诊断的进步，尤其是MRI及PET的实际应用和普及，诊断的阳性率大大提高。提高疗效、提高生存质量是基础和临床研究的最终目的和终极追求，国内外取得较大进展。自80年代以来，很多学者除提出能量代谢、兴奋性氨基酸毒、自由基、钙超载等学说外，最近也注意到脑缺血与细胞因子、炎症反应、粘附分子等密切相关，相应的药物也应运而出，如钙拮抗剂、兴奋性氨基酸受体拮抗剂和自由基清除剂等，但能否治疗急性缺血性脑卒中仍不能完全肯定。迄今尚无较理想的治疗药物。近年来许多学者提出早期或超早期溶栓治疗是缺血性脑血管疾病最有希望的治疗方法，并取得一些成果，但再灌注损害及脑出血增加却给该项治疗带来不利影响。由于脑缺血损伤在初始和迟发性损伤过程中为多因素参与的过程，如在治疗上只解决复杂过程中的一个方面，不会收到好的效果。因此在治疗上去阻断脑缺血病理过程的多个环节是很重要的。中药具有多组分，多途径，多靶点的特点，恰可以作用于多个病理环节，通过调整机体的自身修复功能而起到治疗作用。中医中药治疗中风病具有独特的优势，这对其开发为抗脑缺血损伤药物具有良好的前景。清开灵注射液是安宫牛黄丸经删减后制成的灭菌水溶液，经过国家多年攻关已经证实它可通过多种不同途径保护缺血损伤的脑组织。但其成分复杂，难以质量控制，对中药走向世界极为不利。血脑屏障对于脑血管疾病治疗具有重要意义。在正常状态下绝大部分物质不能透过血脑屏障。脑缺血可破坏血脑屏障，使血脑屏障通透性增加，造成脑水肿，保护血脑屏障的完整性可能是减轻脑损伤的重要措施。同时因为血脑屏障的存在，使很多药物在脑中不能达到有效的治疗浓度，是治疗中枢神经系统疾病的主要障碍。而中风病疗效的关键靶点应在中枢神经系统。中枢神经系统由神经元和神经胶质细胞构成。神经胶质细胞是神经元数目的10倍左右，对神经元起着重要的营养、支持、保护作用。近年来的研究发现，神经胶质细胞在脑缺血后异常活跃。脑缺血所激发的星形胶质细胞和小胶质细胞反应极其复杂，已远远超出了对神经元的营养支持和清除变性坏死组织的作用范围。更有研究发现半暗带神经元死亡的原因可能首先是胶质细胞功能异常。脑缺血后胶质细胞的反应有独特的时间和空间模式，并出现复杂的分子水平变化，它对缺血性神经元损伤有双向作用。星形胶质细胞能合成多种营养因子如bFGF、NGF、BDNF等，这些因子对神经组织的存活和创伤修复起到积极作用。同样，小胶质细胞作为神经组织的清道夫，能清除细胞碎片及细胞代谢产物，在损伤、炎症、缺血条件下也能反应性地增多，它所分泌的一些活性因子如IL-1、TNF-α加重脑缺血损伤。中药绝大部分是大分子物质，在病理状态下能否透过血脑屏障达到有效的治疗浓度? 治疗的靶点在于神经元还一是神经胶质细胞？因此，系统研究药物在脑釉状态下对血脑屏障通透性对于阐明药物的作用机制，提高临床疗效辅重要的理贼临床意义。 中医学认为中风发病的机制包括三个方面：气血逆乱，发病在脑；脉络闭阻，半身不遂；脏腑气机失调。这些理论一直指导着临床辨证施治脯，具有一定的临床疗效，古今病例积累很多，但疗效不甚满意且重复性很差。针对毗，导师王永炎院士指出，提高其疗效的突破口就中医而言，应重视病因病理学说的发展，认为“毒邪”和“络病”可以作为深少J歼究的切入点。综弧方研究成果，我们认为，中风病病变的核心在于“脑络”，“脏腑功能虚损、气血逆乱、邪盛成毒、犯脑损络”则是中风病病理发展、演变的主要机制。针对病机提出俗毒通络”的治法，解毒以对抗损害因素是为祛邪，通络以畅行气血是为扶正。 拥究的基本思路可概括为：首先分析能通驷脑屏障的有效成分，以神经元和神经顺细胞为靶点，研究其对脑锄后细胞因子网络的干撇制，揭示“毒损脑络”病似清开灵“解毒通络”治法及“祛邪扶正”的毗生物学内涵。进而为脑晌损伤提供成分明确、靶点及作用机制明确、安全高效符合国际标准的新型绿色中药，为中医药现代化研究提供理论与实践上有益尝试。1目的1．1理论研究 主要探讨中风病“毒损脑络”理论框架；1．2实验研究 以中风病为代表，顺证结合，方剂有效成分与靶点相关的角度，运用复杂性科学原理，采用微观分析与宏观分析、腿论与整体论相结合的方法，通过对复杂方剂及复杂机体的有机的拆分，拆分方剂组分（黄岑戒、桅子戒、绿原酸）与作用靶点（神经元、神经懈细胞），通雌测并确定清开灵及其有效成分雌肋脑屏障的情况，观察缺血损伤神经元及神经胶质细胞存活率的影响，探讨清开灵及其有效成分与中枢神经系统中两个重要而密切靶点神经元及神经胶质细胞的相关性；腑统神经保护剂及调控内源性?更多还原

【Abstract】 Windstroke is a coromon and difficult disease. The TCM treatment based on the differentiation of syndromes owns the superiority in the therapeutic effect. The following is the study on the pathogenesis of windstroke ?Heat-toxic Affecting Brain Collaterals and the mechanism of Qingkailing in treatment of windstroke ?Removing Heat-toxic from Brain Collaterals.1. PurposeTo study the pathogenesis of windstroke ?heat-toxic affecting the brain collaterals. To observe the formula compositions ?baicalin, gardenoside, and chlorogenic acid and their targets ?neurons and neuroglia cells, to research the effect of the compositions of Qingkailing on the survival rate of neurons and neuroglia cells damaged by ischemia, to discuss the interrelations between the compositions and the two important target neurons and neuroglia cells of the central nervous system, with the application of microcosmic and macroscopic analysis and by way of the combination of reduction and entirety and the organic separation of the complicated formulas and organism. In addition, to study the biological