【作者】 纪超；

【导师】 左萍萍； 宋采； 刘雁勇； 杨楠；

【作者基本信息】 中国协和医科大学 ， 神经药理学， 2009， 博士

【摘要】 阿尔茨海默病（Alzheimer’s disease,AD）是一种以老年斑（senile plaque,SP）形成、神经纤维缠结（neurofibrillary tangles,NFT）及神经元丢失为主要病理特征的神经系统退行性疾病,主要表现为进行性的认知功能下降。有关AD发病机制的研究虽然取得了一些成果,提出了一系列关于AD病理学的假说,但是这些假说尚未能完全解释AD的发病机制。大量研究表明AD是多病因相互作用的结果。其中,β-淀粉样蛋白（β-amyloid protein,Aβ）的生成、代谢及毒性作用是AD病理机制的中心环节。遗传（如基因缺陷）等病因引发淀粉样前体蛋白（amyloid precursor protein,APP）代谢异常导致Aβ蓄积,Aβ通过兴奋性毒性,线粒体功能障碍,能量代谢异常,钙离子稳态破坏,细胞凋亡,氧化应激反应及神经炎症反应等多个途径诱发神经损伤,导致神经元机能障碍或死亡。目前,对AD的治疗尚缺乏有效手段,而越来越多的研究将重点转移到中草药,原因是中草药是天然来源,比合成药物更安全。中草药的成分复杂,对于病理机制复杂的疾病,可能具有更好的疗效。此外,中草药的代谢谱对于药物活性成分筛选具有重要价值,为药物的研发及作用机制阐述提供了线索。新疆草花（Gossypiumherbaceam L.,GH）是新近开发的一种产于新疆、用于治疗记忆力减退的民间药物。该药物的治疗作用仅限于民间经验,对其改善学习记忆损伤的药效及确切机制尚无报道。在本研究中,我们将新疆草花提取物（Gossypium herbaceam L.extracts,GHE）用于治疗Aβ_25-35诱导的急性学习记忆损伤大鼠,同时使用多奈哌齐（donepezil）作为阳性对照药,观察GHE能否对抗Aβ诱导的损伤作用。在整体水平评价GHE的神经保护作用及抑制细胞凋亡效果,并对其抗Aβ诱导神经炎症的机制进行阐述,为开发安全有效的神经保护新药提供依据。随着对AD病理机制研究的深入,炎症反应已经受到越来越多的关注。大量研究证明,AD的发病及进展过程中都伴随着炎症反应的发生。其中,胶质细胞（小胶质细胞和星形胶质细胞）在AD的神经炎症机制中发挥了重要作用。Aβ的病理性聚积会激活胶质细胞（尤其是小胶质细胞）,诱导炎症介质释放增加,继而将诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）激活,产生一氧化氮（nitricoxide,NO）及活性氧簇（reactive oxygen species,ROS）,后者又可以与活性氮簇（reactive nitrogen species,RNS）作用,生成过氧化亚硝酸盐。这种过氧化物能对细胞产生很强的毒性,从而引发细胞凋亡。此外,小胶质细胞和星形胶质细胞的相互作用还会进一步增加炎症因子前体、补体以及氧化物的产生。这些炎症介质和氧化物的产生会导致Aβ的进一步聚积,从而将细胞内炎症因子介导的级联反应进一步扩大,使之进入恶性循环。另一方面,胶质细胞,尤其是星形胶质细胞,在神经退行性病变过程中还发挥了神经保护的作用。有研究证明,两种胶质细胞被激活后还可以通过释放神经营养因子（neurotrophic factors,NTFs）及抗炎细胞因子（如IL-4,IL-10等）对抗炎症反应,起到细胞保护的作用。这就是胶质细胞的“双刃剑”效应。基于上述理论,本文采用目前国际上公认的更接近于AD病理机制的长期侧脑室注射Aβ_25-35诱导的慢性学习记忆损伤模型,观察并比较在AD病理进程中胶质细胞发挥的炎症损伤和神经保护两方面的作用,阐述Aβ激活胶质细胞致学习记忆损伤的神经通路,为今后的药物靶点筛选及新药研发奠定了基础。第一部分新疆草花提取物（GHE）对Aβ致痴呆大鼠模型的保护作用1.GHE由很多成分组成,指纹图谱显示主要成分的保留时间分别为18.36,18.46,18.56,19.51,20.46,20.79,21.99,22.26及23.37。2.成年雄性SD大鼠随机分为6组:假手术对照组（Control）,侧脑室单次注射生理盐水加灌胃给予蒸馏水14天;模型组（Model）,侧脑室单次注射Aβ_25-35加灌胃给予蒸馏水14天;阳性药多奈哌齐组（Donepezil）,侧脑室单次注射Aβ_25-35加灌胃给予多奈哌齐0.6 mg/kg 14天;GHE低剂量组（GHE-L）,侧脑室单次注射Aβ_25-35加灌胃给予GHE 35 mg/kg 14天;GHE中剂量组（GHE-M）,侧脑室单次注射Aβ_25-35加灌胃给予GHE 70 mg/kg 14天;GHE高剂量组（GHE-H）,侧脑室单次注射Aβ_25-35加灌胃给予GHE 140 mg/kg 14天。3.水迷宫测试结果证明,与假手术对照组相比,模型组大鼠寻台潜伏期明显延长（P＜0.01）,而给药多奈哌齐（P＜0.01）及GHE 35 mg/kg和GHE 70 mg/kg（P＜0.05）后大鼠寻台潜伏期明显缩短,但结果不具有剂量依赖关系。4.避暗测试结果显示,多奈哌齐和GHE（70 mg/kg）给药能够显著改善Aβ诱导的恐惧记忆损伤,降低大鼠避暗实验的错误次数。此外,GHE给药在一定程度上延长了避暗实验的潜伏期,但是差异没有达到统计学意义。5.Nissl染色结果证明,与假手术对照组相比,Aβ_25-35侧脑室注射显著下调大鼠海马CA1区健康神经元密度,神经元活力明显降低（P＜0.05）,而GHE 70 mg/kg连续给药14天能够逆转Aβ诱导的损伤,且结果具有统计学意义（P＜0.05）。6.TUNEL结果证实,模型组大鼠海马齿状回区出现较多的可以检测到的凋亡细胞,而给药各组大鼠海马齿状回区仅偶尔可见凋亡神经元。7.生化检测结果表明,与假手术对照组相比,模型组大鼠海马区GSH-Px和CAT的酶活力显著降低,给药GHE 35 mg/kg和GHE 70 mg/kg后,GSH-Px和CAT的酶活性显著增加。但GHE对SOD的活性和MDA的含量没有影响。8.ELISA和western blotting检测结果显示,侧脑室注射Aβ_25-35显著诱导大鼠海马区IL-1β含量升高（P＜0.01）,而IL-1RA的表达也有一定程度的升高,但IL-1RA/IL-1β的比率显著降低（P＜0.01）,说明IL-1炎症系统激活。而给药GHE70 mg/kg和140 mg/kg后,IL-1β含量显著降低,同时IL-1RA/IL-1β的比率显著升高,说明GHE能够有效对抗Aβ诱导的IL-1系统的炎症反应。9.本文分别采用western blotting和EMSA方法对炎症因子NF-κB的核转位及活性进行了检测。结果证明,GHE能够有效抑制Aβ_25-35诱导的NF-κB核转位及激活。机制是给药GHE 70和140 mg/kg后能够显著抑制Aβ_25-35诱导的IκB-α的降解（P＜0.05）。