【作者】 赵光峰；

【导师】 彭胜权；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2009， 博士

【摘要】 缺血性脑水肿是脑血管病伴发的最常见的死亡原因,是脑梗死必然且重要的病理过程,对其机制的研究及防治对策,一直是神经科的研究热点之一。其发病机制的主要环节为血脑屏障(BBB)受损。目前对脑水肿的治疗主要有高渗性脱水剂,利尿剂,糖皮质激素以及外科手术减压等,这些治疗虽然能一定程度减轻脑水肿,但都没有解决其发病机制的主要环节即BBB受损的问题。中药及复方对缺血性脑损伤的治疗具有一定的优势,其药理作用的多靶点、多途径性引起了医学界的广泛重视。而醒脑开窍法作为治疗中风的一种重要方法,虽然近年来广泛应用于临床,但一直未能引起足够的重视。本课题通过文献研究,总结并阐述了缺血性中风急性期使用芳香开窍法的必要性,中医药尤其是麝香和冰片对BBB的作用,脑缺血再灌注后BBB损伤的分子机制。根据导师刘亚敏教授等的工作基础,按线栓法造成大鼠大脑中动脉闭塞,建立局灶性脑缺血再灌注模型,造成缺血性脑水肿,采用电镜、免疫组化、RT-PCR等方法,围绕缺血性脑水肿中血脑屏障(BBB)损伤的主要环节,探讨麝香配伍冰片对脑微血管内皮细胞、基质及胶质细胞足突三个环节的作用,系统地分析麝香配伍冰片对缺血后BBB损伤的影响,阐明其作用的分子机制。为临床合理应用开窍醒脑法治疗急性缺血性中风提供科学的依据,对临床应用也具有重要的指导意义和应用前景。1文献研究1.1缺血性中风的病机为脑窍痹阻、神机失用,醒脑开窍是缺血性中风的基本治则。醒脑开窍法是中风急性期的重要方法,应及早使用。使用开窍药不仅可醒脑开窍、恢复神机,且可兼顾化痰、活血、息风止痉、清热等,一举多得。开窍法在治疗偏瘫、失语、痴呆等后遗症方面也有一定的优势。1.2中医药对BBB的影响目前研究较广泛,芳香开窍药麝香、冰片广泛应用于神经系统疾病,二者透过BBB迅速,麝香可抑制脑缺血后炎症、抗自由基损伤、减轻脑水肿、增加血流量等,对缺血性神经元有保护作用。冰片可改善BBB的通透性,使其他药物能更快更多地进入中枢神经系统发挥作用。1.3脑缺血再灌注后BBB的超微结构损伤,包括内皮细胞肿胀、吞饮小泡增多、紧密连接开放、基底膜破坏及胶质细胞水肿等。BBB损伤分子机制涉及炎性细胞因子、粘附分子、基质金属蛋白酶、水通道蛋白、自由基、花生四烯酸代谢产物、血管内皮生长因子、纤溶酶、凝血酶等,这些因素相互作用,互为因果。2实验研究2.1麝香配伍冰片对缺血再灌注后大鼠病理及BBB超微结构影响的研究目的:通过观察缺血再灌注后大鼠神经元病理及BBB超微结构变化的影响探讨麝香配伍冰片对BBB的保护作用。方法:建立大鼠局灶性脑缺血再灌注模型,光镜下观察各组动物神经元病理变化,电镜下观察BBB超微结构。结果:麝香配伍冰片可以有效减轻缺血再灌注后神经元坏死、水肿,BBB内皮细胞肿胀、紧密连接疏松、基底膜结构模糊及胶质细胞水肿等。结论:麝香配伍冰片通过上述作用减轻BBB损害、脑水肿,减轻缺血再灌注损伤。2.2麝香配伍冰片对缺血再灌注后大鼠ICAM-1、LFA-1表达的影响目的:研究缺血再灌注后BBB内皮细胞ICAM-1、LFA-1表达情况,探讨麝香配伍冰片对BBB内皮细胞的保护作用。方法:RT-PCR方法研究ICAM-1mRNA、LFA-1 mRNA表达,免疫组化方法研究ICAM-1蛋白表达变化。结果:麝香配伍冰片、安宫牛黄丸可显著降低缺血再灌注后脑组织中ICAM-1 mRNA及其配基LFA-1 mRNA的表达(P＜0.01),冰片组各时间段ICAM-1 mRNA和缺血2h再灌注48h时间段的LFA-1mRNA也有明显降低(P＜0.05)。蛋白表达方面,麝香配伍冰片组、安宫牛黄丸组、冰片组ICAM-1蛋白表达量较模型组明显下降(P＜0.05),其中麝香配伍冰片组下降最多。结论:麝香配伍冰片可以降低缺血再灌注后粘附分子ICAM-1及其配基LFA-1的表达,从而减轻炎性反应对BBB的损伤而起脑保护作用。2.3麝香配伍冰片对缺血再灌注后大鼠MMP-9、TIMP-1表达的影响目的:研究缺血再灌注后BBB基底膜MMP-9、TIMP-1表达情况,探讨麝香配伍冰片对BBB基底膜的保护作用。方法:RT-PCR方法研究MMP-9 mRNA、TIMP-1 mRNA表达,免疫组化方法研究MMP-9蛋白表达变化。结果:麝香配伍冰片组、安宫牛黄丸组、冰片组各缺血再灌注时间段脑组织MMP-9 mRNA表达均受到明显抑制;与假手术组比较,三组TIMP-1 mRNA表达明显升高,在缺血2h再灌注24h时间段,麝香组也有相同变化趋势。MMP-9蛋白表达方面,麝香配伍冰片组、冰片组、安宫牛黄丸组较模型组表达下降明显(P＜0.05)。结论:麝香配伍冰片可通过抑制MMP-9表达、提升TIMP-1表达而减轻基底膜细胞外基质的降解和BBB通透性。2.4麝香配伍冰片对缺血再灌注后大鼠AQP4表达的影响目的:研究麝香配伍冰片对缺血再灌注后BBB胶质细胞AQP-4表达情况,探讨麝香配伍冰片对BBB胶质细胞的保护作用。方法:分别用RT-PCR方法、免疫组化方法研究AQP-4 mRNA、AQP-4蛋白表达的变化。结果:在缺血再灌注各时间段,麝香配伍冰片、安宫牛黄丸、冰片均可明显抑制缺血脑组织中AQP-4 mRNA的表达,其中麝香配伍冰片、安宫牛黄丸组降低幅度最大。蛋白表达方面,麝香配伍冰片组、冰片组、安宫牛黄丸组下降明显(P＜0.05)。结论:麝香配伍冰片通过抑制缺血再灌注后脑组织AQP-4 mRNA和蛋白的表达而降低BBB通透性、减轻脑水肿而起到脑保护作用。3本研究的创新性从古至今,绝大多数医家均认为中风急性期出现意识不清才使用“开窍法”,我们以大鼠局灶性脑缺血再灌注模型为基础,研究“芳香开窍”代表药物麝香、冰片药对缺血再灌注后BBB损伤的影响机制,深入研究其作用机理,为临床治疗缺血性中风即使未出现意识不清也可应用芳香开窍法提供依据。过去对麝香、冰片多进行单味药的药理研究,我们以成分比较清楚的、且能通过BBB的两个单味中药麝香、冰片配伍组合进行系统的研究,有别于传统的大复方研究,不仅有利于药物作用的分析及其应用时增效减毒作用的研究,而且便于新药的开发,具有潜在的应用价值。探讨麝香配伍冰片抗缺血性脑损伤的作用机理,对揭示该药对配伍规律及其“芳香开窍”作用机理具有理论意义。现有的研究未能系统阐明该药对抗缺血性脑损伤的机制,因而未能较清晰地认识其作用环节,限制了该药对临床的合理使用。本研究从多角度探讨该药对配伍抗脑缺血损伤的作用及机理。这对于深入认识该药对的配伍原理和临床合理应用均具有指导作用。围绕缺血性脑水肿中BBB损伤的主要环节,从内皮细胞损伤、基质破坏和胶质细胞损伤三个环节动态研究麝香配伍冰片抗缺血性脑水肿的作用和分子机制,从而系统地研究其作用环节。更多还原

