【作者】 傅风华；

【导师】 张岫美；

【作者基本信息】 山东大学 ， 医学基础药理学， 2008， 博士

【摘要】 研究目的:脑血管疾病是危害人类生命健康的常见病和多发病,具有发病率高、致残率高、死亡率高和复发率高的特点,是中老年人致死、致残的主要疾病,已成为危害人类健康的重要原因,造成了极大的经济和社会负担。血管性痴呆是以认知损害为特征的一种综合征,主要表现为认知功能障碍,包括学习、记忆、思维功能的降低等。脑血管疾病,特别是缺血所致的脑损伤是导致血管性痴呆(vascular dementia,VD)的重要原因。药理学和临床研究证明,包括多奈派齐在内的胆碱酯酶抑制剂可明显缓解痴呆病人的症状,改善生活质量。但由于胆碱酯酶抑制剂可引起以胃肠道症状为主的严重不良反应,其临床使用受到了很大的限制。脑缺血损伤后产生的炎症和水肿,以及随之发生的神经损伤与血管性痴呆发生密切相关。七叶皂苷钠是从中药娑罗子中提取的三萜皂苷类化合物,具有很强的抗炎抗水肿作用,临床上广泛用于脑血栓的治疗。脑血栓形成后,抗炎治疗是否能够减少脑缺血损伤,降低缺血所致的痴呆的发生,以及七叶皂苷钠作为抗炎抗水肿是否适合用于脑缺血损伤的治疗;若七叶皂苷钠可以改善脑缺血损伤引起的学习记忆障碍,其机制是什么?胆碱酯酶抑制剂多奈派齐早期给药是否可以改善脑缺血所致的学习记忆障碍,M—受体阻断剂是否可以降低多奈派齐的消化道不良反应,而不影响多奈派齐对痴呆的治疗作用(理论上,M—受体阻断剂进入中枢可以加重痴呆的症状)。药物在脑缺血损伤时,对炎症相关基因和神经递质受体及调控因子基因,又产生什么影响?本论文以七叶皂苷钠和多奈哌齐为代表,研究抗炎和胆碱酯酶抑制剂对脑缺血损伤及血管性痴呆的作用,并对这两种药物使用中可能存在的不良反应及其防治进行研究。研究方法:本论文分为两大部分。第一部分研究七叶皂苷钠对缺血性脑损伤的作用及其机制;第二部分研究胆碱酯酶抑制剂多奈哌齐的神经保护作用及不良反应的防治。一、七叶皂苷钠对缺血性脑损伤的作用及机制研究1.采用两种大鼠缺血性损伤模型研究七叶皂苷钠对缺血致神经损伤的影响。第一种采用经典栓线致大鼠大脑中动脉阻塞(MCAO)方法造成局灶性脑缺血再灌注损伤,以Longa’s神经功能学评分法检测动物神经损伤程度;以氯化四唑(TTC)染色法测定脑梗死面积百分比,观察七叶皂苷钠对脑缺血性损伤的保护作用。第二种采用双侧颈总动脉永久结扎大鼠脑缺血模型,结扎后,静脉注射七叶皂苷钠,每天1次,连续给药3天,进行学习记忆观察;另取大鼠手术后,连续给药3天,末次给药6小时后,麻醉取脑,观察线粒体结构功能变化。2.采用小鼠短暂性缺血致脑损伤(记忆障碍)模型研究七叶皂苷钠对血管性痴呆学习记忆的影响。实验时分离双侧颈总动脉,用动脉夹夹闭同时放血。假手术组只分离颈总动脉,不夹闭和放血。手术次日开始给药,假手术组给予生理盐水,阳性药对照组给予多奈哌齐,实验组分别给予不同剂量的七叶皂苷钠。连续给药3天后,开始Morris迷宫训练,连续5天,记录每只小鼠的记忆潜伏期等,评价学习记忆成绩。取海马,以比色法测定乙酰胆碱酯酶活性。3.以脑含水量评价七叶皂苷钠对大鼠缺血再灌注致脑水肿的影响。证明七叶皂苷钠的抗缺脑水肿作用后,以大鼠足跖肿模型和小鼠腹腔毛细血管通透性模型研究其抗炎抗水肿作用特点;以去肾上腺大鼠研究七叶皂苷钠抗炎作用和肾上腺皮质激素的关系。4.以基因芯片,研究观察缺血和给药对NFκB通路基因和神经递质受体及调控因子基因水平的影响。以蛋白芯片,观察缺血对NFκB通路蛋白表达的影响。二、胆碱酯酶抑制剂多奈哌齐的神经保护作用及其不良反应防治胃肠运动功能亢进是胆碱酯酶抑制剂引起消化道不良反应的主要原因。胃排空和肠推进常被用作观察动物胃肠运动的指标。1.以胃排空和小肠推进实验,评价M-受体阻断剂溴丙胺太林对胆碱酯酶抑制剂多奈哌齐增强胃肠运动所致的消化道不良反应的影响。2.以能降低多奈哌齐胃肠道不良反应的溴丙胺太林的剂量,观察溴丙胺太林对多奈哌齐治疗效应的影响。采用小鼠短暂性脑缺血致损伤(记忆障碍)模型,研究多奈哌齐对血管性痴呆学习记忆的影响。手术次日开始给药,假手术组动物给予生理盐水,实验组分别给予阳性药对照药多奈哌齐,溴丙胺太林,溴丙胺太林和多奈哌齐。连续给药3天后,开始Morris迷宫训练,连续5天,记录每只小鼠的记忆潜伏期。取固定区域的皮层,测定乙酰胆碱酯酶活性。以学习记忆指标,评价多奈哌齐对血管性痴呆的治疗作用,以及溴丙胺太林对多奈哌齐治疗作用的影响。3.以基因芯片,研究缺血、胆碱酯酶抑制剂多奈哌齐或溴丙胺太林和多奈哌齐联用,对神经递质受体及调控因子基因水平的影响。结果:1.在大鼠大脑中动脉阻塞(MCAO)局灶性脑缺血再灌注损伤模型,七叶皂苷钠可以明显降低缺血所致神经功能评分的升高,降低缺血致坏死面积,表明七叶皂苷钠对缺血再灌注损伤具有一定的保护作用。2.在大鼠双侧颈总动脉结扎模型,七叶皂苷钠改善脑缺血损伤所致的学习记忆障碍。这种保护与改善线粒体呼吸功能有关。连续3天静脉注射七叶皂苷钠0.9mg/kg可以抑制线粒体膜结构和呼吸能力受损,升高呼吸能力(S3),降低基础氧耗(S4),升高呼吸链活性(RCR)。3.小鼠短暂性脑缺血致脑损伤模型研究显示,七叶皂苷钠可明显改善缺血所致的学习记忆障碍。七叶皂苷钠对短暂性脑缺血损伤有一定的防护作用,但该作用与脑内乙酰胆碱酯酶活性无关。4.七叶皂苷钠具有明显的抗缺血致脑水肿作用。5.在大鼠足跖肿模型和小鼠毛细血管通透性模型,七叶皂苷钠均具有较强的抗炎作用,其作用起效相对缓慢,但维持时间长达24 h。七叶皂苷钠无致胃肠粘膜损伤作用,对应激性溃疡形成具有一定的抑制作用。七叶皂苷钠对体液免疫、细胞免疫均无抑制作用。在去肾上腺大鼠,七叶皂苷钠不显示抗炎作用,表明七叶皂苷钠的抗炎作用依赖于肾上腺的存在;但静脉注射七叶皂苷钠并不升高大鼠体内皮质酮的水平;低剂量的皮质酮和七叶皂苷钠均无抗炎作用,而低剂量联合给药显示明显的协同效应,表明七叶皂苷钠不是通过刺激肾上腺皮质激素的的释放发挥抗炎效应,而可能是通过放大糖皮质激素的抗炎效应而显示抗炎作用。6.基因芯片研究显示,缺血可致部分神经递质和受体基因发生明显变化,七叶皂苷钠可在一定程度上调节这些变化。特别是NFκB通路相关的基因,七叶皂苷钠下调致炎性和致脑损伤相关的基因,上调脑损伤保护相关的基因水平。在缺血的晚期,对NFκB蛋白表达无直接抑制作用。7.