免疫组化染色结果进一步证明,Aβ_25-35能够显著上调大鼠海马CA1和CA3区细胞核内NF-κB的表达（P＜0.01）。GHE和多奈哌齐阳性药能够不同程度地抑制胞核内NF-κB的表达。第二部分长期侧脑室注射Aβ致痴呆模型的炎症机制探讨1.成年雄性Long Evans大鼠随机分为2组:假手术对照组（NS）,侧脑室注射生理盐水,每天5μl,连续14天;Aβ模型组（Aβ）,侧脑室注射Aβ_25-35,每天300 pmol（5μl）,连续14天。2.水迷宫测试结果显示,与假手术对照组组相比,长期侧脑室注射Aβ_25-35可导致大鼠的寻台潜伏期明显延长（P＜0.01）。3.免疫组织化学结果证明,与假手术对照组相比,Aβ模型组大鼠海马CA1、CA3和DG区的GFAP染色较深且阳性细胞显著增多,说明GFAP的表达增加,星形胶质细胞大量激活,统计结果表明此差异具有统计学意义（P＜0.01）;而小胶质细胞标志物Iba1染色阳性细胞在海马CA1区显著增多（P＜0.05）,说明长期侧脑室注射Aβ_25-35也诱导海马区小胶质细胞激活。此外,real-time PCR结果进一步证实,星形胶质细胞（P＜0.01）和小胶质细胞标记物（P＜0.05）的基因转录水平在Aβ模型组大鼠海马区均呈现出显著增加的趋势。4.通过细胞因子检测技术Bio-plex对接受Aβ_25-35长期侧脑室注射的大鼠脑海马区9种细胞因子进行了检测,结果表明,IL-1α（P＜0.01）,IL-1β（P＜0.01）和TNF-α（P＜0.05）三种细胞因子受Aβ_25-35的诱导出现显著上调;抗炎因子IL-4出现明显下调,但由于其标准差较大,此差异没有达到统计学意义。5.采用western blotting和ELISA检测方法对大鼠海马区NTFs及其受体的表达进行检测,结果发现,长期侧脑室注射Aβ_25-35能够显著上调BDNF（P＜0.01）和GDNF（P＜0.05）的蛋白表达水平;同时,显著下调NGF（P＜0.01）和p75^NTR（P＜0.05）的蛋白表达。Real-time PCR结果证明,受Aβ_25-35的诱导,NTFs及其受体的基因转录水平呈下降的趋势,其中BDNF（P＜0.05）和Trk B（P＜0.05）的下调具有统计学意义。6.对大鼠血清中皮质酮水平的检测结果表明,长期侧脑室注射Aβ诱导血清中皮质酮浓度显著升高（P＜0.05）。7.Western blotting和real-time PCR结果均显示,Aβ能够诱导大鼠海马区APP蛋白表达水平和基因转录水平出现一定程度的变化,但此差异没有统计学意义。更多还原

【Abstract】 Alzheimer’s disease（AD）,one of the most prominent neurodegenerative diseases,is characterized by progressive loss of cognitive abilities.The neuropathological hallmarks of the disease are senile plaque（SP）,neurofibrillary tangles（NFT） and loss of neurons. There have been a lot of studies providing some hypotheses on the pathophysiology of AD,but the mechanisms of AD remain unclear.Most of the studies suggested that AD was resulted from a lot of different etiopathogenisis and their interactions.And the formation,metabolism and toxicity ofβ-amyloid protein（Aβ） have been suggested to play a central role in the pathogenesis of AD.Some causes,such as gene defect,might induce abnormal metabolism of Aβprecursor protein（APP）,which can contribute to Aβaccumulation.As a consequence,Aβwould induce the damage of neurons through several different pathways,including excitatory toxicity,dysfunctions of mitochondria, abnormal energy metabolism,disequilibrium of calcium homeostasis,apoptosis, oxidative stress,neuroinflammation process,and so on.At the present,there is no definitive treatment or cure for AD.A large segment of the public finds solace in herbs,in part believing that herbs are natural and hence safer than synthetic drugs,and that a complex mixture of herbs can effectively treat complex diseases.In addition,the metabolite profile of herbal medicine is important for screening its active constituents,thus providing a valuable contribution to the drug discovery process and elucidation of the underlying mechanism of action.Gossypium herbaceam L. extracts（GHE） is an active standardized extract obtained from Gossypium herbaceam L., an ethical herb used by Uygur people in Xinjiang,China,to treat mental retardation.In the present study,the neuroprotective effect of GHE against Aβ_25-35 induced