【Abstract】 Ischemic brain edema is the most common cause of death following cerebrovascular disease.It is the necessary and important pathological process.It is one of the focal points in neurological territory.Injuried Blood Brain Barrier(BBB) is the major pathogenesy.Treatments on brain edema currently are hyperisotonic dehydrater,diuretic,glucocorticoid and surgical intervention to reduce intracranial pressure.Although these treatments could relieve brain edema to a certain degree,its major pathogenetic link that BBB is injured is still remain to be resolved.Traditional Chinese drugs and complex prescriptions have certain advantages on treating ischemic brain injuries,it has aroused extensive attention because of the multi-target, multi-channel pharmacological effects.As an important tool of treating stroke, brain waking-up therapy has not been able to arouse enough attention although in recent years is widely Used in clinical.Through studying literatures,this project summed up and expounded the necessity of using resuscitation-inducing therapy with fragrant drugs during the acute stage of ischmic stroke,traditional Chinese Medicine on the role of BBB,particularly the musk and borneol,the molecular mechanism of BBB injury after cerebral ischemia-reperfusion.According to the basis of the work of Professor Liu Yamin,we established the focal cerebral ischemia-reperfusion model by suture middle cerebral artery occlusion in rats.Then Focusing on ischemic brain edema in the BBB injury of the major aspects,we Explored the influence of Borneol with musk on cerebral microvascular endothelial cells, stromal cells and glial foot using electron microscopy,immunohistochemistry, RT-PCR and other methods.The role of musk with Borneol BBB and its molecular mechanism after ischemic injury was systematicly analyzed.This could provide scientific basis for clinical treating acute ischemic stroke with brain waking-up therapy.1 Documents study1.1 Obstruction in the brain orifice and its functions failing to work is the leading pathogenesis for ischemic stroke,so brain waking-up therapy is the basic rule to cure ischemic stroke.It is an an important means of acute phase of stroke and should be used as soon as possible.Resuscitation-inducing drugs could wake up brain to resume its functions,eliminate sputum,promote blood flow,relieve spasm by calming endogenous wind,clear heat and so on. Resuscitation-inducing therapy also has advantages when treating hemiparalysis,anepia,dementia and other residuals.1.2 The impact of Traditional Chinese Medicine on BBB is researched widely right now.Musk and borneol are widely used in diseases of the nervous system,they could traverse BBB quickly.Musk could restrain inflammation, resist injuries because of free radical,extenuate brain edema and increase blood flow after brain ischemia.Borneol could improve permeability of BBB so as to help other drugs enter central nervous system more and fasterly.1.3 The ultrastructure injury of BBB after cerebral ischemia-reperfusion includes endothelial cell swelling,increased pinocytotic vesicles,tight junctions open,membrane damage and edema and glial cell swelling and so on. The molecular mechanisms of BBB injury involves inflammatory cytokines, adhesion molecules,matrix metalloproteinases,water channel proteins,free radicals,arachidonic