【作者】 侯艳；

【导师】 刘耕陶； 罗远； 张建军； 叶菲；

【作者基本信息】 中国协和医科大学 ， 药理学， 2009， 博士

【摘要】 随着世界人口平均寿命的增加,老年人口逐年递增,伴随衰老而来的神经退行性疾病如阿尔茨海默病（Alzheimer’s disease,AD）和帕金森氏病（Parkinson’s disease,PD）的发病率亦相应增加。因而全世界开始面临老龄化社会带来的诸多问题。为了延长人类的预期寿命,提高老年人的生命质量,抗衰老和防治衰老相关疾病受到了越来越多的重视。其中,脑衰老和衰老相关的神经退行性疾病是老年医学中最急需解决的问题之一。关于衰老机制的假说甚多,其中包括生殖系统衰老导致的性激素缺乏对脑衰老性病理改变的发展具有促进作用。雌激素是一种具有神经调节和神经保护作用的激素。雌激素通过激活其受体调控多种基因的表达而发挥神经保护作用,另外,它还具有抗氧化作用。绝经后雌激素水平的迅速下降可以导致认知和记忆能力的减退,增加神经退行性疾病老年痴呆和帕金森病的发病率。雄激素同样具有神经调节和神经保护作用。男性的睾酮水平在中年以后开始减退,老年男性的睾酮低水平导致认知记忆功能的减退,睾酮缺乏是AD的重要危险因素。在衰老过程中,脑组织暴露于多种应激刺激中,损伤的蛋白质逐渐积累。错误折叠蛋白质的聚集,对细胞的存活造成严重的威胁,促进神经退行性病变的发生。热休克蛋白（heat shock protein,HSP）作为分子伴侣,与错误折叠的蛋白质结合,促进其正确折叠或清除。热休克蛋白可以抵抗多种应激刺激导致的组织损伤,因此被视为内源性细胞保护因子。在多种应激刺激作用下,两种重要的热休克蛋白HSP70和HSP27,在大脑的许多区域存在明显的诱导表达。在衰老脑组织中,应激刺激诱导的热休克蛋白表达减少,导致内源性防御机制减弱。阿尔茨海默病是老年人常见的,以进行性的记忆和认知功能损伤为特征的神经退行性病变。老年斑的形成是AD最重要的特征性病理学改变。老年斑的主要成分淀粉样蛋白（Amyloidβ,Aβ）Aβ_1-40和Aβ_1-42,在脑组织中的进行性沉积可以引发一系列神经损害性反应,促进神经退行性病变的发展。Aβ是由淀粉样前体蛋白（Amyloid precursor protein,APP）经α-,β-和γ-内分泌酶酶切加工而生成的。APP的酶切加工存在两种相互排斥的不同途径:淀粉样蛋白生成途径系APP被β-内分泌酶和γ-内分泌酶切割生成Aβ;非淀粉样蛋白生成途径系APP被α-内分泌酶切割生成sAPPα。抑制β-内分泌酶以减少Aβ沉积是抗AD药物研究的靶点。研究表明,增加α-内分泌酶的表达可以减少AD转基因小鼠海马的Aβ沉积,改善学习记忆功能。因此,促进α-内分泌酶对APP的酶切加工成为治疗AD的新手段。FLZ是人工全合成的番荔枝酰胺新衍化物。既往的研究证实FLZ具有明显的神经保护作用:保护Aβ导致的SH-SY5Y细胞的损伤;改善脑室注射Aβ导致的小鼠学习记忆能力的损害并且减轻海马病理学损伤。因此,FLZ具有研发为抗AD新药的前景,已申请专利保护。为了解性激素与衰老和神经退行性疾病发病的关系以及寻找抗AD新药,本论文主要从以下三个方面进行研究:第一部分:性腺切除加速小鼠脑功能衰老模型的建立为建立性腺切除促进脑功能衰老的模型,在本研究中,将2月龄年轻雄性和雌性ICR小鼠的性腺切除,继续饲养10个月至12月龄。实验用小鼠分为4组:雌性假手术组（female-sham,12月龄）、雌性切除卵巢组（female-OVX,12月龄）、雄性假手术组（male-sham,12月龄）、雄性切除睾丸组（male-ORX,12月龄）。评价各项指标时,临时领取雌性年轻小鼠（female-young,2月龄）和雄性年轻小鼠（male-young,2月龄）,与上述各组进行对比分析。小鼠脑衰老相关指标的检测包括学习记忆功能及脑组织相关部位的神经病理学指标的变化。在水迷津和Morris水迷宫实验中,结果显示,与2月龄年轻小鼠比较,性腺切除小鼠空间学习记忆能力下降;免疫组织化学染色和Western blot结果显示,性腺切除小鼠脑组织Aβ、BACE1和phospho-Tau表达增加;而NGF、BDNF、NT3和TrkA表达水平减低,p75NTR表达水平增高。尼氏染色结果显示,性腺切除小鼠海马和皮层神经元尼氏体数量减少。与同性别年轻小鼠相比较,卵巢切除小鼠脑组织的上述衰老相关指标的改变比睾丸切除小鼠更加明显。假手术组小鼠的上述指标则与同性别年轻小鼠相比没有明显差异。由此表明,性腺切除导致的性激素长期缺乏可以加速小鼠脑衰老。表现为学习记忆功能的减退,脑组织Aβ沉积增多,而神经营养因子减少。第二部分:雌激素对卵巢切除小鼠脑组织热休克蛋白表达的调节热休克蛋白是一种内源性细胞保护因子,老年人耐受酷暑天气能力差,可能与热休克蛋白表达调控的失调有关。而热休克蛋白与AD和PD的发展亦有关系,为了解卵巢切除对小鼠脑组织热休克蛋白表达的影响,以及雌激素对热休克蛋白表达的调控作用,研究了小鼠切除卵巢后雌激素对高热刺激诱导的脑组织热休克蛋白表达的调节作用和相关机制。2月龄雌性小鼠经双侧卵巢切除或假手术后,继续饲养10个月。小鼠分成4组:年轻组（young,2月龄）、切除卵巢组（old-OVX,12月龄）、假手术组（old-sham,12月龄）、雌激素补充组（OVX+E2,12月龄）。雌激素补充组小鼠隔日皮下注射雌二醇0.5mg/kg,持续两周,以恢复其血清雌激素生理水平。将小鼠置于44℃烤箱中接受1h高热刺激后,将小鼠取出,置于室温环境中,3h后将小鼠处死。Western blot和RT-PCR结果显示,切除卵巢组小鼠脑组织高热刺激后HSP70蛋白的表达和mRNA的转录均明显低于年轻组小鼠。凝胶迁移阻滞实验检测调控热休克蛋白表达的关键因子,结果显示,切除卵巢组小鼠脑组织热休克因子和热休克元件的结合能力明显减弱。并且发现切除卵巢组小鼠在高热刺激后脑组织出现明显的DNA片断化损伤和caspase-3活性增高,线粒体细胞色素C和凋亡诱导因子的释放亦均增加。与切除卵巢组小鼠相比,雌激素补充组小鼠脑组织中,高热刺激后HSP70蛋白的表达和mRNA的转录明显增高,热休克因子和热休克元件的结合能力明显恢复;高热刺激导致的DNA片断化损伤、caspase-3活性的增高和线粒体细胞色素C和凋亡诱导因子的释放均明显减轻。由此可见,切除卵巢小鼠脑组织在高热刺激后HSP70蛋白的诱导表达能力减低,其机制与热休克因子和热休克元件的结合能力减弱有关。而补充雌激素可明显恢复高热刺激诱导的切除卵巢小鼠脑组织HSP70蛋白表达,并且保护高热刺激导致的脑组织损伤。这些结果均属新发现。第三部分:番荔枝酰胺衍生物FLZ通过促进α-内分泌酶介导的APP酶切加工而减少Aβ生成如前所述,增加α-内分泌酶的活性可以减少APP切割为Aβ,从而可能是治疗AD的新策略。