【作者】 王张；

【导师】 张艺； 孟宪丽；

【作者基本信息】 成都中医药大学 ， 中药学， 2009， 博士

【摘要】 民族药在治疗心脑血管等复杂疑难疾病方面相对于单一化学成分药物有其独到之处。但是对民族药有效性和安全性的现代认识和研究成为制约其发展的关键问题,尤其应在民族药的有效性评价和作用机制研究的思路和方法等方面进行总结和提炼,建立一套体现民族医药特点、系统论和还原论相结合的研究新方法。苗药灯盏细辛和藏药沙棘分别是临床上用于防治缺血性中风和胸痹心痛的疗效确切的常用药,二者的应用历史悠久,在民族药中占有十分重要的地位,如很多藏药成方制剂中均含有沙棘膏,且均被药典收载,具有较高的市场占有率,此外二者的药效物质基础都是业已证明对心脑血管疾病具有明显治疗作用的黄酮类有效成分,故以苗药灯盏细辛和藏药沙棘为例进行研究,具有较好的代表性和推广价值。目的在民族医药学理论体系的指导下,结合系统化学生物学的思路和代谢组学的方法,以治疗缺血性中风的苗药灯盏细辛和防治胸痹心痛证的藏药沙棘为例,尝试建立基于代谢组学的民族药防治心脑血管疾病的有效性评价和作用机制研究的新方法。方法应用线栓法阻塞大脑中动脉致大鼠局灶性脑缺血再灌注损伤模型和肾上腺素加冰水致急性血瘀证大鼠模型,分别进行苗药灯盏细辛和藏药沙棘的药效学研究；同时基于超高效液相色谱结合四极杆飞行时间串联质谱(UPLC-QTOF/MS)和主成份分析、偏最小二乘法等模式识别技术对造模和给药前后大鼠的尿液进行代谢谱型分析,开展基于代谢组学的民族药有效性评价和作用机制研究。结果(1)药效学研究：灯盏细辛注射液能明显地减少脑梗死面积,改善大鼠脑组织形态学病变,并能促进尼氏小体数量的增加,这可能是通过减少海马区神经细胞凋亡促进因子——Bax蛋白表达,对抗缺血再灌注后诱导的细胞凋亡,从而发挥脑保护作用的；亦能明显促进神经功能缺失体征的提前恢复；综合各指标的结果提示,量-效关系不规则,疗效由强到弱依次为：低剂量>高剂量>中剂量。沙棘提取物能够明显降低急性血瘀动物的血液粘稠度和红细胞浓度,减弱红细胞的聚集能力和血液的凝固能力,最终改善急性血瘀大鼠血液流变学的“浓、粘、凝、聚”等异常特征；其中又以沙棘提取物高剂量和低剂量的作用最明显。(2)基于代谢组学的有效性评价：灯盏细辛注射液和沙棘提取物能使模型动物的代谢谱型向正常动物回归,其中低剂量优于高剂量,后者又优于中剂量。(3)基于代谢组学的作用机制研究：灯盏细辛注射液减弱或取消了外界刺激因素“线栓法阻塞大脑中动脉”对机体的作用,使模型大鼠的代谢谱型向正常动物回归,其作用机制可能与犬尿喹啉酸、琥珀酰鸟氨酸和亮氨酰脯氨酸等潜在生物标志物有关,它们又有可能涉及“色氨酸-犬尿喹啉酸”和“琥珀酰鸟氨酸-尿素循环-NO”等代谢途径。沙棘提取物减弱或取消了外界刺激因素“肾上腺素结合冰水”对机体的作用,使模型大鼠的代谢谱型向正常动物回归,其作用机制可能与苯丙氨酸、色氨酸、琥珀酰鸟氨酸、犬尿喹啉酸、鹅去氧胆酸和胆酸等潜在生物标志物有关,它们有可能涉及“苯丙氨酸-酪氨酸-儿茶酚胺类”、“色氨酸-5-羟色胺”、“琥珀酰鸟氨酸-尿素循环-NO”和“胆固醇-胆汁酸”等代谢途径。结论本文证实了代谢组学对苗药灯盏细辛和藏药沙棘有效性的评价与传统药效学研究结果的一致性；代谢组学还揭示了民族药所调控的机体潜在生物标志物及其代谢途径,丰富了民族药的作用机制研究；此外本文揭示了急性血瘀“证”本质的生化物质基础,故代谢组学可以作为民族药的有效性评价和作用机制研究的新方法,亦拓宽了民族药现代研究的思路。创新点(1)本文揭示了脑缺血再灌注损伤模型大鼠的代谢谱型及灯盏细辛注射液调控的潜在生物标志物；(2)本文揭示了急性血瘀“证”的生化物质基础为一系列的潜在生物标志物,并阐释了沙棘提取物对其的影响；(3)探索了代谢组学结合药效学的手段来进行民族药有效性评价的新方法。更多还原

