Aβ损伤对大鼠脑内神经甾体合成代谢的影响及孕酮保护作用及机制研究
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摘要
阿尔茨海默病（Alzheimer’s disease，AD）又称老年痴呆症，是老年期痴呆症最常见的类型。AD临床表现为进行性记忆障碍，认知功能损伤，以及日常生活能力减退，同时可伴有幻觉、妄想、行为紊乱、人格异常等多种精神症状和行为障碍。AD常发生于65岁以上的老年人，发病率随年龄增长而迅速增加。据世界卫生组织统计，目前全球以AD为主的痴呆症患者约有3560万，并以每年770万的速度递增；预计到2050年，每85人中将有1人罹患此病。因此，对AD进行有效的预防和治疗不仅为医学界所重视，同时也倍受全社会的关注。AD典型病理改变为细胞外β-淀粉样蛋白（β-amyloid，Aβ）过度沉积形成老年斑（senile plaque，SP），胞内Tau蛋白过度磷酸化形成神经纤维缠结（neurofibrillary tangles，NFT），以及弥漫性神经元变性、缺失和全脑萎缩。在AD病例中，家族遗传性病例约占5%，其他绝大部分散发病例的致病机理尚不明确。研究认为，AD与Aβ过度沉积、Tau蛋白异常修饰、早老素（presenilin，PS）基因突变等有关。其中，Aβ作为AD发病始动因子的观点得到广泛认可，即：淀粉样蛋白斑块的主要成分Aβ在AD发生过程中发挥至关重要的作用。Aβ是淀粉样蛋白前体蛋白（amyloid precursor protein，APP）经过β-分泌酶（BACE-1）和γ-分泌酶（γ-secretase）顺序剪切后的产物。正常情况下，Aβ为可溶解状态，不具有毒性；而在病理状态下，Aβ聚集形成寡聚体、多聚体、原纤维，进一步沉积形成斑块，产生损伤作用。Aβ产生损伤作用的机制包括：引起氧化应激，引发炎症反应，诱导细胞凋亡等。胞外Aβ过量生成、过度沉积是邻近胶质细胞过度活化，神经元死亡，进而导致AD临床症状的主要原因。因此，减轻Aβ的损伤作用是治疗AD的策略之一。
神经甾体（neurosteroid）是中枢神经系统中的甾体类物质及其代谢产物的总称，可由神经元或胶质细胞在中枢神经系统合成，也可由外周腺体合成后通过血脑屏障进入中枢神经系统，神经甾体在脑组织中的含量高于外周血中的含量。神经胶质细胞和神经元合成的神经甾体主要包括：孕烯醇酮（pregnenolone，PREG）、孕烯醇酮硫酸酯（pregnenolone sulfate，PREGS）、脱氢表雄酮（dehydroepiandrosterone，DHEA）、脱氢表雄酮硫酸酯（dehydroepiandrosterone sulfate，DHEAS）、孕酮（progesterone，PROG）、别孕烯醇酮（allopregnanolone，ALLO），以及雌二醇（estradiol，E2）等。研究显示，神经甾体具有镇静催眠、抗惊厥、抗焦虑、抗精神分裂症、改善学习记忆，以及神经保护等作用，在中枢神经系统功能调节中发挥广泛作用。研究显示，神经甾体的表达调控改变与包括AD在内的多种神经退行性疾病的发生有关，AD时神经甾体含量及合成代谢酶表达发生改变。对AD患者神经甾体水平变化的研究多为神经甾体水平与AD易患倾向关系的分析研究，其研究对象多为AD患者外周血、脑脊液或尸脑，研究对象来源有限。AD患者血浆或脑组织中神经甾体水平的变化为长时间、多种因素共同作用的结果，而在AD进程中，神经甾体含量的变化与学习记忆能力受损的关系尚不明确。
目的：本研究采用单一损伤因素Aβ25-35诱导AD样学习记忆障碍大鼠模型，通过液液萃取结合高效液相色谱-串联质谱（high performanceliquid chromatography-tandem mass spectrometry，HPLC-MS/MS）方法分析Aβ损伤大鼠学习记忆相关脑区（前额叶皮质及海马）中主要神经甾体的含量，通过RT-PCR、 Western blot和免疫组织化学（immunohistochemistry，IHC）分析神经甾体合成代谢酶的表达；筛选与学习记忆障碍关系密切的神经甾体，观察其对Aβ损伤大鼠学习记忆能力的改善作用，探讨其学习记忆保护作用的机制。利用原代培养的海马神经元，进一步在细胞水平研究Aβ25-35对海马神经元细胞活力的影响及神经甾体孕酮对抗Aβ损伤的保护作用。本实验为神经甾体尤其是孕酮在AD等学习记忆障碍类疾病中的应用提供理论依据及实验基础。
方法：
1Aβ所致学习记忆障碍大鼠模型的制备
将30只体重210-230g雄性Spague-Dawley（SD）大鼠随机分为3组，分别为：假手术组、溶剂对照组（假手术+蒸馏水）、以及Aβ损伤组，每组10只。Aβ损伤模型制备方法如下：动物经腹腔注射麻醉后，固定于脑立体定位仪，沿颅骨中线切开皮肤，暴露前囟。参照图谱海马CA1区位置，于前囟后3.5mm，中线左右2.0mm处，开直径1mm的骨窗，垂直颅骨表面进针2.7mm，缓慢注入5μL（2g/L）Aβ25-35溶液，留针10min使注射物充分扩散，缓慢撤针，缝合切口。溶剂对照组注入等体积蒸馏水。术后7天-12天进行Morris水迷宫行为学测试，观察大鼠空间学习记忆能力。行为学测试结束后，动物经多聚甲醛心脏灌流进行预固定，之后取脑组织，外固定，石蜡包埋，制备病理切片，通过硫堇尼氏体染色进行组织病理学分析。
行为学测试：术后7天-12天进行Morris水迷宫（the Morris watermaze）行为学测试。实验开始前将动物置于行为学测试房间，使其适应环境1h。行为学测试于每天14:30开始，室温25℃，水温22℃-24℃，动物测试顺序保持不变，测试开始象限随机选取。Morris水迷宫行为学测试历时6天，前5天进行定位航行实验（the navigation trial），每天训练4次。记录大鼠入水至寻找到隐匿平台所需的时间，即逃避潜伏期（escapelatency）。第6天进行空间探索实验（the probe trial），撤去平台，将大鼠从平台相对的象限面壁放置入水，记录动物90s内穿越平台区的次数（number of platform crossings）以及在平台所在象限（第四象限）停留的时间百分比（Ⅳ time ratio）等行为学指标。
组织病理学分析：预固定的脑组织经4%多聚甲醛固定至少48h，石蜡包埋，常规脑组织切片（5μm），硫堇尼氏体染色观察海马组织学改变。选取相同海马截面的切片进行硫堇尼氏体染色。每张切片随机选取CA1区3个400倍视野，计数海马CA1区每1mm区段内细胞膜完整、胞核饱满、核仁清晰的锥体细胞数目，每张切片海马各计数3个区段取平均数。
2Aβ损伤大鼠学习记忆相关脑区（前额叶皮质及海马）神经甾合成代谢的改变
将90只体重210-230g雄性SD大鼠随机分为3组，分别为：假手术组、溶剂对照组、以及Aβ损伤组，每组30只。动物处理同上。分别于术后7天及12天断头处死每组中的10只动物，迅速取脑，冰浴分离前额叶皮质及海马，-70℃存放，通过HPLC-MS/MS分析神经甾体孕烯醇酮、孕酮、别孕烯醇酮、及17β–雌二醇的含量；其余动物于术后12天取材，每组中的5只动物断头处死，迅速取脑，冰浴分离海马，-70℃存放，通过RT–PCR及Western blot分析神经甾体合成代谢酶CYP11A1、3β-HSD及aromatase的表达改变；其余动物经多聚甲醛心脏灌流进行预固定，之后取脑组织，外固定，石蜡包埋，制备病理切片，通过免疫组化分析神经甾体合成代谢酶aromatase的表达改变。