FLZ在多种模型中具有神经保护作用。本文从α-内分泌酶介导的APP酶切加工角度对FLZ的作用进行研究。在本研究中,我们发现FLZ可以减少N2a（APPswe/PS1Δ9）细胞的Aβ生成,同时增加培养基中sAPPα的分泌。Westernblot结果显示,FLZ可以增加细胞膜表面ADAM10和APP的表达。免疫细胞化学染色显示FLZ可以增加ADAM10和APP在细胞膜表面的共定位。预先给与细胞蛋白运输抑制剂brefeldin能阻断FLZ介导的ADAM10和APP向细胞膜的运输,逆转FLZ抑制Aβ生成和增加sAPPα分泌的作用。在TgAPP/PS1转基因小鼠中,FLZ能减少海马Aβ的生成,增加ADAM10的表达。上述结果表明,FLZ通过促进α-内分泌酶介导的APP酶切加工而减少Aβ的生成。这为FLZ用于治疗AD提供了新的理论依据。综上所述,建立衰老动物模型对研究衰老机理、研发延缓衰老和防治衰老相关神经退行性疾病的药物具有重要意义。衰老研究的最终目的之一是为防治神经退行性疾病提供新策略。本文发现:（1）性腺切除小鼠与同性别年轻小鼠相比,空间学习记忆能力减退;脑组织Aβ、BACE1和phospho-Tau表达增加;海马和皮层神经元尼氏体数量减少;脑组织NGF、BDNF、NT3和TrkA表达减低,p75NTR表达水平增高。这些新发现提示:性腺切除导致的长期性激素缺乏可以加速脑衰老。（2）切除卵巢小鼠脑组织在高热刺激后HSP70蛋白的诱导表达减少,其机制与热休克因子和热休克元件的结合能力减弱有关。由此可见,雌激素的长期缺乏可加重小鼠脑组织内源性防御功能的减退。补充雌激素可使切除卵巢小鼠脑组织在高热刺激后HSP70蛋白的诱导表达功能恢复,并且保护高热刺激所致的脑组织损伤。因此,雌激素对热休克蛋白诱导表达的调节作用为其神经保护作用提供了新的依据。（3） FLZ可以通过促进α-内分泌酶介导的APP酶切加工而减少Aβ的生成,这为FLZ的神经保护作用提供了新的依据。此外,为寻找治疗肝功能衰竭新药,对抗肝炎新药双环醇的衍化物WLP-S-10对扑热息痛所致小鼠急性肝衰竭的保护作用及机制进行了研究,结果如下:急性肝衰竭是一种以肝功能迅速恶化为特征的严重急症。扑热息痛过量是药物中毒性急性肝衰竭的最常见原因。双环醇是我国自主研发的新型药物,在临床上广泛用于治疗病毒性肝炎,药物和化学毒物导致的肝损伤。药理学研究证明,双环醇对多种动物肝损伤模型导致的肝损伤具有保护作用。但是,双环醇属脂溶性化合物,不溶于水。目前临床只能口服,无法注射用药。在本研究中,我们合成了一系列水溶性双环醇衍化物。使用CCl₄导致的小鼠肝功能损伤模型,扑热息痛和D-氨基半乳糖加脂多糖所致的急性肝衰竭模型,对其中10种化合物进行筛选,发现其中的一种化合物（WLP-S-10）作用最明显。我们进而研究了该化合物对扑热息痛所致急性肝衰竭的保护作用和机制。提前1小时给小鼠腹腔注射WLP-S-10 200mg/kg,然后腹腔注射扑热息痛450mg/kg。结果显示,WLP-S-10可以降低小鼠死亡率,降低血清ALT和AST;防止线粒体GSH及ATP的耗竭;减轻线粒体肿胀和膜电位崩溃;减少线粒体细胞色素C和AIF的释放。HPLC结果显示,WLP-S-10可以抑制肝微粒体对扑热息痛的代谢。WLP-S-10在体内代谢转化为双环醇和蛋氨酸。结果表明,双环醇衍化物WLP-S-10对扑热息痛导致的小鼠急性肝衰竭有明显的保护作用。其作用的主要机制是抑制扑热息痛在肝脏的毒性代谢和增加肝脏GSH水平,从而保护线粒体功能,减轻扑热息痛导致的肝细胞死亡。本文已发表于Liver International,2008 Nov;28（9）:1226-35。更多还原

【Abstract】 Over several decades,with the rise of average life expectancy,aging population gradually increased,which results in increased incidence of neurodegenerative disease, such as Alzheimer’s disease（AD） and Parkinson’s disease（PD）.The public has to face the problems which aging society brings to the whole world.In order to extend the lifespan and improve the living quality of aging people,researches on aging and age related disease have raised more concerns in recent years.Brain aging and age related neurodegenerative disease is the focus of neuroscience gerontology.There are several hypotheses about mechanism of aging.Aging of reproductive system is considered to accelerate brain aging and increase the incidence of neurodegenerative diseases.Substantial evidence suggests that estrogen is a neuroprotective and neuromodulatory hormone.Besides its anti-oxidative effect, estrogen can regulate expression of a variety of genes by activation of estrogen receptors. The sharp decline of estrogen level after menopause may lead to impaired memory and cognition,and increases the incidence of AD and PD.Testosterone also has neuroprotective and neuromodulatory effect.The decline of testosterone level after middle age could lead to impaired memory and cognition and increased incidence