acid metabolites,vascular endothelial growth factor, plasminogen,thrombin,etc. 2 Experimental research2.1 Studies about the influence of Musk with Borneol on pathology and BBB ultrastructure after ischemia-reperfusionObjective:Discuss the protection effect of Musk with borneol on BBB by observation of neuron pathology and BBB ultrastructure after ischemia-reperfusion.Methods:Focal cerebral ischemia-reperfusion model(MCAO) rat was set up first of all,then observation of neuron pathology using light microscopy and BBB ultrastructure using electron microscopy was done.Results:Musk with borneol could effectively reduce neuronal necrosis, edema,BBB endothelial cell swelling,loose tight junctions,basement membrane structure and glial cell line-fuzzy edema after ischemia-reperfusion.Conclusion:Musk with Borneol reduced BBB damage,brain edema and ischemia-reperfusion injury through these mentioned roles above.2.2 Studies about the influence of Musk with Borneol on the expression of ICAM-1 and LFA-1 after ischemia-reperfusionObjective:Discuss the protection effect of Musk with borneol on BBB endothelial cell by investigating the expression of ICAM-1 and LFA-1 on the rats with focal cerebral ischemia-reperfusion injury.Methods:ICAM-1 and LFA-1 mRNA expression were measured utilizing real-time PCR,ICAM-1 protein expression was evaluated utilizing immunohistochemistry.Results:ICAM-1 mRNA,LFA-1 mRNA expression in musk with borneol group and Angong Niuhuang Pill group significantly decreased(P＜0.01),ICAM-1 mRNA expression each time and LFA-1 mRNA expression after reperfusion for 48h in borneol group also significantly reduced(P＜0.05).ICAM-1 protein expression in musk with borneol group,Angong Niuhuang Pill group and borneol group decreased significantly compared with model group(P＜0.05),it decreased at most in musk with borneol group.Conclusion:Musk with borneol could protect BBB against inflammatory response through reducing the expression of adhesion molecule ICAM-1 and its ligand LFA-1 after ischemia-reperfusion.2.3 Studies about the influence of Musk with Borneol on the expression of MMP-9 and TIMP-1 after ischemia-reperfusionObjective:Discuss the protection effect of Musk with borneol on BBB basement membrane by investigating the expression of MMP-9 and TIMP-1 on the rats with focal cerebral iscbemia-reperfusion injury.Methods:MMP-9 and TIMP-1 mRNA expression were measured utilizing real-time PCR,MMP-9 protein expression was evaluated utilizing immunohistochemistry.Results:MMP-9 mRNA expression in musk with borneol group,borneol group and Angong Niuhuang Pill group were significantly inhibited(P＜0.01).TIMP-1 mRNA expression significantly increased compared with the sham-operated group in those three groups and musk group after 24h reperfusion.MMP-9 protein expression in musk with borneol group,Angong Niuhuang Pill group and borneol group decreased significantly compared with model group(P＜0.05).Conclusion:Musk with borneol could reduce the degradation of extracellular matrix basement membrane and BBB permeability by inhibiting the expression of MMP-9 and elevating the