神经甾体提取：参考本实验室已有方法略加改进，通过液液萃取提取神经甾体。脑组织样品（双侧海马组织或前额叶皮质）加入PBS及MT（内标），制备组织匀浆。乙酸乙酯/正己烷（9:1）萃取3次，转移并合并有机相，50℃水浴下N2吹干；60℃水浴中用丹酰氯衍生40min，离心后取上清，4℃保存。
神经甾体含量测定：色谱条件：色谱柱Agilent XDB C18分析柱（4.6mm×50mm）；保护柱Agilent XDB C18（4.6mm×12mm）；柱温40℃；流动相：A水（含0.1%甲酸），B甲醇（含0.1%甲酸）；流速：0.5mL/min；洗脱梯度：0-6.5min，36%A；6.5-6.6min，30%A；6.6-17min，10%A；17-18min，36%A。进样量30μL，每针运行18min。质谱条件：离子化方式：大气压化学离子源（atmospheric pressure chemical ionization，APCI）；喷雾电压：4500V；鞘气（N2）压力：30L/min；辅助气（N2）压力：5L/min；离子检测方式：正离子多反应监测；用于定量分析的离子反应分别为：(m/z)299.03→(m/z)158.86、280.9（PREG），(m/z)315.03→(m/z)97、109.01（PROG），(m/z)301→(m/z)188.9、282.85（ALLO），(m/z)254.97→(m/z)132.9、158.9（17β-E2），(m/z)303.1→(m/z)97.04、109.06（MT）。
RT-PCR：按照Invitrogen公司产品手册，采用TRIzol一步法，提取组织总RNA。每50-100mg脑组织加入1mL TRIzol试剂，匀浆器内冰浴研磨、裂解组织。将裂解液移至0.1%DEPC处理并高压灭菌的1.5mL离心管中，加入0.5mL氯仿，剧烈震荡15s，室温孵育，离心，静置，加等体积异丙醇，颠倒混匀，室温静置，离心，弃上清，75%乙醇洗沉淀，室温晾干，20μL DEPC水溶解。按照Promega逆转录试剂盒说明，取等量组织总RNA，70℃水浴10min，之后迅速置于冰上；加入逆转录体系各组分，涡旋混匀，瞬时离心，42℃孵育1h，合成cDNA，95℃加热3min终止反应。参考Genbank中mRNA序列设计引物，以反转录产物为模板，进行PCR扩增。PCR扩增产物经1.5%琼脂糖凝胶电泳分离，扫描成像。凝胶成像分析软件进行相对定量分析。
组织总蛋白提取与Western blot分析：组织加入RIPA裂解液，制备组织匀浆。离心取上清（即蛋白提取液），分装，-20℃保存。以牛血清白蛋白为标准物，采用考马斯亮蓝蛋白测定试剂盒进行蛋白定量。取等量蛋白提取液，RIPA裂解液补齐体积，与5×SDS上样缓冲液混合，沸水浴加热使蛋白变性，冷却后上样。稳压电泳，溴酚蓝移动至凝胶底部时，停止电泳。切去浓缩胶，剪取与凝胶相同大小的PVDF膜，甲醇活化后，与滤纸、凝胶一起浸入转膜缓冲液，平衡15min。依次放置滤纸、PVDF膜、凝胶、滤纸，20V恒压半干转膜。将PVDF膜置于含5%脱脂奶粉的TTBS中，37℃封闭2h。之后进行一抗结合反应，4℃缓慢摇动过夜。洗膜后，进行二抗结合反应。洗膜后，用化学发光试剂盒检测PVDF膜上抗原抗体结合区带。凝胶成像分析软件进行相对定量分析。
免疫组织化学染色分析：脑组织标本经4%多聚甲醛固定后，梯度乙醇脱水，石蜡垂直定向包埋，5μm厚度连续切片。SP三步法进行免疫组织化学染色，即：石蜡切片脱蜡至水，微波抗原修复，自然冷却；滴加H2O2去离子水，室温孵育10min，消除内源性过氧化物酶影响；PBS冲洗，滴加山羊血清工作液，封闭非特异结合位点；倾去血清，滴加以PBS适当比例稀释的一抗，4℃过夜；PBS冲洗，滴加生物素化二抗工作液，室温孵育15min，PBS冲洗；滴加辣根酶标记链霉卵白素工作液，室温孵育15min，PBS冲洗；滴加DAB试剂显色，镜下控制显色时间，自来水冲洗终止反应；苏木精复染，常规脱水、透明、封片。阴性对照以PBS替代一抗，其余步骤同上。显微镜下随机选取3个视野，计数阳性细胞数。以细胞中出现棕褐色浓染颗粒作为阳性细胞的判定标准。
3孕酮对Aβ损伤大鼠行为学及组织病理学改变的影响
将50只体重210-230g雄性SD大鼠随机分为5组，分别为溶剂对照组（假手术+蒸馏水）、Aβ损伤组、以及Aβ损伤+孕酮低、中、高剂量保护组。溶剂对照组大鼠双侧海马CA1区注射5μL蒸馏水，其余组大鼠注射5μL（2g/L）Aβ25-35溶液，模型制备方法同第一部分。孕酮保护组于术后6天-12天13:30皮下注射不同剂量（4mg/kg，8mg/kg，16mg/kg）的孕酮，溶剂对照组和Aβ损伤组皮下注射等体积注射用油。术后7天-12天进行Morris水迷宫行为学测试。行为学测试结束后取材，每组中的5只动物断头取脑，冰浴分离海马，-70℃存放，通过RT-PCR及Western blot分析炎症因子TNF-α及IL-1β表达改变；其余动物经多聚甲醛心脏灌流进行预固定，之后取脑组织，外固定，石蜡包埋，制备病理切片，进行组织病理学分析。
4孕酮对Aβ所致原代培养海马神经元损伤的保护作用
海马神经元原代培养：取新生SD大鼠，75%酒精消毒，无菌条件下断头取脑，分离双侧海马组织。去除被膜，剪碎组织，37℃水浴中胰酶消化15-20min，用含10%FBS（fetal bovine serum）的高糖DMEM（dulbecco minimum essential medium）终止消化。吹打重悬消化后的细胞团，至细胞团基本消失。调整细胞密度为1×106个/mL，接种于多聚赖氨酸预先包被的96孔板中，细胞培养箱中孵育，8h后换为含2%B27neuromix的Neurobasal-A培养液继续培养。培养72h后加入阿糖胞苷（终浓度5μg/mL）抑制胶质细胞生长，孵育48h后完全换液（含2%B27neuromix的Neurobasal-A培养液），之后隔日半量换液。体外培养7天-9天的细胞用于实验。
实验分组及处理：将1mg Aβ25-35溶于471.6μL蒸馏水中，得2mMAβ25-35溶液；混匀、分装、-20℃冻存。用前将Aβ25-35溶液（2mM）于37℃孵育一周，使其老化、聚合。体外培养7天-9天的细胞，分别加入不同终浓度的Aβ25-35（1μM，10μM，50μM），继续孵育48h，或加入终浓度10μM Aβ25-35孵育不同时间（12h，24h，48h），确定Aβ25-35对体外培养的海马神经元的损伤作用。不同浓度孕酮（0.1μM，0.5μM，1μM）预处理1h或与Aβ25-35同时加入，继续孵育48h，观察孕酮对Aβ细胞损伤作用的影响。
细胞活力分析：接种于96孔板中的神经元给予不同处理因素后，每孔加入20μL MTT（5mg/mL），于37℃孵育4h，弃去培养基，每孔各加入150μL DMSO，混匀，酶标仪测定各组细胞490nm波长处的吸光度（OD值），以此表示细胞的相对活力。
5统计学处理
各组数据以均数±标准误（mean±SEM）表示，实验数据均采用SPSS13.0统计软件进行处理。定位航行实验数据采用重复测量进行方差分析（repeated-measure analysis of variance），其他数据采用单因素方差分析（One-WayANOVA）继以LSD post hoc test进行统计分析，以P <0.05为差异有统计学意义。
结果：
1Aβ所致学习记忆障碍大鼠模型的制备
1.1A25-35对大鼠空间学习能力的影响
