【作者】 尹天雷；

【导师】 蔡光先；

【作者基本信息】 湖南中医药大学 ， 中医内科学， 2007， 博士

【摘要】 背景:缺血性脑血管病是临床常见病、多发病,有病死率高、致残率高的特点,脑缺血后神经元损害是其病理机制的核心部分,因而探寻神经元保护剂是缺血性脑血管病的研究热点。补阳还五汤是治疗该类疾病的常用方剂之一,对缺血性脑血管病的治疗具有显著疗效和一定特色。目的:(1)通过对超微补阳还五汤与传统补阳还五汤的有效成分的对比研究,探讨超微复方制剂的特点,为中药新型饮片的研制作出有益的探索。(2)在建立局灶性脑缺血大鼠模型基础上,通过动物实验,探讨超微补阳还五汤对局灶性脑缺血大鼠神经元损伤的保护作用及其机制,为治疗脑梗塞的临床用药提供实验依据。方法:(1)采用一系列试验对超微补阳还五汤与传统汤剂的有效成分进行对比研究。对当归、赤芍、川芎、红花、桃仁的主要有效成分进行薄层鉴别研究,并对主要成分进行含量测定;采用《中国药典》收录的相关方法检测重金属、砷盐含量以明确其安全性;对水溶性浸出物和正丁醇浸出物进行含量测定对比研究。(2)采用栓线法复制MCAO大鼠模型,随机分成六组灌胃给药;在1天、3天、7天、14天四个时点观察各相应指标以探求超微补阳还五汤对脑缺血神经元损伤的保护作用及机制。对大鼠脑缺血损伤的保护作用选用行为学变化及组织形态学变化等指标,从对氧自由基的影响对其进行验证;对作用机制的探求从NGF及其受体(TRKA)的表达和信号转导机制(PI3K/PKB)两个方面进行。具体方法则为检测血浆SOD、MDA的活性变化,用免疫组化法和分子生物学方法观察对脑组织中NGF、TRKA及PI3K、CREB、AKT的表达的影响。结果:(1)TLC测定各组供试样品在色谱相应的位置上,显相同颜色的斑点,且复方超微补阳还五汤浸泡液中主要有效成分斑点较传统汤剂更明显,清晰;对其主要成分的含量测定显示超微补阳还五汤中黄芪甲苷含量与传统汤剂相当;对砷盐、重金属检测未发现其含量超标;对浸出物的测定显示超微补阳还五汤水溶性浸出物为传统汤剂水溶性浸出物的1.22倍,正丁醇浸出物为其传统汤剂的1.26倍,均高于传统汤剂。(2)超微补阳还五汤可降低脑缺血损伤大鼠神经功能评分,并改善脑组织缺血的病理损伤,其作用明显优于模型组及假手术组;超微高剂量组作用优于传统汤剂组。(3)超微补阳还五汤可提高脑缺血后血中SOD活性并降低MDA,其作用明显优于模型组及假手术组;超微高剂量组作用优于传统汤剂组。(4)超微补阳还五汤可上调脑缺血后脑组织NGF及其受体TRKA的表达,并增强PI3K/AKT信号转导的表达,其作用明显优于模型组及假手术组;超微高剂量组与传统汤剂比较其影响有明显增高的优势。结论:(1)补阳还五汤及其超微制剂可以改善局灶性脑缺血大鼠神经功能缺损评分及脑组织病理变化,提高脑缺血后血液SOD活性并降低MDA活性以减弱氧自由基的毒性反应,对脑缺血神经元损伤具有保护作用。(2)超微补阳还五汤保持了传统补阳还五汤中的有效成分,且有效成分的溶出相对传统汤剂更具优势。(3)超微补阳还五汤可提高脑缺血后脑组织神经营养因子PI3K/PKB信号转导途径的表达,提高神经生长因子及其受体表达水平,这可能是超微补阳还五汤对脑缺血神经元损伤的保护作用机制之一。(4)超微补阳还五汤以1/2等效剂量时即可达与传统汤剂相同的作用效果,以等效剂量时其作用效应有明显增加的趋势。综上,本论文采用药物化学、药理学方法以及免疫组化、分子生物学方法,以超微技术对补阳还五汤复方制剂与传统补阳还五汤标准汤剂进行对照研究,论证了超微技术在促进中药复方制剂(补阳还五汤)有效成分溶出上的优势,并从神经营养因子通路(即信号转导机制)角度揭示了超微补阳还五汤的作用机制,具有显著的创新性。更多还原