learning and memory impairment was observed in vivo.We used GHE to treat the rats injected with Aβ_25-35,and used donepezil as the positive control.The results suggested GHE could be considered as a potential agent for preventing or retarding the development or progression of AD.Additionally,recent evidences showed that inflammatory disorders were involved in the onset and progression of AD.Glial cells（microglia and astrocytes） played a major role in the neuroinflammatory process in AD.The activation of microglia,in response to Aβaccumulation,leaded to enhancements in the production of inflammatory mediators. As a consequence,inducible nitric oxide synthase（iNOS） was activated,which produced nitric oxide（NO）,and reactive nitrogen specie（RNS）.Moreover,toxic reactive oxygen species（ROS） were produced,which could also react with RNS to form peroxynitrite that was highly toxic to cells and caused apoptosis.Additionally,interactions between microglia and astrocytes have been observed in the brain,which might further result in the increased production of pro-inflammatory cytokines,complement proteins,and oxidants.Both types of glial cells might contribute to neuronal damage and the accumulation of Aβ.Aβcould further induce a cascade of cellular mechanism,including activation of both glial cells to release inflammatory mediators.In contrast to the neurotoxic effects,neuroprotective effects of glial cells,especially astrocytes,in the process of neurodegeneration have been observed.Some investigations supported that both microglia and astrocytes,while responding to pro-inflammatory agents,could protect neuronal cells by producing neurotrophic factors（NTFs）,and anti-inflammatory mediators（i.e.IL-4,IL-10）.Thus,in the present study the chronic animal model of AD induced by Aβwas used to evaluate the relationship between neuroinflammatory process and neuroprotective effect resulted from glial cells activation.And it could provide some valuable evidences for the drug discovery process in the future.PartⅠ:The neuroprotective effects of Gossypium herbaceam L.extracts （GHE） on AD rat induced by Aβ1.There were many complex components in GHE.The retention times of main components were 18.36,18.46,18.56,19.51,20.46,20.79,21.99,22.26,and 23.37, respectively. 2.SD rats were randomly divided into 6 groups:rats treated with i.c.v,normal saline and i.g.distilled water were used as control group;rats treated with i.c.v.Aβ_25-35 and i.g. distilled water were used as model group;rats treated with i.c.v.Aβ_25-35 and i.g. donepezil were used as positive control group;rats treated with i.c.v.Aβ_25-35 and i.g. GHE 35 mg/kg were used as GHE low dose group;rats treated with i.c.v.Aβ_25-35 and i.g.GHE 70 mg/kg were used as GHE medium dose group;rats treated with i.c.v. Aβ_25-35 and i.g.GHE 140 mg/kg were used as GHE high dose group.3.Morris water maze data showed that Aβ_25-35-treated animals exhibited obviously longer