定位航行实验结果显示，各组大鼠逃避潜伏期均随学习天数增加而逐渐缩短；各组大鼠平均游泳速度之间无统计学差异；假手术组大鼠与溶剂对照组大鼠学习成绩无统计学差异；与溶剂对照组相比，Aβ25-35损伤组大鼠逃避潜伏期显著增加（P <0.01）。
1.2Aβ25-35对大鼠空间记忆能力的影响
空间探索实验结果显示，假手术组与溶剂对照组大鼠穿越平台区的次数及在平台所在象限停留时间百分比无统计学差异；与溶剂对照组相比，Aβ25-35损伤组大鼠穿越平台区的次数及在平台所在象限停留时间百分比均显著减少（P <0.01）。
1.3Aβ25-35对大鼠海马CA1区神经元形态及数量的影响
硫堇尼氏体染色显示，假手术组及溶剂对照组大鼠海马CA1区神经元有3-4层，排列整齐、紧密，细胞形态清晰、完整，可见大量突起，尼氏体呈紫色，含量丰富，细胞核不着色，核仁深染、清晰、居中；Aβ25-35损伤组大鼠海马CA1区神经元层数减少，排列紊乱，细胞失去原有形态，可见断裂的突起和不完整的胞体。细胞计数结果显示，假手术组与溶剂对照组大鼠海马CA1区完整锥体细胞的数量无统计学差异；与溶剂对照组相比，Aβ25-35损伤组大鼠海马CA1区完整锥体细胞数量显著减少（P <0.05）。
2Aβ损伤大鼠学习记忆相关脑区神经甾体合成代谢的改变
2.1Aβ损伤大鼠前额叶皮质及海马内神经甾体含量的改变
Aβ25-35注射后7天，前额叶皮质中孕酮显著下降（P <0.01），17β-雌二醇显著升高（P <0.05），孕烯醇酮及别孕烯醇酮含量无统计学差异；Aβ25-35注射后12天，前额叶皮质中孕酮显著下降（P <0.01），孕烯醇酮、别孕烯醇酮及17β-雌二醇含量无统计学差异。
Aβ25-35注射后7天，海马组织别孕烯醇酮显著下降（P <0.05），孕酮显著下降（P <0.01），17β-雌二醇显著升高（P <0.05）。Aβ25-35注射后12天，海马组织孕烯醇酮显著下降（P <0.05），孕酮显著下降（P <0.01），17β-雌二醇显著升高（P <0.05）。
2.2Aβ损伤大鼠海马内CYP11A1、3β-HSD、aromatase表达的改变
RT-PCR结果显示，与溶剂对照组相比，Aβ损伤大鼠海马内CYP11A1、3β-HSD、aromatase表达均显著（P <0.01）增加。Western blot结果与RT-PCR结果一致。免疫组化结果显示，假手术组与溶剂对照组大鼠海马组织中aromatase主要表达于胶质细胞，aromatase阳性细胞数较少，Aβ损伤大鼠海马内aromatase阳性细胞数显著增加（P <0.01）。
3孕酮对Aβ损伤大鼠空间学习记忆能力的改善作用及机制
3.1孕酮对Aβ损伤大鼠空间学习记忆能力的改善作用
定位航行实验结果显示，各组大鼠逃避潜伏期均随学习天数增加而逐渐缩短。孕酮部分逆转Aβ损伤引起的逃避潜伏期增加，作用呈浓度依赖性。空间探索实验结果显示，孕酮浓度依赖性改善Aβ损伤大鼠平台区穿越次数减少及平台所在象限停留时间百分比减少的现象。同时，定位航行及空间探索实验显示，孕酮不影响正常大鼠的学习记忆能力。提示，孕酮特异性改善Aβ损伤大鼠的空间学习记忆能力。
3.2孕酮对Aβ损伤大鼠海马CA1区神经元形态及数量的影响
硫堇尼氏体染色结果显示，Aβ损伤大鼠海马CA1区域可见断裂的突起和不完整的胞体，细胞数减少，尼氏体减少。孕酮浓度依赖性改善Aβ损伤大鼠海马锥体细胞的损伤，增加其CA1区锥体细胞存活数（P <0.01）。
3.3孕酮对Aβ损伤大鼠海马组织炎症因子表达的影响
RT-PCR结果显示，Aβ损伤大鼠海马组织内炎性因子TNF-α及IL-1β表达显著增加（P <0.01）。孕酮可抑制Aβ损伤大鼠TNF-α及IL-1β表达上调的现象，作用呈浓度依赖性。Western blot结果与RT-PCR结果一致，即：孕酮抑制Aβ损伤大鼠海马组织内TNF-α及IL-1β表达上调。
4孕酮对Aβ所致原代培养海马神经元损伤的保护作用
4.1Aβ以浓度、时间依赖的方式降低原代培养海马神经元活性
MTT结果显示，不同浓度的Aβ作用于体外培养7天的海马神经元48h后，神经元活力下降，其中10μM及50μM Aβ使神经元活力显著下降（P <0.05，P <0.01）。10μMAβ作用于海马神经元不同时间后神经元活力下降，其中作用24h及48h后，细胞活力显著下降（P <0.05）。
4.2孕酮对抗Aβ的细胞损伤作用
MTT结果显示，10μM Aβ作用于神经元48h后，细胞活力显著下降（P <0.05）；孕酮可对抗Aβ引起的海马神经元活力下降，作用呈浓度依赖性。与Aβ损伤组相比，1μM孕酮处理组海马神经元活力显著增加（P<0.05）；与Aβ损伤组相比，0.5μM及1μM孕酮预处理组细胞活力显著增加（P <0.05，P <0.01）。
结论：
1双侧海马CA1区注射Aβ25-35损伤大鼠海马CA1区锥体细胞，引起大鼠空间学习记忆能力显著下降，Aβ所致学习记忆障碍大鼠模型制备成功。
2Aβ损伤大鼠脑组织中孕烯醇酮及孕酮含量降低，17β–雌二醇含量增加，同时，神经甾体合成代谢酶CYP11A1、3β-HSD、aromatase表达上调。推测，Aβ上调3β–HSD及aromatase表达，孕烯醇酮及孕酮分解加快，引起二者含量下降，其代谢产物雌二醇含量上升；机体通过增加神经甾体合成限速酶CYP11A1的表达代偿孕烯醇酮及孕酮含量的下降，Aβ促进神经甾体代谢向生成雌二醇的方向进行。
3外源性给予孕酮改善Aβ损伤大鼠的空间学习记忆能力，对抗Aβ损伤大鼠海马CA1区锥体细胞损伤，抑制Aβ损伤大鼠海马组织中的炎症反应。抑制炎症反应，发挥细胞保护作用可能是孕酮改善Aβ损伤大鼠学习记忆能力的机制之一。
4孕酮浓度依赖性对抗Aβ引起的原代培养海马神经元活力下降，从细胞水平进一步证实孕酮对Aβ损伤具有保护作用。
Alzheimer’s disease (AD), characterized clinically by progressivelycognitive impairment and pathologically by the appearance of senile plaquesand neurofibrillary tangles, is the most prevalent senile dementia. Amyloid β(Aβ), a β-sheet peptide fragment produced by the proteolytic cleavage ofamyloid precursor protein (APP) by γ-and β-secretases, is the majorcomponent of senile plaques. Both in vivo and in vitro studies have shown thataccumulation of Aβ fibrils trigger neurodegeneration, supporting the view thatAβ aggregation is of importance in AD. Aβ causes oxidative damage,inflammatory responses, and memory impairment, all of which may lead tothe neuronal dysfunction and degeneration responsible for the cognitivedeficits observed in AD.