fundamentals of the mechanism of Qingkailing in removing heat-toxic from the brain collaterals for the treatment of windstroke from the viewpoints of traditional nervous protective agents and the regulation of endogenous activated substances (NGF and TNF-a) in order to reveal the reasonability of compatibility of formulas and to clarify the superiority and features of Chinese medicine and pharmacology.2. MethodsTheoretical method: The literature and logical analysis are adopted in the research of "heat-toxic" and "collateral" in the duration of windstroke.Experimental method: The focal cerebral ischemia of the rat is produced by ligation. HPLC is applied to determine the volume of the effective compositions of Qingkailing permeated the blood-brain barrier (BBB) under the pathological conditioa The pharmacodynamic experiment on cells is done with the permeated compositions by means of MTT, ft-TDR incorporation, radioimmunoassay, ELISA, and immuno-fluorescence to observe the effect of the total formula and each composition of the formula on the cortex and the survival rate and reproduction rate of the hippocampus neurons and the neuroglia cells damaged by ischemia, the effect on the neuroglia cells secreting theendogenous active substance (NGF and TNF-a), and the action pattern of the medicine on cells. 3. Results3.1. The theory study: According to Prof. Wang Yongyan, the dysfunction of zangfu is the fundamental and the disordered qi and blood are the inducing factors of windstroke, in which the heat-toxic affects the collaterals of brain. He puts forward that the basic principle of treatment for windstroke is to remove the heat-toxic from the collaterals on the basis of modern biology. 3. 2. The experimental study:3.2.1. Study on the compositions of Qingkailing permeated the BBB under the condition of ischemic damage.In the normal and pseudo-operation groups there is a small amount of baicalin, gardenoside and chlorogenic acid permeated the BBR The quantity of baicalin, gardenoside, and chlorogenic acid permeated the BBB in the ischemic group is remarkably more than that of the normal and pseudo-operation groups (P<0.01). It means that the effective compositions enter the central nerve system, reach the damaged area, and act on the neurons and the neuroglia cells. The substantial pharmacodynamic bases of Qingkailing are baicalin, gardenoside, and chlorogenic acid.3.2.2. The effect of the compositions of Qingkailing on the survival rate of hippocampus and cortex neurons damaged by ischemia.Three days after ischemia, the survival rate of hippocampus and cortex neurons is decreased (P<0. 05). The survival rate of hippocampus neurons in the total formula and baicalin, gardenoside and chlorogenic acid groups is higher than that of the ischemic model group. There is an obvious difference among the groups(P<0. 05). The survival rate of cortex neurons in the total formula and baicalin and gardenoside groups is higher更多还原