小鼠肠推进实验显示,多奈哌齐能够明显提高肠推进速度,而溴丙胺太林明显减慢肠推进速度;以不同剂量的溴丙胺太林与多奈哌齐联合使用,溴丙胺太林2mg/kg给药时,可明显缓解多奈哌齐所致的胃肠运动亢进,表明适当剂量的M-受体阻断剂可以降低胆碱酯酶抑制剂对胃肠运动的影响,降低消化道不良反应的发生。8.溴丙胺太林与多奈哌齐联合使用对多奈哌齐治疗效应影响研究显示,短暂性脑缺血损伤可致小鼠学习记忆障碍:多奈哌齐对缺血致记忆障碍具有一定的改善作用;溴丙胺太林单独使用对学习记忆障碍无明显影响,与多奈哌齐联合使用,不降低多奈哌齐的治疗作用。脑缺血损伤后第8天,脑皮层的胆碱酯酶活性升高,多奈哌齐抑制胆碱酯酶的活性,溴丙胺太林不改变多奈哌齐对胆碱酯酶的抑制程度。表明多奈哌齐用药的初始阶段,选择不易透过血脑屏障的M-受体阻断剂与胆碱酯酶抑制剂联合使用,既可降低胆碱酯酶抑制剂胃肠道不良反应,又不影响治疗痴呆的药理效应。9.在短暂性脑缺血小鼠,使用胆碱酯酶抑制剂多奈哌齐、M-受体阻断剂溴丙胺太林,或联合使用两种药物,对神经递质受体及调控因子基因产生复杂的调节。缺血和药物对胆碱酯酶系统的调节表现为,不影响乙酰胆碱酯酶基因水平;对胆碱能受体基因的调节十分复杂。短暂性脑缺血和药物对胆碱能系统以外的受体如多巴胺、甘丙肽、神经肽Y等也具有显著的调节作用。10.基因芯片研究的结果还显示,缺血和药物对递质受体及调控因子基因发生复杂的调节作用。溴丙胺太林与多奈哌齐联用与多奈哌齐单独使用相比,基因水平上显示了一定的差异。结论:1.七叶皂苷钠对缺血性脑损伤具有保护作用,对脑缺血所致的学习记忆障碍具有改善作用。该作用可能与抗脑水肿,线粒体保护,减轻神经损伤有关;而与乙酰胆碱酯酶活性无关。2.七叶皂苷钠对缺血海马组织炎症损伤因子如补体C3、趋化因子、TNF和CD40基因的下调和损伤保护因子GM-CSF基因的上调,可能是其神经损伤保护的重要机制。3.七叶皂苷钠的抗炎作用与糖皮质激素相似。其抗炎作用依赖于肾上腺的存在,但并不刺激体内糖皮质激素的释放,放大糖皮质激素抗炎效应可能是七叶皂苷钠抗炎作用的重要机制。七叶皂苷钠在发挥抗炎作用的同时,既不抑制机体的免疫功能,又不引起消化道粘膜的损伤,且对应激性溃疡有一定的防治作用,因此适合用于脑缺血疾病的治疗。4.多奈哌齐可改善缺血所致的学习记忆障碍。不易透过血脑屏障的季胺类M-受体阻断剂溴丙胺太林,可明显降低多奈哌齐的消化道不良反应,而对其治疗学效应无明显影响。脑缺血、多奈哌齐、多奈派齐和溴丙胺太林两者联合使用对神经递质受体及调控因子基因产生复杂的影响。本研究的意义与创新点是:本研究明确了临床常用于缺血性脑中风治疗的抗炎抗水肿药物七叶皂苷钠能够降低缺血致痴呆的发生,明确了其抗炎作用的特点和初步机制,为科学合用药奠定了理论基础;明确了老年性痴呆治疗药物多奈哌齐早期给药具有降低缺血致痴呆的作用,提出了解决胆碱酯酶抑制剂消化道不良反应的方案,为更合理地应用胆碱酯酶抑制剂治疗血管性痴呆提供了实验依据。主要创新点:(1)首次明确七叶皂苷钠的神经保护作用可以减少脑缺血引起的血管性痴呆的发生;(2)首次明确七叶皂苷钠可以同时上调神经损伤保护因子的基因,下调致炎症损伤因子的基因;(3)首次提出七叶皂苷钠放大糖皮质激素抗炎效应抗炎作用的新机制;(4)证明了不能透过血脑屏障的M-受体阻断剂溴丙胺太林既可降低多奈哌齐的不良反应,又不影响多奈哌齐的疗效。更多还原

【Abstract】 Objective::Cerebrovascular diseases have the characteristics of high morbidity, high rate of mutilation and high mortality.They are the main causes resulting in cripplehood and death in middle and elder man,therefore has given rise to an enormous socioeconomic burden.Vascular dementia is a degenerative cerebrovascular disease that leads to a progressive decline in memory and cognitive functioning.It occurs when the blood supply carrying oxygen and nutrients to the brain is interrupted by a blocked or diseased vascular system.Signs and symptoms are varied and usually reflect increasing difficulty to perform everyday activities,such as eating,dressing,shopping, etc.Alzheimer’s disease(AD) and vascular dementia(VD) are the two main causes of dementia.The incidence of VD is the second most common form of dementia in the elderly after AD.Pharmacological experiments and controlled clinical trials with cholinesterase inhibitors,such as donepezil,galantamine and rivastigmine in VD and AD,have demonstrated improvements in cognition,behavior and activities of daily living.However,the cholinesterase inhibitor agents may cause a broad spectrum of adverse events-nausea,vomiting,and diarrhoea,and so on,which make many patients withdrawn themselves from taking CHI agents.Vascular dementia is associated with the