Steroids hormones and their metabolites within the central nervoussystem (CNS) are commonly defined as neuroactive steroids or neurosteroids.They can be either synthesized de novo in the CNS by glial cells and neuronsfrom cholesterol or synthesized in the periphery by the adrenals and gonads.The concentration of neurosteroid was higher in CNS than in periphery.Neurosteroids mainly include pregnenolone (PREG), dehydroepiandrosterone(DHEA), their sulfate derivatives pregnenolone sulfate (PREGS) anddehydroepiandrosterone sulfate (DHEAS), and progesterone (PROG),5α-dihydroprogesterone (5α-DHP),3α,5α-tetrahydroprogesterone(allopregnanolone/ALLO), deoxycorticosterone (DOC),tetrahydrodeoxycorticosterone (THDOC), estradiol (E2), etc. Neurosteroidsplay an important role as endogenous modulators in altering neuronalexcitability rapidly; however, no specific receptor has been reported to datefor neurosteroids. Most of their actions in the nervous tissue were reported tomodulate membrane neurotransmitter receptors, such as GABAA, NMDA, and sigma1receptors, and thus affect neuronal plasticity, anxiety, responses tostressful stimuli, and neuropsychiatric symptoms represented during AD.Some neurosteroids were reported to improve learning and memory abilityand protect against Aβ peptide-induced neurotoxicity, thus exerting neuronalprotection. However, changes in the metabolic pathway of neurosteroidsduring AD and their role in Aβ-mediated impairment remain relativelyelusive given the limitations in sample sizes and analysis methods.
Objective: We have reported previously that the concentration ofprogesterone decreased in the media of Aβ25-35-induced cortical neurons, andprogesterone treatment inhibited the declination of cell viability as well as theapoptosis induced by Aβ25-35. The present study was designed to investigatethe levels of neurosteroids in encephalic regions related to learning andmemory (the prefrontal cortex and hippocampus) of Sprague-Dawley (SD)rats injected with Aβ25-35as well as the expression of neurosteroidogenesisenzymes. Furthermore, the effect of progesterone administration againstAβ25-35induced impairment was investigated in vivo and in vitro. The presentstudy provided a potential therapeutic strategy for disorders with learning andmemory impairment, such as AD.
Material and Methods:
1Effect of Aβ micro-injection on the spatial learning and memory of ratsTo investigate the effect of Aβ25-35administration on spatial learning andmemory of rats, a total of thirty SD rats were divided into three groups (shamgroup, vehicle group, and Aβ group) randomly with ten in each. Afteranesthetized with2%pentobarbital sodium (40mg/kg weight, intraperitonealinjection), the animals were placed in stereotaxic apparatus (Jiangwai typeⅠ).Five microliters aggregated Aβ25-35or vehicle (sterile distilled water, forvehicle group) were injected slowly over10min into bilateral CA1hippocampus (anterior/posterior:-3.5mm, media/lateral:±2.0mm, anddorsal/ventral:-2.7mm ventral to the skull surface) with a10μLmicrosyringe, followed by10min remaining to allow the diffusion ofinjection content. The rectal temperature was maintained at36℃to37℃for all animals throughout the surgery. Six-day Morris water maze behavioraltask was employed to test the spatial learning and memory of the animalssince the seventh day after surgery. After the behavioral test, all animals wereanesthetized with pentobarbital sodium and perfused through the ascendingaorta with normal saline followed by4%paraformaldehyde. The brain tissueswere then dissected and fixed for histological examination.
Behavioral Tests: The Morris water maze (JLBehv-MWM, Shanghai,China) consisted of a black pool (180cm diameter,60cm high) filled withwater (40cm,23±1℃), and was divided into four quadrants with fourequidistant release points around the edge, with a circular black platform (10cm diameter) submerging1cm below the water surface and remaining thesame position of the target quadrant. The behavior of rats in the pool wastracked with a camera which allowing us to measure the swim distance andtime to find the platform. Rats were trained four trials each day, with aninterval of20min at least, for five consecutive days. The starting quadrantwas randomized every day with all animals according to the same order. Theanimals were faced towards the pool wall before being released, and allowedto stay on the platform for10s after finding the platform within60s, orguided to the platform to stay for10s if failed to reach the platform in60s.The time needed to reach the platform (escape latency) was analyzed asindices of spatial learning. The probe trial was conducted on the sixth day,when each subject allowed90s to search the water from which the platformhad been removed. The number of times the animal crossed the platform(number of platform crossings) and time ratio in platform quadrant (Ⅳ timeratio) were used as index of spatial memory retention. A camera was used totrack the time to find the platform as well as to measure the swim speed anddistance travelled.
Histological Examination: The brain tissues were fixation in4%paraformaldehyde in phosphate buffer (pH7.4), dehydration in a series ofethanol, embedded in wax and sectioned into5-micrometer-thick sections.Tissue sections were then deparaffinized, dehydrated, and stained with thionin, followed by differentiation, dehydration and clearance before they weremounted. The number of intact pyramidal cells (per1mm linear length of thesame section of CA1hippocampus) was then counted by three experimentersand analyzed.
2The metabolism alteration of neurosteroids in the prefrontal cortex andhippocampus of Aβ treated rats
To investigate the effect of Aβ25-35on neurosteroids level in brain, a totalof ninety SD rats were divided into three groups (sham group, vehicle group(sterile distilled water), and Aβ group) randomly with thirty in each group andtreated the same as above. Ten rats in each group were sacrificed (viadecapitation) during16:00-18:00on the seventh day and the twelfth dayafter Aβ injection respectively, with the prefrontal cortex and hippocampusremoving (on ice) and stored in-70℃until neurosteroids detectionthroughhigh performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. On the twelfth day after Aβ injection, five rats in eachgroup were sacrificed (via decapitation) with hippocampus removed (on ice)and stored at-70℃for steroidogenesis enzymes analysis through RT-PCR,Western blot, and immunohistochemisty. The rests were anesthetized withpentobarbital sodium and perfused through the ascending aorta with normalsaline followed by4%paraformaldehyde. The brain tissues were thendissected and fixed for histological examination.
Sample Preparation and HPLC-MS/MS Analysis: Samples (about100mg) were added with10μL30ng/mL MT (internal standards), homogenizedin1mL of phosphate buffered saline (PBS) using an ultrasonic homogenizer(Heidolph, Germany), mixed with2mL ethyl acetate/n-hexane (9:1, v/v)and centrifuged at12000rpm for10min. The pellet was extracted three timeswith2mL ethyl acetate/n-hexane (9:1, v/v), and the organic phases werecombined and dried with a gentle stream of nitrogen in a50℃water bath.The samples were derivatized with dansyl chloride in a60℃water bath for40min, concentrated at12000rpm for10min and then transferred inautosampler vials before the high performance liquid chromatography- tandem mass spectrometry (HPLC-MS/MS) analysis.
The HPLC-MS/MS system (Thermo Fisher Scientific, Waltham, MA,USA) consisted of Surveyor MS Pump Plus, Surveyor AS Plus, TSQQuantum Access triple quadrupole mass spectrometer and Xcalibur DataSystems (Thermo Fisher Scientific). Separation was achieved on a XDB C18analytical column (4.6mm×50mm, Agilent, Palo Alto, CA, USA) fittedwith a XDB C18guard column (4.6mm×12mm, Agilent). The HPLC mobilephases were (A) H2O/0.1%formic acid and (B) MeOH/0.1%formic acid,and the flow rate was0.5mL/min. The gradient was as follows:0-6.5min，36%A;6.5-6.6min,30%A;6.6-17min,10%A;17-18min,36%A.The column temperature was40℃and the injection volume was30μL. AMSD quadrupole mass spectrometer equipped with atmospheric pressurechemical ionization (APCI) source (Thermo Fisher Scientific) was used forthe detection of analytes in the positive ion mode. The optimized conditionswere as follows: the spray voltage,4500V; the vaporizer temperature,400℃; the sheath gas,30L/min; the aux gas,5L/min; the capillary temperature,270℃. The quantification was performed using multiple-reaction monitoring(MRM) method with the transitions of (m/z)299.03→(m/z)158.86and280.9for PREG,(m/z)315.03→(m/z)97and109.01for PROG,(m/z)301→(m/z)188.9and282.85for ALLO,(m/z)254.97→(m/z)132.9and158.9for17β-E2,(m/z)303.1→(m/z)97.04and109.06for MT, respectively.
