【作者】 王婷；

【导师】 陈建国；

【作者基本信息】 华中科技大学 ， 药理学， 2009， 博士

【摘要】 第一部分二苯乙烯苷对缺血性脑损伤的保护作用及其机制目的：缺血性脑损伤因其高致残率和致死率被认为是威胁人类生命健康最主要的原因之一。研究表明，缺血再灌注过程中活性氧/氮族（reactive oxygen/nitrogen species，ROS/RNS）的过度产生导致的氧化应激是介导神经细胞损伤的主要机制之一。氧化应激可以通过破环细胞膜正常功能，引起胞内钙离子超载，激活胞内氧化应激相关的信号通路如c-Jun氨基末端激酶（c-Jun N-teminal Kinase，JNK），引起线粒体功能紊乱等，导致凋亡相关因子的释放和表达，从而导致细胞的凋亡坏死。缺血再灌注损伤过程中产生的ROS还能活化重要的核转录因子-κB （nuclear transcription factor-κB，NF-κB），增强诱导型一氧化氮合酶（inductible nitric oxide synthase，iNOS）的表达，从而使氮自由基如一氧化氮（nitrogen oxide，NO）、亚硝酸盐的产生进一步增多。二苯乙烯苷（tetrahydroxystilbene glucoside，TSG），是从中药何首乌中提取的一种具有多酚结构的活性成分，已证实其具有清除自由基、抗氧化、抗炎的作用。但TSG是否对脑缺血损伤有保护作用，目前尚未见报道。本研究旨在从细胞和整体水平考察TSG对脑缺血再灌注损伤的保护作用及其相关机制。方法：建立原代培养的皮层神经元氧-糖剥夺（oxygen-glucose deprivation，OGD）模型，采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐（3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazoliumbromide，MTT）比色法和乳酸脱氢酶（lactate dehydrogenase，LDH）测定法检测细胞活性；Hoechst染色检测细胞凋亡；细胞免疫荧光和流式细胞术检测胞内ROS水平、线粒体膜电位（mitochondrial membrane potential，MMP）变化和核转录因子NF-κB的激活程度；钙离子成像技术检测胞内钙离子水平；免疫印迹（western blotting）和实时定量PCR（real-time PCR）技术检测凋亡相关蛋白（Bcl-2，Bax）、应激活化蛋白JNK、抗衰老蛋白沉默信息调节因子类型1（silent informationregulator type 1，SIRT1）及iNOS表达水平。采用大脑中动脉栓塞法（middle cerebralartery occlusion，MCAO）建立小鼠局灶性脑缺血损伤再灌注模型，2,3,5-三苯基氯化四氮唑（2,3,5-triphenyltetrazolium chloride，TTC）染色法测定脑梗死面积和体积，Tunel法检测皮层组织神经元凋亡程度。结果：细胞分别经过1 h，2 h，4 h OGD再灌注24 h后，与对照组相比，MTT法显示细胞存活率呈时间依赖性降低。因此我们选择OGD 2 h再灌注24 h（oxygen-glucose deprivation following reperfusion，OGD-R）作为后续的离体细胞缺血再灌注损伤模型。与对照组相比，细胞经OGD-R处理后，细胞存活率降低，LDH释放增加；Hoechst染色表明凋亡细胞增多，细胞核呈固缩突亮或碎块状致密浓染。经过不同浓度的TSG（5μM，10μM，25μM，50μM，100μM）预处理细胞后能减轻OGD-R所致的细胞损伤，减少胞内ROS积聚，逆转MMP的驱散；减轻H₂O₂所致的胞内钙超载。Western blotting和Real-time PCR结果显示，TSG能减少OGD-R处理后促凋亡蛋白Bax及应激活化蛋白p-JNK的水平，增加抗凋亡蛋白Bcl-2的表达。TSG孵育细胞可增强SIRT1蛋白表达，能减弱OGD-R所致的NF-κB活化，减少OGD-R所致的iNOS基因的表达。SIRT1抑制剂烟酰胺（nicotinamide）削弱了TSG对NF-κB激活和iNOS表达增加的抑制作用。TTC染色法显示，与假手术组比较，模型组脑梗死面积和体积增加，TSG组脑梗死面积和体积均减少。Tunnel法显示，TSG组皮层组织神经元凋亡程度减轻。结论：二苯乙烯苷可通过清除自由基，减轻钙超载，下调促凋亡蛋白Bax表达和应激活化蛋白JNK的磷酸化水平，增加抗凋亡蛋白Bcl-2表达，激活抗衰老蛋白SIRT1和抑制NF-κB激活等途径发挥脑缺血再灌注损伤的保护作用。第二部分二苯乙烯苷对小鼠海马突触可塑性及学习记忆的影响及其机制目的：随着人口老龄化问题逐渐加重，衰老相关的神经退行性疾病如阿尔茨海默病（Alzheimer’s disease，AD）、血管性痴呆（vascular dementia，VD）及帕金森病（Parkinson’s disease，PD）等伴随的认知功能损伤和学习记忆能力的下降，成为影响人民生活质量的重大问题。何首乌是我国一种沿用已久的用于抗衰老治疗的名贵中药。二苯乙烯苷（tetrahydroxystilbene glucosid，TSG）是从何首乌中提取的一种具有多酚结构的活性成分。最近的研究表明，TSG对衰老大鼠和APP转基因的AD模型鼠海马区突触超微结构和学习记忆能力均有所改善。但TSG是否对正常动物的突触可塑性及学习记忆能力有调节作用，其具体的分子机理是什么，目前尚未见报道。本研究旨在从脑片水平观察TSG对正常小鼠海马CA1区长时程增强（long-term potentiation，LTP）的调节及对学习记忆能力的影响。方法：急性分离小鼠海马脑片，采用细胞外场电位记录和western blotting的方法研究TSG对海马CA1区LTP的影响及其机制。采用行为学方法观察灌胃给予TSG对小鼠学习记忆能力的影响。结果：本实验所用浓度的TSG（0．1μM，1μM，5μM，10μM）对海马CA1区N-甲基-D-天冬氨酸（N-methyl-D-aspartic acid，NMDA）受体依赖的LTP的增强作用呈钟形分布，且在1μM时作用最明显，但对基础突触传递和反映突触前反应的双波易化（paired pulse facilitation，PPF）和输入-输出反应（input-output curve，I/Ocurve）均无影响。TSG增强LTP的作用需要钙/钙调蛋白激酶Ⅱ（calcium/calmodulin-dependent protein kinaseⅡ，CaMKⅡ）和细胞外信号调节激酶（extracellular signal-regulated kinases，ERKs）的激活。行为学结果显示，TSG处理的小鼠在Morris水迷宫和跳台实验的表现优于对照组。透射电镜的结果显示，TSG增加小鼠海马CA1区辐射层的突触后密度（postsynaptic density，PSD）面积。结论：TSG增强正常小鼠海马CA1区NMDA受体依赖的LTP和学习记忆能力，这些增强作用可能与其激活学习记忆相关的关键分子CaMKⅡ和ERK1/2以及改变突触的超微结构有关。更多还原