inflammation,edema and neural lesion resulted from cerebral ischemia.Escin(Sodium aescinate) is prescribed to treat ischemic stroke and traumatic brain injury.Donepezil,a cholinesterase inhibitor,is administered to the patients with dementia in the world.In this study,we investigated the effects of both escin and donepezil on cerebral ischemic injury and dementia in rodent animals,and the prevention of adverse action of these drugs.Methods:There are two parts of our study.One is about the effects and it’s mechanism of escin on cerebral ischemic injury;the other is about the effects of donepezil on vascular dementia,and prevention of adverse action of donepezil.1.The effect of escin on cerebral ischemic injury and dementiaIn rats,two animal models were used to study the effect of escin on the cerebral ischemic injury and dementia.The first rat model,typical rat thread model,performed by middle cerebral artery occlusion(MCAO),was used to induce the reversible focal cerebral ischemia/reperfusion injury.Focal cerebral ischemia/reperfusion injury group which were subject to 2 hours of ischemia followed by 22 hours of reperfusion.The behavioral tests were used to evaluate the damage to central nervous system.2,3, 5-triphenyl tetrazolium chloride(TTC) staining method was used to assess the percentage of brain infarct area.H&E staining was used to observe the pathologic histological changes of hippocampus.The second rat model,rats were subjected to 2VO surgery.Briefly,under anesthesia,the bilateral common carotid arteries of rats were exposed and carefully separated from the carotid sheath and cervical sympathetic and vagus nerves through a ventral cervical incision,and ligated with silk thread.The animals in sham group received the same surgical operation without ligation of the carotid arteries served as sham-operated controls.The drugs were administered and Morris performance was carried out.In another experiment,six hours after occlusion,escin was given by vein every day for 3 days.To isolate brain mitochondria,rats were anesthetized with 10% chloral hydrate and killed by cardiac perfusion.The forebrain tissue was removed immediately and homogenized.The homogenate was immediately centrifuged to separate mitochondria.Mitochondrial swelling was assayed by measuring the decrease in absorbance at 540 nm.Mitochondrial respiratory control ratio(RCR) was measured. In mice,transient cerebral ischemia was induced by bilateral common carotid occlusion.The mice were anesthetized.Then the common carotid arteries were exposed and occluded with artery clips for 20 min.While the arteries were clamped, 0.3 ml of blood was withdrawn from the tail vein.Sham-operated mice were subjected to the same procedure