RT–PCR: Total RNA was isolated from cells using TRIzol Reagent(Invitrogen, CA, USA) and equal amounts were reverse transcribed intocDNA using the oligo dT primer, then the cDNAs were used as DNAtemplates for PCR. GAPDH was used to ensure equal loading. The PCRproducts were separated on1.5%agarose gel and visualized by ethidiumbromide.
Western Blot: Lysates from brain tissue were prepared with lysis buffer(1%Triton X-100,150mM NaCl,10mM Tris-HCl, pH7.4,1mM EDTA,pH8.0,0.2mM Na3VO4,0.2mM phenylmethylsulfonyl fluoride, and0.5%NP-40). Equal amounts of protein (40μg) were separated by10%SDS- PAGE, and electrotransferred to a PVDF membrane (Millipore, Billerica, MA,USA). Membranes were blocked with5%BSA for2h at room temperature,and incubated with the primary antibodies,1:1000mouse anti-TNF-α(Abcam),1:500rabbit anti-IL-1β (Abcam),1:500rabbit anti-CYP11A1(anbobio,),1:500rabbit anti-3β-HSD (Epitomics),1:500rabbit anti-aromatase (Abcam), and1:5000rabbit anti-β-actin (Santa Cruz) overnight,respectively, and then with the respective secondary antibody for2h. Proteinswere detected using the Chemiluminescence Plus Western blot analysis kit(Santa Cruz). The experiments were replicated three times.
Immunohistochemistry: Five-micrometer-thick sections weredeparaffinized, dehydrated, followed by Antigen retrieval (microwave). Thensections were in turn incubated with3%H2O2for10min, with primaryantibody overnight at4℃, with diluted biotinylated IgG secondary antibody,with DAB, followed by being differentiated, dehydrated, cleared, andmounted.
3The effect of progesterone on behavioral and cellular impairment in Aβtreated rats
To investigate the effect of PROG on cognition impairment induced byAβ25-35CA1hippocampus injection, a total of fifty SD rats were dividedrandomly into five groups: vehicle group (sterile distilled water), Aβ group, PLgroup (Aβ+progesterone4mg/kg weight), PMgroup (Aβ+progesterone8mg/kg weight), and PHgroup (Aβ+progesterone16mg/kg weight). During6-12days after Aβ injection described above, animals were injected withdifferent dosages of progesterone (PL,PM,and PHgroup) or vehicle (sesame oilfor vehicle group and Aβ group) daily at13:30. Behavioral tests wereconducted during7-12days after Aβ injection beginning at14:30.Immediately after behavior tests, five rats in each group were sacrificed (viadecapitation) with hippocampus removed (on ice) and stored at-70℃forinflammatory factors analysis through RT–PCR and Western blot. The restanimals were anesthetized with pentobarbital sodium and perfused through theascending aorta with normal saline followed by4%paraformaldehyde. The brain tissues were dissected and fixed for histological examination.
Behavioral tests, RT–PCR, Western blot and histological examinationwere conducted as mentioned above.
4The effect of PROG on Aβ25-35induced cell impairment in vitro
Hippocampus cells of newborn SD rat were cultured and treated asdescribed. In brief, bilateral hippocampus newborn rat were dissociated with0.125%trypsin/HBSS for15min at37℃, followed by trypsin quenchingwith DMEM-HG containing10%FBS. Cell suspensions were centrifuged(5min,200×g), resuspended, dissociated by repeated passage through a fire-polished Pasteur pipette, and then filtered through a sterile cell strainer. Thedissociated hippocampus cells plated on poly-L-lysine (Sigma,50μg/mL)coated96-well plates at a density1×106cells/mL were grown in Neurobasalmedium-A supplemented with5units/mL penicillin,5mg/mL streptomycin,1%GlutaMAX I and2%B27neuromix at37℃in humidified5%CO2/95%air atmosphere for7days to9days before experimentation.
The cultures were treated with or without Aβ (1μM,10μM, or50μM)for48h, or10μM Aβ for12h,24h, or48h. In the following experiment,cultures were pre-treated with or without PROG (0.1μM,0.5μM, or1μM)for60min followed by exposure to10μM aggregated Aβ for48h, or treatedsimultaneously with different dosages of PROG and Aβ for48h. Cellviability was assessed by MTT analysis. The results were presentedgraphically as a percentage of live cells in the control group.
Assessment of Cell Viability: Loss of cell viability was measured by theMTT (methyl thiazolyl tetrazolium) assay. In brief,2×105cells per well wereseeded in triplicate onto96-well plates in Neurobasal-A medium and allowedto grow for7days. The cells were treated according to the experimentaldesign. After drug treatment, cells were incubated with5mg/mL MTT for2h,and subsequently solubilized in dimethyl sulfoxide (DMSO). The absorbencyat570nm was then measured using ELIASA (BMG Labtech, Germany). Theresults were expressed as the mean of the absorbance relative to the controlgroup. The experiments were replicated three times.
5Statistical Analysis
The data were expressed as mean±standard error of the mean (SEM).The mean escape latency of the Morris water maze behavioral tests collectedduring the training days were analyzed by repeated-measure analysis ofvariance (ANOVA). Other data were statistically analyzed by one-wayANOVA, followed by between-group comparisons using LSD post hoc test.Statistical significance was concluded with a value of P <0.05for all analyses.Data analyses were performed with the SPSS software version13.0.
Results:
1Effect of Aβ micro-injection on the spatial learning and memory of rats
The Morris water maze behavioral task was employed to examinehippocampus-dependent spatial learning and memory. The mean swimspeeds did not differ across the groups. The mean escape latency in the Morriswater maze behavioral task was significantly (P <0.01) higher in Aβ group(47.9±2.3,39.1±3.0,37.4±2.9,33.3±2.6, and29.4±3.0; Day1to Day5,respectively) compared with those of the vehicle group (47.3±1.3,28.6±2.7,21.1±2.7,15.8±1.9, and12.8±1.4; Day1to Day5, respectively.).Moreover, the number of platform crossings (48.7%) and the staying timeratio in platform quadrant (32.7%) of Aβ group in the probe test wereconsiderably (P <0.01) lower compared with those of vehicle group, thusconfirming spatial learning and memory impairment in Aβ25-35treated rats.Overall, no difference was observed between sham and vehicle group.
Histological examination showed that Aβ25-35significantly (P <0.01)decreased (37.6%) the intact pyramidal cell number in the CA1hippocampuscompared with the vehicle group.
2The metabolism alteration of neurosteroids in the prefrontal cortex andhippocampus of Aβ treated rats
High performance liquid chromatography-tandem mass spectrometrywas employed to simultaneously quantify the level of neurosteroids indifferent encephalic region. Levels of neurosteroids were altered in theprefrontal cortex and hippocampus of Aβ treated rats7and12days after surgery. Levels of PREG in the hippocampus of Aβ group were significantly(P <0.05) lower (66.1%and70.8%)7days and12days after surgerycompared with those of vehicle group, while levels of PREG in the prefrontalcortex did not differ compared with those of vehicle group on either timepoint. Furthermore, levels of PROG were significantly (P <0.01) reduced inboth the prefrontal cortex and hippocampus (63.8%and61.8%for7daysand12days after surgery in the prefrontal cortex;44.3%and42.2%for7days and12days after surgery in hippocampus, respectively) compared withthose of vehicle group. However, the hippocampus of Aβ treated ratscontained significantly (P <0.05) elevated (146.2%and137.9%) levels of17β-E27days and12days after surgery compared with those of vehiclegroup, while the prefrontal cortex of Aβ treated rats contained significantly (P<0.05) higher (171.8%) levels of17β-E2only on7days after surgerycompared with those of vehicle group. The level of ALLO did not differ ineither brain regions on both time points. The level of PREG, PROG and17β-E2did not differ between sham and vehicle group in either brain region onboth time points.