escape latencies in probe test than that of the control（P＜0.01）.The GHE （70mg/kg and 140mg/kg） significantly shortened escape latencies（P＜0.05）,and ameliorated memory impairment due to Aβ_25-35,as did donepezil（P＜0.01）.However, there was no dose dependence observed among each GHE treatment group.4.Step through test suggested that treatment with donepezil and GHE（70 mg/kg） effectively reduced Aβ-induced increase of numbers of errors in the passive performance test.GHE intragastricly administrated tended to increase the step-through latency,but the improvement did not achieve statistical significance.5.Nissl staining analysis indicated Aβ_25-35 significantly decreased the healthy cell density in CA1 region of hippocampus in rats,as compared to that in the control group（P＜0.05）,and treatment with GHE-M（70 mg/kg） for following 14 days markedly reversed the Aβ_25-35-induced changes（P＜0.05） in the CA1 region.6.TUNEL results suggested apoptotic neurons in dentate gyrus in model rats were markedly increased compared to those in control rats,and positive cells were occasionally seen in dentate gyrus of rats treated with donepezil and GHE.7.The activity of GSH-Px and CAT in the hippocampus tissues of rats treated by Aβ_25-35 alone decreased in comparison with those in the control group.The most significant benefits exerted on improvement of GSH-Px and CAT activities in the hippocampus tissue were observed in GHE-L and GHE-M groups.Unfortunately,no significant differences existed in the activity of SOD and the level of MDA in hippocampus between the model group and each GHE group. 8.ELISA and western blotting results showed that Aβ_25-35 i.c.v,injection could significantly induce the enhancement of IL-Iβconcentration in hippocampus（P＜0.01）,while to some extent,the IL-1RA expression showed a trend of increase as well. However,the ratio of IL-1RA/IL-1βobviously increased in hippocampus of rats treated with Aβ_25-35 alone（P＜0.01）.Which suggested the activity of IL-1 system in model group is higher than that in the control group.Besides,GHE（70 mg/kg and 140 mg/kg） could reverse the changes induced by Aβ,which indicated that GHE could play an important role against the inflammation induced by IL-1 system.9.The nuclear translocation and activation of NF-κB were analyzed by western blot and EMSA,respectively.Western blot analysis proved that GHE,especially at the dose of 70mg/kg administration（P＜0.05）,could effectively inhibit the Aβ_25-35 induced translocation of NF-κB into cell nucleus in hippocampus of rats,and EMSA result indicated that treatment of GHE could prevent the activation of NF-κB due to Aβ_25-35. Aβeffectively caused degradation of IκB-α（P＜0.05）,which could be significantly inhibited by treatment with GHE at the doses of 70 and 140 mg/kg（P＜0.05）. Immunohistochemistry assay data also showed that the levels of NF-κB in cell nucleus in CA1 and CA3 region of hippocampus were significantly increased