without carotid clamping and withdrawal of blood.Mice alive 24 hr after operation were used in the Morris water maze task.Dally learning consisted of four trials in which the mouse was placed in the water from four different starting points and the latency of escape onto the platform was recorded. This was conducted for 5 consecutive days.A maximum of 60 sec was allowed during which the mouse had to find the platform and climb onto it.When the mouse reached the platform,it was allowed to remain on it for 20 sec.If the mouse failed to find the platform within 60 sec,it was removed from the water and placed on the platform for 20 sec.All latency for a given day was calculated by averaging the four trials.For cholinesterase activity assay,mice were killed by decapitation after the Morris water maze task had been carried out.The cerebral cortex from the right hemisphere was dissected on ice and homogenized.Cholinesterase activity in the homogenates was measured using the spectrophotometric methods.The brain edema was evaluated by the water content in the brain.The anti-inflammation characteristics of escin administered by intravenous injection,was observed by carrageenan-induced paw edema model in rats and vascular permeability induced by acetic acid in mice.The relationship between the effect of escin and adrenal gland was investigated by observing the anti-inflammatory effect of it in rats with adrenalectomy.The neurotransmitter receptors and regulators microarray were used to observe the change of gene regulation in the course of inflammation and anti-inflammation. The signal pathway of NFκB was also observed by ELISA.2.Neuroprotection of Acetylcholinesterase inhibitor donepezil and prevention of it’s adverse actionIn mice,transient cerebral ischemia was induced by bilateral common carotid occlusion as above. Mice alive 24 hr after operation were used in the Morris water maze task.Daily learning consisted of four trials in which the mouse was placed in the water from four different starting points and the latency of escape onto the platform was recorded. This was conducted for 5 consecutive days.A maximum of 60 sec was allowed during which the mouse had to find the platform and climb onto it.When the mouse reached the platform,it was allowed to remain on it for 20 sec.If the mouse failed to find the platform within 60 sec,it was removed from the water and placed on the platform for 20 sec.All latency for a given day was calculated by averaging the four trials.For cholinesterase activity assay,mice were killed by decapitation after the Morris water maze task had been carded out.The cerebral cortex from the right hemisphere was dissected on ice and homogenized.Cholinesterase