The expression of neurosteroidogenesis enzymes was altered inhippocampus of Aβ treated rats. RT-PCR showed that gene expression ofCYP11A1,3β-HSD, and aromatase in hippocampus were significantly (P <0.01) higher in Aβ group (149.0%,247.6%, and159.5%) compared withthose of vehicle group. The up-regulation of CYP11A1,3β-HSD, andaromatase were confirmed when the protein level of CYP11A1,3β-HSD,and aromatase were significantly (P <0.01) increased in Aβ group (142.3%,219.9%, and145.0%) compared with those of vehicle group, showed byWestern blot. Results of immunohistochemisty analysis confirmed the up-regulation of aromatase in hippocampus of Aβ treated rats (113.6%, P <0.01,compared with vehicle group).
3The protective effect of progesterone against the behavioral and cellularimpairment in Aβ treated rats
Administration of PROG enhanced the cognitive performance of Aβ treated rats in a dose-dependent manner (4mg/kg,8mg/kg,16mg/kg for PL,PM, PHgroups, respectively). The mean swim speeds did not differ across thegroups. The mean escape latency of PL, PMand PHgroups were significantly(P <0.05for PL; P <0.01for PMand PH) lower compared with Aβ group. Therestoration of cognitive impairment was confirmed when the number ofplatform crossings of the PMand PHgroup was significantly increased by170.6%(P <0.05) and217.7%(P <0.01), compared with those of Aβ group.Meanwhile, the Ⅳ time ratio of PL, PM, and PHgroup was significantly (P <0.01) increased by170.6%,190.13%, and217.7%, compared with Aβgroup.
PROG reversed Aβ25-35-induced cell loss in CA1hippocampus dosedependently. Compared with vehicle group, the intact pyramidal cell numberof Aβ group significantly (P <0.01) decreased by35.9%. Compared with Aβgroup, the intact pyramidal cell number of PL, PM, and PHgroups weresignificantly (P <0.01) increased by130.9%,190.1%, and241.3%,respectively.
On the cellular level, PROG reversed Aβ25-35injection induced up-regulation of TNF-α and IL-1β. RT-PCR showed that that gene expressionof TNF-α and IL-1β in hippocampus were significantly (P <0.01) higher inAβ group (176.4%and176.4%) compared with those of vehicle group. Theup-regulation of TNF-α and IL-1β were confirmed when the protein levelof TNF-α and IL-1β were significantly (P
神经甾体（neurosteroid）是中枢神经系统中的甾体类物质及其代谢产物的总称，可由神经元或胶质细胞在中枢神经系统合成，也可由外周腺体合成后通过血脑屏障进入中枢神经系统，神经甾体在脑组织中的含量高于外周血中的含量。神经胶质细胞和神经元合成的神经甾体主要包括：孕烯醇酮（pregnenolone，PREG）、孕烯醇酮硫酸酯（pregnenolone sulfate，PREGS）、脱氢表雄酮（dehydroepiandrosterone，DHEA）、脱氢表雄酮硫酸酯（dehydroepiandrosterone sulfate，DHEAS）、孕酮（progesterone，PROG）、别孕烯醇酮（allopregnanolone，ALLO），以及雌二醇（estradiol，E2）等。研究显示，神经甾体具有镇静催眠、抗惊厥、抗焦虑、抗精神分裂症、改善学习记忆，以及神经保护等作用，在中枢神经系统功能调节中发挥广泛作用。研究显示，神经甾体的表达调控改变与包括AD在内的多种神经退行性疾病的发生有关，AD时神经甾体含量及合成代谢酶表达发生改变。对AD患者神经甾体水平变化的研究多为神经甾体水平与AD易患倾向关系的分析研究，其研究对象多为AD患者外周血、脑脊液或尸脑，研究对象来源有限。AD患者血浆或脑组织中神经甾体水平的变化为长时间、多种因素共同作用的结果，而在AD进程中，神经甾体含量的变化与学习记忆能力受损的关系尚不明确。
目的：本研究采用单一损伤因素Aβ25-35诱导AD样学习记忆障碍大鼠模型，通过液液萃取结合高效液相色谱-串联质谱（high performanceliquid chromatography-tandem mass spectrometry，HPLC-MS/MS）方法分析Aβ损伤大鼠学习记忆相关脑区（前额叶皮质及海马）中主要神经甾体的含量，通过RT-PCR、 Western blot和免疫组织化学（immunohistochemistry，IHC）分析神经甾体合成代谢酶的表达；筛选与学习记忆障碍关系密切的神经甾体，观察其对Aβ损伤大鼠学习记忆能力的改善作用，探讨其学习记忆保护作用的机制。利用原代培养的海马神经元，进一步在细胞水平研究Aβ25-35对海马神经元细胞活力的影响及神经甾体孕酮对抗Aβ损伤的保护作用。本实验为神经甾体尤其是孕酮在AD等学习记忆障碍类疾病中的应用提供理论依据及实验基础。
方法：
1Aβ所致学习记忆障碍大鼠模型的制备
将30只体重210-230g雄性Spague-Dawley（SD）大鼠随机分为3组，分别为：假手术组、溶剂对照组（假手术+蒸馏水）、以及Aβ损伤组，每组10只。Aβ损伤模型制备方法如下：动物经腹腔注射麻醉后，固定于脑立体定位仪，沿颅骨中线切开皮肤，暴露前囟。参照图谱海马CA1区位置，于前囟后3.5mm，中线左右2.0mm处，开直径1mm的骨窗，垂直颅骨表面进针2.7mm，缓慢注入5μL（2g/L）Aβ25-35溶液，留针10min使注射物充分扩散，缓慢撤针，缝合切口。溶剂对照组注入等体积蒸馏水。术后7天-12天进行Morris水迷宫行为学测试，观察大鼠空间学习记忆能力。行为学测试结束后，动物经多聚甲醛心脏灌流进行预固定，之后取脑组织，外固定，石蜡包埋，制备病理切片，通过硫堇尼氏体染色进行组织病理学分析。