of neurodegenerative diseases in men.During aging,brain is exposed to various stressors,which leads to accumulation of damaged proteins.The aggregation of misfolded proteins becomes a threat to cell survival,and promotes pathogenesis of neurodegenerative diseases.Heat shock proteins （HSP） serve as molecular chaperons,binding to misfolded proteins,assisting to refold misfolded proteins and eliminate irreversibly damaged proteins.Therefore,heat shock proteins are considered as endogenous cytoprotective factors,with function to protect cells against a multitude of stresses.Two of the most important heat shock proteins Hsp70 and Hsp27,can be significantly induced in many areas of brain after stresses. During aging,stress-induced expression of heat shock proteins declines in the brain, which leads to impairment of endogenous defense system.AD is a neurodegenerative disease characterized clinically by progressive impairment of memory and cognition.One of the hallmarks of AD is the presence of senile plaques in the hippocampus,which are composed primarily of aggregated extracellular deposition of Aβ_1-40 and Aβ_1-42.The progressive accumulation of Aβaggregates is widely believed to be fundamental to initiate neurodegenerative pathogenesis.Aβis generated by proteolysis of amyloid precursor protein（APP） by three specific enzymes,α-,β-,andγ-secretases.APP is enzymatically processed by two distinct and mutually exclusive pathways:in amyloidogenic pathway,cleavage of APP byβ-secretase produces Aβ.Alternatively,in nonamyloidogenic pathway,cleavage of APP byα-secretase generates sAPPαInhibition ofβ-secretase to decrease Aβproduction has been taken as target for AD drug development.In addition,substantial evidence indicates that AD transgenic mice with over-expression ofα-secretase display improved learning and memory,and less Aβdeposition in the hippocampus.Therefore,upregulation ofα-secretase mediated APP processing may be novel therapeutic applications in AD.FLZ is a synthetic cyclic analogue of natural squamosamide.Previous studies demonstrated that FLZ had significant neuroprotective effect.FLZ protected against Aβ-induced toxicity in SH-SY5Y cells.It also ameliorated the impairment of learning and memory and pathological damage to the hippocampus induced by ventricular injection of Aβin mice.Therefore,FLZ could be a promising candidate for AD treatment,and it has applied for patent.In order to elucidate the relationship between sex hormones and aging related neurodegenerative diseases,and develop novel drugs for AD treatment,three parts of study were performed and are reported in this dissertation as following:PartⅠ:Establishment of gonadectomy-aecelerated brain aging model in miceIn this study,we aim to develop a gonadectomy-accelerated brain aging model. Male or female mice of 2-month old were orchiectomized（ORX） or