activity in the homogenates was measured using the spectrophotometric methods.In order to investigate how attenuate the side reaction of CHI agent while without affecting its efficacy,the effects of propantheline bromide(PB) co-administered with donepezil,such as the gastric emptying(GE) and gastrointestinal transit(GIT),the markers of gastrointestinal motility,and brain cholinesterase(ChE) activities,maze tasks of mice with vascular dementia induced by transient ischemia,were observed.The effects of ischemia and drugs(donepezil,propantheline alone,or co-administration of them) on the gene associated with neurotransmitter and receptor were investigated by real time PCR.Results:1.Compared with sham-operate group,rats with MCAO showed significant increase of neurological scores,significant increase of infarct area ratio.Escin could attenuate these changes.It suggested that escin could have a protection against ischemic injury.Twenty eight days after occlusion,in rats subjected to 2VO surgery,the memory of rat was decreased by ischemia and ameliorated by escin.Six hour after occlusion, escin could protect the respiratory energy system and increase the RCR.Mice subjected to transient cerebral ischemia took a longer time to locate the hidden platform than the sham-operated control mice during the learning trials, although the ischemia did not affect the swimming ability of the mice in the pretraining trial in the water maze.Treatment of mice with escin shortened the latency of escape onto the platform during the learning,as compared with the ischemia model control.Escin could attenuate the brain edema significantly for 12 h.In acute inflammatory animal models,escin inhibited carrageenan-induced paw edema in rats and vascular permeability induced by acetic acid in mice.The results demonstrated that the anti-inflammatory effect of escin was similar to dexamethasone,beginning from 4 h to 24 h after it was given.The anti-inflammatory effect of escin would be abolished by adrenalectomy in rats.The Corticosterone in sera was not increased by escin administered intravenously. These findings suggest the effect of escin depend on the adrenal gland and may amplify,the anti-inflammatory effect of glucocorticoids in vivo.2.Neuroprotection of acetylcholinesterase inhibitor donepezil and prevention of adverse action of donepezilGIT was accelerated significantly by donepezil 0.625 mg/kg during 30-60 min after it was given.The maximal acceleration of 26%was achieved 60 min after administration.Thus in experiments the effects of donepezil were observed 60 min after it was administered.Donepezil increased GIT,but GIT was inhibited by PB. When donepezil and PB were administered simultaneously,PB