行为学测试：术后7天-12天进行Morris水迷宫（the Morris watermaze）行为学测试。实验开始前将动物置于行为学测试房间，使其适应环境1h。行为学测试于每天14:30开始，室温25℃，水温22℃-24℃，动物测试顺序保持不变，测试开始象限随机选取。Morris水迷宫行为学测试历时6天，前5天进行定位航行实验（the navigation trial），每天训练4次。记录大鼠入水至寻找到隐匿平台所需的时间，即逃避潜伏期（escapelatency）。第6天进行空间探索实验（the probe trial），撤去平台，将大鼠从平台相对的象限面壁放置入水，记录动物90s内穿越平台区的次数（number of platform crossings）以及在平台所在象限（第四象限）停留的时间百分比（Ⅳ time ratio）等行为学指标。
组织病理学分析：预固定的脑组织经4%多聚甲醛固定至少48h，石蜡包埋，常规脑组织切片（5μm），硫堇尼氏体染色观察海马组织学改变。选取相同海马截面的切片进行硫堇尼氏体染色。每张切片随机选取CA1区3个400倍视野，计数海马CA1区每1mm区段内细胞膜完整、胞核饱满、核仁清晰的锥体细胞数目，每张切片海马各计数3个区段取平均数。
2Aβ损伤大鼠学习记忆相关脑区（前额叶皮质及海马）神经甾合成代谢的改变
将90只体重210-230g雄性SD大鼠随机分为3组，分别为：假手术组、溶剂对照组、以及Aβ损伤组，每组30只。动物处理同上。分别于术后7天及12天断头处死每组中的10只动物，迅速取脑，冰浴分离前额叶皮质及海马，-70℃存放，通过HPLC-MS/MS分析神经甾体孕烯醇酮、孕酮、别孕烯醇酮、及17β–雌二醇的含量；其余动物于术后12天取材，每组中的5只动物断头处死，迅速取脑，冰浴分离海马，-70℃存放，通过RT–PCR及Western blot分析神经甾体合成代谢酶CYP11A1、3β-HSD及aromatase的表达改变；其余动物经多聚甲醛心脏灌流进行预固定，之后取脑组织，外固定，石蜡包埋，制备病理切片，通过免疫组化分析神经甾体合成代谢酶aromatase的表达改变。
神经甾体提取：参考本实验室已有方法略加改进，通过液液萃取提取神经甾体。脑组织样品（双侧海马组织或前额叶皮质）加入PBS及MT（内标），制备组织匀浆。乙酸乙酯/正己烷（9:1）萃取3次，转移并合并有机相，50℃水浴下N2吹干；60℃水浴中用丹酰氯衍生40min，离心后取上清，4℃保存。
神经甾体含量测定：色谱条件：色谱柱Agilent XDB C18分析柱（4.6mm×50mm）；保护柱Agilent XDB C18（4.6mm×12mm）；柱温40℃；流动相：A水（含0.1%甲酸），B甲醇（含0.1%甲酸）；流速：0.5mL/min；洗脱梯度：0-6.5min，36%A；6.5-6.6min，30%A；6.6-17min，10%A；17-18min，36%A。进样量30μL，每针运行18min。质谱条件：离子化方式：大气压化学离子源（atmospheric pressure chemical ionization，APCI）；喷雾电压：4500V；鞘气（N2）压力：30L/min；辅助气（N2）压力：5L/min；离子检测方式：正离子多反应监测；用于定量分析的离子反应分别为：(m/z)299.03→(m/z)158.86、280.9（PREG），(m/z)315.03→(m/z)97、109.01（PROG），(m/z)301→(m/z)188.9、282.85（ALLO），(m/z)254.97→(m/z)132.9、158.9（17β-E2），(m/z)303.1→(m/z)97.04、109.06（MT）。
RT-PCR：按照Invitrogen公司产品手册，采用TRIzol一步法，提取组织总RNA。每50-100mg脑组织加入1mL TRIzol试剂，匀浆器内冰浴研磨、裂解组织。将裂解液移至0.1%DEPC处理并高压灭菌的1.5mL离心管中，加入0.5mL氯仿，剧烈震荡15s，室温孵育，离心，静置，加等体积异丙醇，颠倒混匀，室温静置，离心，弃上清，75%乙醇洗沉淀，室温晾干，20μL DEPC水溶解。按照Promega逆转录试剂盒说明，取等量组织总RNA，70℃水浴10min，之后迅速置于冰上；加入逆转录体系各组分，涡旋混匀，瞬时离心，42℃孵育1h，合成cDNA，95℃加热3min终止反应。参考Genbank中mRNA序列设计引物，以反转录产物为模板，进行PCR扩增。PCR扩增产物经1.5%琼脂糖凝胶电泳分离，扫描成像。凝胶成像分析软件进行相对定量分析。
组织总蛋白提取与Western blot分析：组织加入RIPA裂解液，制备组织匀浆。离心取上清（即蛋白提取液），分装，-20℃保存。以牛血清白蛋白为标准物，采用考马斯亮蓝蛋白测定试剂盒进行蛋白定量。取等量蛋白提取液，RIPA裂解液补齐体积，与5×SDS上样缓冲液混合，沸水浴加热使蛋白变性，冷却后上样。稳压电泳，溴酚蓝移动至凝胶底部时，停止电泳。切去浓缩胶，剪取与凝胶相同大小的PVDF膜，甲醇活化后，与滤纸、凝胶一起浸入转膜缓冲液，平衡15min。依次放置滤纸、PVDF膜、凝胶、滤纸，20V恒压半干转膜。将PVDF膜置于含5%脱脂奶粉的TTBS中，37℃封闭2h。之后进行一抗结合反应，4℃缓慢摇动过夜。洗膜后，进行二抗结合反应。洗膜后，用化学发光试剂盒检测PVDF膜上抗原抗体结合区带。凝胶成像分析软件进行相对定量分析。
免疫组织化学染色分析：脑组织标本经4%多聚甲醛固定后，梯度乙醇脱水，石蜡垂直定向包埋，5μm厚度连续切片。SP三步法进行免疫组织化学染色，即：石蜡切片脱蜡至水，微波抗原修复，自然冷却；滴加H2O2去离子水，室温孵育10min，消除内源性过氧化物酶影响；PBS冲洗，滴加山羊血清工作液，封闭非特异结合位点；倾去血清，滴加以PBS适当比例稀释的一抗，4℃过夜；PBS冲洗，滴加生物素化二抗工作液，室温孵育15min，PBS冲洗；滴加辣根酶标记链霉卵白素工作液，室温孵育15min，PBS冲洗；滴加DAB试剂显色，镜下控制显色时间，自来水冲洗终止反应；苏木精复染，常规脱水、透明、封片。阴性对照以PBS替代一抗，其余步骤同上。显微镜下随机选取3个视野，计数阳性细胞数。以细胞中出现棕褐色浓染颗粒作为阳性细胞的判定标准。
3孕酮对Aβ损伤大鼠行为学及组织病理学改变的影响
将50只体重210-230g雄性SD大鼠随机分为5组，分别为溶剂对照组（假手术+蒸馏水）、Aβ损伤组、以及Aβ损伤+孕酮低、中、高剂量保护组。溶剂对照组大鼠双侧海马CA1区注射5μL蒸馏水，其余组大鼠注射5μL（2g/L）Aβ25-35溶液，模型制备方法同第一部分。孕酮保护组于术后6天-12天13:30皮下注射不同剂量（4mg/kg，8mg/kg，16mg/kg）的孕酮，溶剂对照组和Aβ损伤组皮下注射等体积注射用油。术后7天-12天进行Morris水迷宫行为学测试。行为学测试结束后取材，每组中的5只动物断头取脑，冰浴分离海马，-70℃存放，通过RT-PCR及Western blot分析炎症因子TNF-α及IL-1β表达改变；其余动物经多聚甲醛心脏灌流进行预固定，之后取脑组织，外固定，石蜡包埋，制备病理切片，进行组织病理学分析。
4孕酮对Aβ所致原代培养海马神经元损伤的保护作用
海马神经元原代培养：取新生SD大鼠，75%酒精消毒，无菌条件下断头取脑，分离双侧海马组织。去除被膜，剪碎组织，37℃水浴中胰酶消化15-20min，用含10%FBS（fetal bovine serum）的高糖DMEM（dulbecco minimum essential medium）终止消化。吹打重悬消化后的细胞团，至细胞团基本消失。调整细胞密度为1×106个/mL，接种于多聚赖氨酸预先包被的96孔板中，细胞培养箱中孵育，8h后换为含2%B27neuromix的Neurobasal-A培养液继续培养。培养72h后加入阿糖胞苷（终浓度5μg/mL）抑制胶质细胞生长，孵育48h后完全换液（含2%B27neuromix的Neurobasal-A培养液），之后隔日半量换液。体外培养7天-9天的细胞用于实验。