【Abstract】 Foreword: Ischemia stroke is a frequent and common disease with high mortality and high disability in clinical work. The study show that neuronal injury following cerebral ischemia is the main part of pathological mechanism’s study. So the searching of drugs for protect cerebral ischemia neuronal injury is a hotspot in this domain. BuYangHuanWu is one of the medicament with positive curative effect and obvious trait for Ischemia stroke.Objective:(1) Investigate the trait of the ChaoWei-BuYangHuanWu by contrasting the main effective element in ChaoWei-BuYangHuanWu and BuYangHuanWu, make some rewarding work for the medicament reform of Chinese traditional medicine.(2) Study the effection of protecting the neuronal injury following cerebral ischemia , and make sure the mechanism by the experimentation in MCAO rat. Offering some experience for the clinical work on Ischemia stroke.Methods:(1) Make sure the effective element of ChaoWei-BuYangHuanWu by series test: Study the essential element of Chinese angelica Root, Red Peony Bark, Chuangxiong Rhizome, Safflower and Peach Kernal by TLC; and measure the content of the main useful element in ChaoWei-BuYangHuanWu; Make sure the content of specifically metal , complex and water-solubility- lixivium \alc-ohol-solubility-lixivium by the ways which was recorded by PharMCAOpoeia Of People’s Republic Of China.(2)Make MCAO rats by special ways, divide the rats to 6 group; and abserve the rats at 1\ 4\ 7 and 14 days, observe and record the given symptoms, give the grade by the symptoms; after the test, kill the rats and take blood , then detect the actives of SOD and MDA in blood by given ways ; after all, take the brain , Immunohistochemical avidin-biotin peroxidase complex method was applied to detect the expression of NGF and his Receptor TRKA; And In Situ Hybridization with Nick-Translated Probes was applied to detect the PI3K/PKB, which was the main Intracellular signal transduction of NGF.RESULTS:(1) showing the same color spot in the corresponding place on the chromato-gram of each sample of the two groups measured by TLC, and the spot is more distinctive in the effective element of ChaoWei-BuYangHuanWu.It is showed that the content of Astragaloside IV in ChaoWei-BuYangHuanWu is equivalent in the traditional decoction, the content of arsenide and specifically metal is not in superscale. It is found that the water-solubility- lixivium is 1.22 times and the alcohol-solubility-lixivium is 1.26 times in ChaoWei-BuYangHuanWu than in the traditional decoction.(2) ChaoWei-BuYangHuanWu can reduce the grade of the the neuronal injury following cerebral ischemia, improve the pathological injury of cerebral ischemia. It is statistically different than the model group and artificial operation group, the effection of high dosage ChaoWei-BuYangHuanWu is statisti- cally better than traditional decoction.(3 ) ChaoWei-BuYangHuanWu can improve the active function of SOD, reduce the active function of MDA. This function is statistically different than the model group and artificial operation group, the effection of high dosage ChaoWei-BuYangHuanWu is statistically better than traditional decoction.(4) ChaoWei-BuYangHuanWu can raise the expression of NGF and his Receptor TRKA after cerebral ischemia, and boost up the signal transduction of PI3K/AKT.This function is statistically different than the model group and artificial operation group, the effection of high dosage ChaoWei-BuYangHuan Wu is statistically better than traditional decoction.CONCLUSION:(1) BuYangHuanWu decoction and it’s ChaoWei-preparation can improve the neuronal function grades and pathological changes of rats with partial cerebral ischemia;It can improve the active function of SOD, reduce the active function of MDA to weaken the toxic of superoxide free radicals. It is protective to the injuried neurocyte of cerebral ischemia.(2) ChaoWei-BuYangHuanWu kept the effctive element, as well as the traditional decoction and it is better than the traditional decoction in solubility -lixivium.(3) ChaoWei-BuYangHuanWu can boost up the signal transduction of PI3K/ PKB that is a kind of Neurotrophic factor. It also can raise the expression of NGF and his Receptor TRKA after cerebral ischemia. This is likely to be one of the protective mechanism of ChaoWei-BuYangHuanWu to neuronal injury following cerebral ischemia.(4) 1/2 equivalent effective dosage of ChaoWei-BuYangHuanWu can make the same effecion of the traditional decoction. It is prone to be effective stronger when used the equivalent effective dosage.Above all, this topic adopted Pharmaceutical method, method of pharMCAOlogy, Immunohistochemical method and molecular organism, demonstrated the asc- endancy of promoting solubility-lixivium of effective element in traditional Chinese medicine (BuYangHuanWu decoction) by contrasting ChaoWei-BuY- angHuanWu in which ultramicro technology was used and traditional BuYang- HuanWu decoction, and revealed the mechanism of action in the view of signal transduction of Neurotrophic factor. So this topic had great new ideas.更多还原