【Abstract】 Considering the treatment of complex diseases, ethnomedicine has unique comparing with drugs of single chemical composition. However, ethnomedicine’s indefinable efficacy and security of modern knowledge and research have led a constriction in its development. It is necessary for us to build up a new mode in the person of the ethnomedicine characteristics, combining system theory and reductionism, summarizing and extracting from the ideas, methods and modes of the efficacy evaluation and mechanism of ethnomedicine.Miao National Herbs Herba Erigerontis and Zang National Herbs Fructus Hippophae are effective drugs which respectively used for prevention and treatment of ischemic stroke and thoracic obstruction. Both of them have a long history as drug and are very important in the ethnomedicine. For example, many Zang National Herbs preparations includes Fructus Hippophae paste.They are also contained by Pharmacopoeia of China and have a high market share.Material basis for efficacy of them is the active ingredients flavonoids which have proved to be obvious therapeutic for cardiovascular and cerebrovascular diseases. Therefore, reseaching Miao National Herbs Herba Erigerontis and Zang National Herbs Fructus Hippophae as examples have better representation and application of the value.ObjectiveUnder the guidance of ethnomedicine theory, combining systems chemical biology’s idea and metabonomics’s method, we chose Miao National Herbs Herba Erigerontis and Zang National Herbs Fructus Hippophae as our research targets, which are well known in curing ischemic apoplexy and cardiac obstruction respectively. Through the research, we attempt to establish a new method of efficacy evaluation and mechanism of action on prevention and treatment of cardiovascular and cerebrovascular diseases using ethnomedicine.MethodsWe carried on Miao National Herbs Herba Erigerontis and Zang National Herbs Fructus Hippophae’s pharmacodynamics research separately by two models, which were focal cerebral ischemia-reperfusion models in rats, induced by the transient occlusion of the middle cerebral artery (MCAO) using fishing line and induced the rats into acute syndrome of blood stasis using epinephrine and ice water. At the same time, based on UPLC-Q TOF/MS and the pattern recognition technology of PCA, PLS-DA and so on, we have researched how it effected the metabolic profiles of model rats by its urine analysis before and after making the mold and administiting medicine. Thus we could study the efficacy evaluation and mechanism of action of ethnomedicine using metabonomics.Results(1) Pharmacodynamics researchHerba Erigerontis can significantly reduce the cerebral infarction area, improves the histomorphology changes in rat brain and promote the number of Nissl’s Body increase, and the reason possibly is through the reduction of apoptosis promoter of Bax to express weakly in brain seahorse area nerve cell, thus displaying brain protective function. Herba Erigerontis can also promote neuroethology restoration ahead of time obviously.The results of various index suggest that the dose-effect relationship is irregularity, and the effect is from strong to weak as follows:Low dose, high dose, middle dose.Extractive of Fructus Hippophae can reduce the acute syndrome of blood stasis rats’s blood stickiness and RBC density obviously, weaken the RBC aggregation ability and blood coagulation ability, improves hemorheology of pathologic status, such as "thick, stick, coagulation, aggregation". The effect is obviously with Low dose and high dose.(2) Metabonomics-based on evaluation the efficacyHerba Erigerontis injection and extractive of Fructus Hippophae can help model rats’s metabonomics return to the control, and the effect is from strong to weak as follows:Low dose, high dose, middle dose.(3) Metabonomics-based on research the mechanism of actionHerba Erigerontis could partially relieve or entirely inhibit the effect of external stimulating factor "the transient occlusion of the middle cerebral artery (MCAO) using fishing line" to the body.The mechanism of action of Herba Erigerontis may be related to potential biomarkers, such as kynurenic acid, N2-succinyl-L-ornithine, and L-leucyl-L-proline.They also possibly participate in the metabolic pathway of "the tryptophan-kynurenic acid"and "succinyl ornithine-urea cycle-NO".Extractive of Fructus Hippophae could partially relieve or entirely inhibit the effect of external stimulating factor "epinephrine and ice water" to the body.The mechanism of action of extractive of Fructus Hippophae may be related to potential biomarkers, such as L-Phenylalanine, L-Tryptophan, N2-succinyl-L-ornithine, kynurenic acid, 3,7-Dihydroxy-12-oxocholanoic acid, cholic acid. They also possibly participate in the metabolic pathway of "the phenylalanine-tyrosine-catecholamines", "the tryptophan-5-hydroxy tryptamines", "succinyl ornithine-urea cycle-NO" and "the cholesterol-bile acid".ConclusionThis study primarily demonstrates the efficacy evaluation and the mechanism of action of Miao National Herbs Herba Erigerontis and Zang National Herbs Fructus Hippophae using metabolimics and its consistency with pharmacodynamics research.The metabonomics study discovered some potential biomarkers and the metabolic pathways that ethnomedicine regulates, and enriched the mechanism of action research for ethnomedicine.In addition this study has promulgated the essential biochemistry material base of the acute syndrome of blood stasis. Therefore, metabonomics is very valuable as to be a new choice as evaluating the efficacy and studying the mechanism of action of ethnomedicine, which expanded the modern research mentality of ethnomedicine.Innovation(1) This study primarily discovered some potential Biomarkers and the metabolic pathway related with MCAO rats and Herba Erigerontis.(2)This study not only discovered that the basis of biochemistry substances of the acute syndrome of blood stasis were series of potential biomarkers, but also expatiated how extractive of Fructus Hippophae influenced on it. (3)This study explored a new method to evaluate the efficacy by combination Metabolomics with Pharmacodynamics.更多还原