【Abstract】 PartⅠProtection by tetrahydroxystilbene glucoside against cerebralischemia injuryBackgroud: Ischemic brain injury is considered as one of the leading causes of death andadult disability for its high mortality rate in many countries. Reactive oxygen/nitrogenspecies （ROS/RNS） have been considered important mediators of the brain damage afterischemia/reperfusion injury. Oxidative stress generation during ischemia/reperfusioncontributes to a disturbed membrane function, results in a critical intracellular calciumaccumulation and leads to cell apoptosis and death by triggering various critical cellularsignal transduction pathways such as c-Jun N-terminal kinase （JNK）, an importantsubgroup of the mitogen-activated protein kinase （MAPK） superfamily which is activatedby oxidative stress. Oxidative stress generation during ischemia/reperfusion also leads toactivation of nuclear transcription factor-κB （NF-κB）, which upregulats expression ofinductible nitric oxide synthase （iNOS） and results in the increase of nitric oxide （NO）production and subsequently the increase of peroxynitrite formation. Tetrahydroxystilbeneglucoside （TSG）, an active component of the rhizome extract from Polygonum Multiflorum,exhibits anti-oxidative and scavenges free radical effects. However, whether TSG hasneuroprotecive effects on cerebral ischemia injury remains unkown. Therefore, in this study,we investigated the neuroprotective effects of TSG on ischemia/reperfusion brain injury aswell as the underlying mechanisms.Methods: An in vitro ischemic model of oxygen-glucose deprivation followed byreperfusion （OGD-R） was established. Colorimetric assay methods of 3-（4, 5-Dimeth-ylthiazol-2-yl）-2, 5-diphenyltetrazoliumbromide （MTT） and lactate dehydrogenase （LDH）and staining method of Hoechst were used to measure the cell viability and apoptosis. Cytoimmunity fluorescence and flow cytometry were employed to evaluate the levels ofROS and mitochondrial membrane potential （MMP） and activation of NF-κB. Intracellularcalcium concentration ([Ca²⁺]_i) was monitored using the fluorescent Ca²⁺ indicatorfura-2/AM by calcium imaging technique. The expressions of apoptosis-related proteins（Bcl-2 and Bax）, stress-activated protein of JNK and anti-aging protein of SIRT1 weremeasured by western blotting and real-time PCR analysis. An in vivo ischemic model ofmiddle cerebral artery occlusion （MCAO） was established. Staining method of2,3,5-triphenyltetrazolium chloride （TTC） was used to assay the area and volume ofcerebral infarct. Tunel assay was used to measure the extent of apoptosis in cerebral cortex.Results: The viability of the cells exposed to OGD for 1 h, 2 h and 4 h followed by 24-hreoxygenation decreased time-dependently compared with the control, respectively. Thus,the protocol of 2-h OGD followed by 24-h reoxygenation was selected for furtherexperiments in the present study. When compared with the control, OGD-R inducedneuronal injury determined by MTT, LDH and Hoechst staining, intracellular ROSgeneration and mitochondrial membrane potential dissipation, which were reversed by TSG.The elevation of H₂O₂-induced [Ca²⁺]_i was also attenuated by TSG. The inhibition of JNKand Bcl-2 family related apoptotic signaling pathway was involved in the neuroprotectionof TSG. Meanwhile, TSG inhibited