expression of TIMP-1.2.4 Studies about the influence of Musk with Borneol on the expression of AQP-4 after ischemia-reperfusionObjective:Discuss the protection effect of Musk with borneol on BBB glial cell by investigating the expression Of AQP-4 on the rats with focal cerebral ischemia-reperfusion injury.Methods:The expression of AQP-4 mRNA were measured utilizing real-time PCR and its protein expression was evaluated utilizing immunohistochemistry.Results:AQP-4 mRNA expression in musk with borneol group,borneol group and Angong Niuhuang Pill group were significantly inhibited,it decreased at most in musk with borneol group and Angongniuhuang Pill group decreased the largest.AQP-4 protein expression in musk with borneol group,Angong Niuhuang Pill group and borneol group decreased significantly compared with model group(P＜0.05).Conclusion:Musk with Borneol could reduce the BBB permeability and brain edema through inhibiting the expression of AQP-4 mRNA and protein after ischemia-reperfusion so as to protect the brain.3 The contributions of this research are as followsFrom ancient times,the vast majority of doctors are of the view that the resuscitation-inducing therapy should be used when conscious disturbance appears at the acute phase of stroke.Musk with borneol is the type of fragnant drugs for inducing resuscitation.We set up the focal cerebral ischemia-reperfusion rat model to study the influence of musk with borneol on BBB injury after ischemia-reperfusion and research deeply its action mechanism.We hope to provide a basis for using resuscitation-inducing therapy with fragrant drugs even if conscious disturbance has not appeared when treating ischemic stroke.In the past,most researches on musk and borneol were one-flavor drug pharmacological study.Having clear constituents and the ability to traverse BBB,musk with borneol was studied Systematically,which is different from the traditional research on large compound.That has potential application value not only because of the advantages to the analysis of drug effect and researches on synergy and attenuation when applied,but also the facility for the development of new drugs.Discussion on the mechanism of musk with borneol against ischemic brain damage has theoretical significance to reveal its compatibility law and mechanism of resuscitation-inducing therapy with fragrant drugs.The mechanism against ischemic brain damage of musk with borneol has not been illuminated in existing researches and the link of its role has not been clearly known,so its clinical application has been restrained.This study discussed the mechanism of musk with borneol against ischemic brain damage from different perspectives:It has guiding role to recognize its compatibility law and apply reasonablly.Around Ischemic brain edema around the BBB injury in the main aspect of BBB injury during ischemic brain edema,the effect against ischemic brain edema and molecular mechanism of musk with borneol was researched dynamically from endothelial cells damage,matrix damage and glial cell injury to study the link of its role systematically.更多还原