in the presence of Aβ_25-35 after two weeks（P＜0.01）.Interestingly,this increase in nucleus in CA1 region was prevented by treatment with donepezil,GHE-M and GHE-H（P＜0.05）. Meanwhile,donepezil,GHE-L,GHE-M and GHE-H could also reduce the level of NF-κB in nucleus in CA3 region of hippocampus in rats,respectively（P＜0.01）.PartⅡ:The study of inflammatory mechanisms involved in AD rat model induced by i.c.v,infusion of Aβ1.Long Evans rats were randomly divided into 2 groups:rats treated with i.c.v. continuous infusion of normal saline（NS） 5μl per day for 14 days were used as control group;rats treated with i.c.v,continuous infusion of Aβ_25-35 300 pmol per day for 14 days were used as model group.2.Morris water maze data showed that compared with the control group,the escape latency obviously prolonged due to Aβ_25-35 i.c.v,infusion（P＜0.01）.3.Immunohistochemistry analysis results indicated that compared with the control group, the number of GFAP immunopositive cells was significantly increased in CA1,CA3 and DG regions in hippocampus of rats in Aβmodel group（P＜0.01）,which suggested that GFAP expression enhanced and astrocytes activated;While the number of Ibal （one of the markers of microglia） immunopostive cells were obviously increased only in CA1 region of hippocampus in rats in Aβmodel group compared with those in the control group,which suggested that microglia could be activated by Aβi.c.v,infusion. In addition,real-time PCR results provided further supports that the transcriptions level of markers for astrocytes and microglia were significantly increased in Aβmodel group compared with those in the control group.4.Bio-plex cytokine assay showed that among the 9 cytokines which were detected, IL-1α（P＜0.01）,IL-1β（P＜0.01） and TNF-α（P＜0.05） were obviously up-regulated by Aβi.c.v,infusion,while the anti-inflammatory cytokine IL-4 was down-regulated by Aβi.c.v,infusion.However the latter did not achieve significant change.5.Western blotting and ELISA data indicated that among some NTFs and their receptors, the BDNF（P＜0.01） and GDNF（P＜0.05） expressions were markedly higher in hippocampus of rats in Aβmodel group.While the NGF（P＜0.01） and p75^NTR（P＜0.05） expressions obviously decreased due to Aβi.c.v,infusion.Real-time PCR results showed that BDNF（P＜0.05） and Trk B（P＜0.05） transcription levels were significantly lower than those in the control group.6.The corticosterone levels in serum in rats were analyzed by EIA test.The data suggested that the corsticosterone level was obviously increased due to Aβi.c.v. infusion in this study（P＜0.05）.7.The protein expression and the transcription level of APP in hippocampus of rats were evaluated by western blotting and real-time PCR analysis respectively.The results showed that to some extent,both of the protein expression and transcription level altered due to Aβi.c.v,infusion in this study,but the differences did not achieve statistical significance.更多还原