ovariectomized （OVX） bilaterally,and were kept for 10 months.All the mice were divided into 4 groups: female-sham（12-month old）,female-OVX（12-month old）,male-sham（12-month old） and male-ORX（12-month old）.2-month old female（female-young） and male（male-young） mice were obtained before determination of brain aging-related parameters.The results of water maze show that spatial learning and memory was impaired in gonadectomized mice as compared with young mice.The results of western blotting and immunostanning show increased expression of Aβ,BACE1 and phospho-Tau,decreased expression of NGF,BDNF,NT3 and TrkA as well as increased expression of p75NTR in gonadectomized mice,as compared with young mice.The result of Nissl staining indicates that the amount of Nissl bodies decreased in the brain of gonadectomized mice. In addition,as compared with young mice of the same gender,the changes of brain aging related parameters were more obvious in ovariectomized mice than that in orchiectomized ones.There are no significant changes of these parameters in the brain of sham-operated mice as compared to young mice.As a result,gonadectomy can accelerate brain aging which is displayed as impaired spatial learning and memory,increased Aβexpression as well as decreased neurotrophins level in the brain.PartⅡ:The role of estrogen in modulating expression of brain heat shock proteins in ovariectomized miceHeat shock proteins serve as endogenous cytoprotective factors.The impaired thermotolerance in old people is probably due to diminished expression of heat shock proteins,and heat shock proteins also play a role in the pathogenesis of AD and PD.In order to elucidate the effect of ovariectomy and the modulatory effect of estrogen on expression of brain heat shock proteins,in this study,we investigated the regulatory effect of estrogen on heat stress-induced brain heat shock protein expression in ovariectomized mice and its underlying mechanism.Female mice of 2 months old were ovariectomized bilaterally or sham operated,and were kept for 10 months.Some ovariectomized mice were supplemented with 17β-estradiol（0.5 mg/kg/day every other day for 14 days,S.C.）.Mice were divided into four groups:young（2-month old）,old-OVX （12-month old）,old-sham（12-month old） and OVX+17β-estradiol（E2）（12-month old）,and were subjected to heat stress at 44℃in an oven for 1 hour followed by 3 hours at room temperature.The results of western blotting and RT-PCR show that in ovariectomized mice,heat stress-induced brain HSP70 expression and mRNA transcription are significantly lower than that of young mice.And the result of gel mobility shift assay shows that the binding activity of heat shock factor with heat shock element was also significantly impaired in the brain of ovariectomized mice.In the brain of mice from OVX+E2 group,supplement of estrogen restored heat stress-induced