精制清开灵抗实验性脑缺血的血管保护机制研究

【作者】 朱晓磊；

【导师】 李澎涛；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2006， 博士

【摘要】 近年来,以发展保护“血管系统(vasculature)”的方法来改善中风疗效的观点成为西方学者关注的热点,其中,以内皮型一氧化氮合酶(eNOS)为靶点的治疗措施为治疗脑缺血损伤拓展出了新的思路和途径。研究缺血后血管损伤的病理生理过程,发展能够提供血管保护的治疗措施有着重要的意义,有可能成为缺血性中风病治疗的新方向。现代医学重视“血管系统”的学术动向与缺血性中风“毒损脑络”病机假说及“解毒通络”治法不谋而合。用于急性期中风治疗已逾二十年的清开灵注射液是由古方安宫牛黄丸改制而成,具有清热解毒、化痰通络、醒神开窍的功效,对中风病的临床疗效显著。国家重点基础研究发展规划(973)项目“清开灵注射液多环节阻抑脑缺血级联反应药效物质与作用机理研究”(课题号G1999054404)利用指纹图谱识别清开灵注射液的组分,通过对其细胞毒性、整体动物毒性研究排查,以清开灵注射液中胆酸、栀子苷、黄芩苷、珍珠母水解液四个有效组分研制而成了增效减毒的新型注射液——精制清开灵。本部分工作以线栓法复制大鼠持久性大脑中动脉栓塞(PMCAO)模型,观察精制清开灵及其组分在持久性脑缺血急性期与亚急性期是否能够阻抑脑缺血后血管损伤和促进血管修复,从而发挥脑保护作用。具体包括以下两部分内容及相关实验:第一部分,以eNOS为着眼点,观察PMCAO 2-72h大鼠脑组织eNOS的表达规律及精制清开灵对其影响,探讨精制清开灵对缺血性中风后血管系统损伤与修复过程的干预趋势。第二部分选择PMCAO 72h这一时段,深入研究精制清开灵及其组分的脑保护作用与它们对血管系统损伤和保护相关指标的干预作用,具体包括五个内容:精制清开灵及其组分对PMCAO 72h大鼠的脑保护作用;精制清开灵及其各组分对PMCAO 72h大鼠脑组织eNOS表达的影响;精制清开灵及其各组分对PMCAO 72h大鼠脑组织血管生成素-1,2(Ang-1,2)及其受体Tie-2表达的影响;精制清开灵及其组分对PMCAO 72h大鼠血清肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)含量的影响。本部分实验在观察精制清开灵及其组分对血管系统损伤与保护相关指标干预作用的同时,着重比较精制清开灵与其各组分对上述指标作用的异同并进行相关性分析,希望能够揭示精制清开灵注射液方剂配伍的部分机制,并探讨中风病“毒损脑络”病机及“解毒通络”治法的现代生物学基础。各部分实验结果如下:一、PMCAO 2-72h大鼠脑组织eNOS表达规律及精制清开灵对其影响PMCAO 2-72h,模型组大鼠脑组织eNOS表达较假手术组大鼠反应性增高。PMCAO后2小时,精制清开灵治疗组大鼠脑缺血半暗带eNOS表达即较模型组动物升高,至PMCAO后72小时仍维持这一趋势。二、精制清开灵及其组分对PMCAO 72h大鼠的脑保护作用观察PMCAO 72h精制清开灵及其组分胆酸、栀子苷、黄芩苷、珍珠母水解液的脑保护效果,可知:⑴栀子苷和精制清开灵具有良好的阻抑血浆vWF含量病理性升高的作更多还原

【Abstract】 Ischemic stroke is a common disease of middle-aged and aged people. Jeopardizing people’s health because of its high attack rate, recurrence rate, mutilation rate and mortality rate, ischemic stroke has been the third main cause of death and has attracted great attention of the medical science circle. In recent years, scholars of western countries has shown solicitudes for methods of protecting vasculature to improve curative effect of stroke. Among these methods, therapeutic measures targeting eNOS has provided new idea and pathway to the treatment of ischemic stroke. It is of great importance to study patho-physiology process of the vascular damage after stroke and to develop therapeutic measures of protecting vasculature, maybe it will be a new direction for the treatment of ischemic stroke.Interestingly, this academic trends partly coincides with the new rising hypothesis of‘toxin hurts brain collaterals’and the therapeusis of‘removing toxin and dredging collaterals’. Qing Kai Ling(QKL) injection is a multicomponent injection derived from Angongniuhuang pills. The effects of QKL injection include clearing heat and removing toxin, dissipating phlegm and dredging collaterals, awaking mind and opening orifice, etc. The injection had been proved to had good effects since it was extensively used to treat acute stroke in 1980s. The research of“Study on the effective substances and functionary mechanisms of Qing Kai Ling injection in blocking cascade of cerebral ischemia damage”, which was funded by National Basic Research Program of China (973 Program, G1999054404), clarified the effective substances of QKL injection in blocking cascade of cerebral ischemia damage, and presented an important innovation of Traditional Chinese Medicine (TCM) prescription, namely, the combination of effective ingredients of Chinese materia medica. And according to the theory, a kind of new chinese medical prescription--Refined Qing Kai Ling (RQKL) injection was invented.We copied permanent middle cerebral artery occlusion (PMCAO) model of rats using filament method. And we explored whether RQKL injection and its individual ingredients manifested neuroprotective effects by inhibiting injury of vasculature and facilitating recovery of vasculature. Studies were performed as follows in two parts:First part, using the techniques of immunohistochemistry and western blots, we examined expression of eNOS in brain tissue of rats undergone PMCAO from 2-72 hours and investigated whether RQKL’s cerebroprotective effects were correlated with the expression of eNOS under ischemic condition.Second part, we further explored the relationship between cerebroprotective effects of RQKL and its individual ingredients and their different influences on harmful and beneficial targets of vasculature, and the part of work can be further divided into four individual experiments:1. Cerebroprotective effects of RQKL and its individual ingredients on rats after 72 hours更多还原