iNOS mRNA expression induced by OGD-R, whichmay be mediated by the activation of SIRT1 and inhibition of NF-κB activation. In vivostudies further demonstrated that TSG significantly reduced the brain infarct volume andthe number of positive cells for Tunel staining in the cerebral cortex when compared toMCAO group.Conclusion: Our study indicated that TSG protected against cerebral ischemia/reperfusioninjury through multifunctional cytoprotective pathways including decrease of ROSgeneration, attenuation of intracellular calcium overload, downregulation of apoptoticprotein Bax expression and stress-activated protein phospho-JNK level, activation ofanti-aging protein SIRT1 and inhibition of NF-κB activation. PartⅡTetrahydroxystilbene glucoside promotes hippocampal synapticplasticity and learning-memory of miceBackgroud: Impaired cognition and memory associated with aging-related neurode-generativediseases such as Alzheimer’s disease （AD）, vascular dementia （VD） andParkinson’s disease （PD） have become a large public issue with the increasing elderlypopulation. Tetrahydroxystilbene Glucoside （TSG）, an active component of the rhizomeextract with polyphenolic structure from Polygonum Multiflorum, which has been widelyused in the Orient as a tonic, anti-oxidative and anti-aging agent since ancient times. Recentstudies have demonstrated that TSG has the ability of changing the ultrastructure ofhippocampal synapses and enhancing learning-memory in both APP transgenic mice andaged rats. However, whether TSG can affect physiological synaptic transmission orimprove learning and memory as well as the related mechanisms in normal animal remainsunknown. In the present study, we investigated the roles of TSG on long-term potentiation（LTP） in the CA1 region of the hippocampal slices and cognitive behavioral performancesin intact adult mice as well as the underlying mechanisms.Methods: Acute isolation of hippocampal slice in mouse, field potentials recordings andwestern blotting analysis were used to evaluate the effects and mechanisms of TSG on LTPin CA1 region of hippocampal slices. Behavioral tests of Morris water maze and step-downfear conditioning were applied to investigate the effects of TSG on learning and memory.Results: TSG enhanced the N-Methyl-D-Aspartate （NMDA） receptor dependent CA1-LTPin a dose-dependent manner, with a maximal effective dose at 1μM. The concentrations ofTSG used in this study did not affect the basic synaptic transmission, PPF and input-outputcurves. The enhancement of TSG-induced LTP required calcium/calmodulin-dependent protein kinaseⅡ（CaMKⅡ） and extracellular signal-regulated kinases （ERKs） activation.Behaviorally, TSG-treated mice performed significantly better than that in the controlgroup in Morris water maze and in step-down fear conditioning. Furthermore, increasedpostsynaptic density was found in TSG-treated group by electron microscopy.Conlusion: Our data demonstrate that TSG promotes LTP in CA1 region of hippocampalslices and enhances memory in mice, which positively correlates with the increase of theactivities of plasticity-related proteins such as CaMKⅡand ERK1/2 and change theultrastructure of hippocampal synapses.更多还原