brain HSP70 expression,and attenuated heat stress-induced brain DNA fragmentation, caspase-3 activation and mitochondrial cytochrorne C and AIF leakage.The results suggest that in ovariectomized mice,heat stress-induced brain Hsp70 expression decreased and the mechanism was related to impaired binding of heat shock factor with heat shock element.Estrogen supplement restored heat stress-induced heat shock protein expression and protected against heat stress-induced brain injury in ovariectomized mice. PartⅢ:A squamosamide derivative FLZ reduces amyloidβproduction by increasingα-secretase mediated non-amyloidogenic APP processingAs mentioned above,upregulation ofα-secretase mediated APP processing leads to decreased Aβproduction,which may be novel therapeutic applications in AD.The neuroprotective effect of FLZ has been proved in several models.In this study,we investigate the effect of FLZ onα-secretase mediated APP processing.We found that Aβproduction was reduced by FLZ in Aβ-expressing N2a（APPswe/PS1Δ9） neuroblastoma cells,which was paralleled with an increase in sAPPαin the medium.Moreover,the result of western blotting show that the active form of ADAM10 and APP expression were elevated at the cell surface of FLZ-treated cells,consistent with immunostaining result showing an enhanced co-localization of ADAM10 and APP at the membrane of FLZ treated cells.Pretreatment with protein trafficking inhibitor brefeldin blocked FLZ-induced translocation of ADAM10 and APP to the cell surface;it also reversed FLZ-induced increase in.sAPPαrelease and decrease in Aβproduction.Furthermore,oral administration of FLZ to APPswe/PS1 transgenic mice significantly reduced Aβlevel and increased ADAM10 level in hippocampus.These findings suggest that decreasing effects of FLZ on Aβproduction may be mediated by its promotion ofα-secretase mediated APP non-amyloidogenic processing,which provides evidence for therapeutic applications of FLZ in AD.Establishing good animal models of aging is very important for the research on aging and neurodegenerative disease.The ultimate goal of research on aging and neurodegenerative disease is to find novel therapeutic applications.To summarize the study:（1） As compared with young mice,gonadectomized mice had impaired spatial learning and memory,increased expression of Aβ,BACE1 and phospho-Tau,decreased Nissl bodies and decreased expression of NGF,BDNF,NT3 and TrkA as well as increased expression of p75NTR.All of these results indicate that long-term sex hormone deficiency by gonadectomy can accelerate brain aging.（2） Heat stress-induced brain Hsp70 expression decreased in ovariectomized mice and the mechanism is related to impaired binding of heat shock factor with heat shock element.It suggests that deficiency of estrogen during aging can exacerbate impairment of endogenous defense system in brain.Estrogen supplement restored heat stress-induced heat shock protein expression and protected against heat stress-induced brain injury in ovariectomized mice.Therefore, the modulatory effect of estrogen on heat shock protein expression provides a new evidence for its neuroprotective effect.