attenuated the GIT promoted by donepezil.At a dose of 2 mg/kg,PB restored GIT to the same level as in the saline group.This dose was equivalent to the therapeutic one in patients based on body surface area.Thus in the measurement of GE,the dose of PB was 2 mg/kg.Donepezil 0.625 mg/kg accelerated GE.PB co-administered with donepezil attenuated the increase of in GE.At dose of 2 mg/kg,PB restored GE to nearly normal level.About 30%of mice with transient cerebral ischemia were died within 24 hr after operation.Mice subjected to transient cerebral ischemia took a longer time to locate the hidden platform than the sham-operated control mice during the learning.(?) although the ischemia did not affect the swimming ability of the mice(?) pretraining trial in the water maze.Treatment of mice with donepezil signifi(?) shortened the latency of escape onto the platform during the learning,especia(?) the 5th learning day,as compared with the ischemia model control.PB alone di(?) have any effect on the latency.Co-administration of PB and donepezil did not(?) the effects of donepezil on the latency of escaping onto the platform in the(?) maze performance,showing that PB had no effect on the therapeutic eff(?) donepezil.After administration of donepezil or both donepezil and PB for 8 days,(?) activity in the brain was decreased compared with that in the ischemia model(?) However,there was no difference between the donepezil alone and donepezil pl(?) groups.This suggested that PB could not change the ability of donepezil to pen(?) through the blood-brain barrier and the inhibitory effects of donepezil on brain(?) activity.The muscarinic receptor blocker PB is unable to penetrate the blood-brain(?) and when co-administered with a cholinesterase inhibitor attenuates its side(?) without affecting its therapeutic effects,and thus may be beneficial for patients(?) VD.The gene of acetylcholinesterase was not changed by drugs mentioned above.(?) effects of co-administered with PB and donepezil was different from that of don(?) given alone,which suggested that clinical trial shoud be carried out to know the(?) effect of co-administration.Conclusion:1.Escin could protect rat brains against ischemia injury and attenuate the vas(?) dementia in the rodemt.2.The anti-inflammatory effect of eacin is similar to dexamethasone.(?) anti-inflammatory mechanism of escin may be associated with amplifying(?) anti-inflammatory effect of glucocorticoids in vivo. 3.The muscarinic receptor blocker PB is unable to penetrate the blood-brain barrier and when co-administered with a cholinesterase inhibitor attenuates its side effects without affecting its therapeutic effects,and thus may be beneficial for patients with VD.更多还原