（3） FLZ decreased Aβproduction by promotingα- secretase mediated APP non-amyloidogenic processing.Our study provides new evidence for therapeutic potential of FLZ for AD.In addition,in order to develop novel drugs for the treatment of acute liver failure,we investigated the protective effect and mechanism of bicyclol derivative WLP-S-10 against acetaminophen-induced acute liver failure in mice.The results are reported as follow:Acute liver failure is a severe emergency with rapid deterioration of liver function. Overdose of acetaminophen is the most common cause of drug-induced liver failure. Bicyclol is a novel anti-hepatitis drug and has been widely used to treat chronic viral hepatitis and drug-induced liver injury in China.Pharmacological investigation demonstrated that bicyclol has protective action against liver injury in several models, however,bicyclol is not water soluble;it is difficult to make injectable preparation for treatment of acute liver failure patients at present.In this study,a number of water soluble bicyclol derivatives were synthesized and screened in mice using CCl₄, acetaminophen or D-galactosamine plus LPS induced liver injury models.Among ten compounds,a methionine derivative of bicyclol（WLP-S-10） was shown to be the most effective one in lowering serum ALT level and reducing the mortality of mice.WLP-S-10 was,therefore,further studied in acetaminophen induced acute liver failure in mice.A single dose of WLP-S-10 200mg/kg was intraperitoneally injected 1 hour before administration of acetaminophen 450mg/kg.Pretreatment of mice with WLP-S-10 200mg/kg significantly reduced mortality of mice due to acute liver failure caused by lethal dosage of acetaminophen.The liver necrosis,serum ALT and AST elevation,and GSH depletion induced by injection of acetaminophen were all reduced.WLP-S-10 200mg/kg inhibited mitochondrial swelling,breakdown of membrane potential,depletion of mitochondrial ATP,and release of cytochrome C and AIF from mitochondria.In addition,the result of HPLC shows that WLP-S-10 200mg/kg inhibited metabolism of acetaminophen in liver microsomes.WLP-S-10 is converted into bicyclol and methionine. All the results indicate that WLP-S-10 is a novel potential compound against acetaminophen induced acute liver failure in mice,and its active mechanism is related to a summation of inhibiting bioactivation of acetaminophen and increasing liver GSH level, and subsequently attenuating mitochondrial dysfunction and hepatocyte death.The paper has been published in Liver International,2008 Nov;28（9）:1226-35.更多还原