实验分组及处理：将1mg Aβ25-35溶于471.6μL蒸馏水中，得2mMAβ25-35溶液；混匀、分装、-20℃冻存。用前将Aβ25-35溶液（2mM）于37℃孵育一周，使其老化、聚合。体外培养7天-9天的细胞，分别加入不同终浓度的Aβ25-35（1μM，10μM，50μM），继续孵育48h，或加入终浓度10μM Aβ25-35孵育不同时间（12h，24h，48h），确定Aβ25-35对体外培养的海马神经元的损伤作用。不同浓度孕酮（0.1μM，0.5μM，1μM）预处理1h或与Aβ25-35同时加入，继续孵育48h，观察孕酮对Aβ细胞损伤作用的影响。
细胞活力分析：接种于96孔板中的神经元给予不同处理因素后，每孔加入20μL MTT（5mg/mL），于37℃孵育4h，弃去培养基，每孔各加入150μL DMSO，混匀，酶标仪测定各组细胞490nm波长处的吸光度（OD值），以此表示细胞的相对活力。
5统计学处理
各组数据以均数±标准误（mean±SEM）表示，实验数据均采用SPSS13.0统计软件进行处理。定位航行实验数据采用重复测量进行方差分析（repeated-measure analysis of variance），其他数据采用单因素方差分析（One-WayANOVA）继以LSD post hoc test进行统计分析，以P <0.05为差异有统计学意义。
结果：
1Aβ所致学习记忆障碍大鼠模型的制备
1.1A25-35对大鼠空间学习能力的影响
定位航行实验结果显示，各组大鼠逃避潜伏期均随学习天数增加而逐渐缩短；各组大鼠平均游泳速度之间无统计学差异；假手术组大鼠与溶剂对照组大鼠学习成绩无统计学差异；与溶剂对照组相比，Aβ25-35损伤组大鼠逃避潜伏期显著增加（P <0.01）。
1.2Aβ25-35对大鼠空间记忆能力的影响
空间探索实验结果显示，假手术组与溶剂对照组大鼠穿越平台区的次数及在平台所在象限停留时间百分比无统计学差异；与溶剂对照组相比，Aβ25-35损伤组大鼠穿越平台区的次数及在平台所在象限停留时间百分比均显著减少（P <0.01）。
1.3Aβ25-35对大鼠海马CA1区神经元形态及数量的影响
硫堇尼氏体染色显示，假手术组及溶剂对照组大鼠海马CA1区神经元有3-4层，排列整齐、紧密，细胞形态清晰、完整，可见大量突起，尼氏体呈紫色，含量丰富，细胞核不着色，核仁深染、清晰、居中；Aβ25-35损伤组大鼠海马CA1区神经元层数减少，排列紊乱，细胞失去原有形态，可见断裂的突起和不完整的胞体。细胞计数结果显示，假手术组与溶剂对照组大鼠海马CA1区完整锥体细胞的数量无统计学差异；与溶剂对照组相比，Aβ25-35损伤组大鼠海马CA1区完整锥体细胞数量显著减少（P <0.05）。
2Aβ损伤大鼠学习记忆相关脑区神经甾体合成代谢的改变
2.1Aβ损伤大鼠前额叶皮质及海马内神经甾体含量的改变
Aβ25-35注射后7天，前额叶皮质中孕酮显著下降（P <0.01），17β-雌二醇显著升高（P <0.05），孕烯醇酮及别孕烯醇酮含量无统计学差异；Aβ25-35注射后12天，前额叶皮质中孕酮显著下降（P <0.01），孕烯醇酮、别孕烯醇酮及17β-雌二醇含量无统计学差异。
Aβ25-35注射后7天，海马组织别孕烯醇酮显著下降（P <0.05），孕酮显著下降（P <0.01），17β-雌二醇显著升高（P <0.05）。Aβ25-35注射后12天，海马组织孕烯醇酮显著下降（P <0.05），孕酮显著下降（P <0.01），17β-雌二醇显著升高（P <0.05）。
2.2Aβ损伤大鼠海马内CYP11A1、3β-HSD、aromatase表达的改变
RT-PCR结果显示，与溶剂对照组相比，Aβ损伤大鼠海马内CYP11A1、3β-HSD、aromatase表达均显著（P <0.01）增加。Western blot结果与RT-PCR结果一致。免疫组化结果显示，假手术组与溶剂对照组大鼠海马组织中aromatase主要表达于胶质细胞，aromatase阳性细胞数较少，Aβ损伤大鼠海马内aromatase阳性细胞数显著增加（P <0.01）。
3孕酮对Aβ损伤大鼠空间学习记忆能力的改善作用及机制
3.1孕酮对Aβ损伤大鼠空间学习记忆能力的改善作用
定位航行实验结果显示，各组大鼠逃避潜伏期均随学习天数增加而逐渐缩短。孕酮部分逆转Aβ损伤引起的逃避潜伏期增加，作用呈浓度依赖性。空间探索实验结果显示，孕酮浓度依赖性改善Aβ损伤大鼠平台区穿越次数减少及平台所在象限停留时间百分比减少的现象。同时，定位航行及空间探索实验显示，孕酮不影响正常大鼠的学习记忆能力。提示，孕酮特异性改善Aβ损伤大鼠的空间学习记忆能力。
3.2孕酮对Aβ损伤大鼠海马CA1区神经元形态及数量的影响
硫堇尼氏体染色结果显示，Aβ损伤大鼠海马CA1区域可见断裂的突起和不完整的胞体，细胞数减少，尼氏体减少。孕酮浓度依赖性改善Aβ损伤大鼠海马锥体细胞的损伤，增加其CA1区锥体细胞存活数（P <0.01）。
3.3孕酮对Aβ损伤大鼠海马组织炎症因子表达的影响
RT-PCR结果显示，Aβ损伤大鼠海马组织内炎性因子TNF-α及IL-1β表达显著增加（P <0.01）。孕酮可抑制Aβ损伤大鼠TNF-α及IL-1β表达上调的现象，作用呈浓度依赖性。Western blot结果与RT-PCR结果一致，即：孕酮抑制Aβ损伤大鼠海马组织内TNF-α及IL-1β表达上调。
4孕酮对Aβ所致原代培养海马神经元损伤的保护作用
4.1Aβ以浓度、时间依赖的方式降低原代培养海马神经元活性
MTT结果显示，不同浓度的Aβ作用于体外培养7天的海马神经元48h后，神经元活力下降，其中10μM及50μM Aβ使神经元活力显著下降（P <0.05，P <0.01）。10μMAβ作用于海马神经元不同时间后神经元活力下降，其中作用24h及48h后，细胞活力显著下降（P <0.05）。
4.2孕酮对抗Aβ的细胞损伤作用
MTT结果显示，10μM Aβ作用于神经元48h后，细胞活力显著下降（P <0.05）；孕酮可对抗Aβ引起的海马神经元活力下降，作用呈浓度依赖性。与Aβ损伤组相比，1μM孕酮处理组海马神经元活力显著增加（P<0.05）；与Aβ损伤组相比，0.5μM及1μM孕酮预处理组细胞活力显著增加（P <0.05，P <0.01）。
结论：
1双侧海马CA1区注射Aβ25-35损伤大鼠海马CA1区锥体细胞，引起大鼠空间学习记忆能力显著下降，Aβ所致学习记忆障碍大鼠模型制备成功。
2Aβ损伤大鼠脑组织中孕烯醇酮及孕酮含量降低，17β–雌二醇含量增加，同时，神经甾体合成代谢酶CYP11A1、3β-HSD、aromatase表达上调。推测，Aβ上调3β–HSD及aromatase表达，孕烯醇酮及孕酮分解加快，引起二者含量下降，其代谢产物雌二醇含量上升；机体通过增加神经甾体合成限速酶CYP11A1的表达代偿孕烯醇酮及孕酮含量的下降，Aβ促进神经甾体代谢向生成雌二醇的方向进行。
3外源性给予孕酮改善Aβ损伤大鼠的空间学习记忆能力，对抗Aβ损伤大鼠海马CA1区锥体细胞损伤，抑制Aβ损伤大鼠海马组织中的炎症反应。抑制炎症反应，发挥细胞保护作用可能是孕酮改善Aβ损伤大鼠学习记忆能力的机制之一。
4孕酮浓度依赖性对抗Aβ引起的原代培养海马神经元活力下降，从细胞水平进一步证实孕酮对Aβ损伤具有保护作用。
Alzheimer’s disease (AD), characterized clinically by progressivelycognitive impairment and pathologically by the appearance of senile plaquesand neurofibrillary tangles, is the most prevalent senile dementia. Amyloid β(Aβ), a β-sheet peptide fragment produced by the proteolytic cleavage ofamyloid precursor protein (APP) by γ-and β-secretases, is the majorcomponent of senile plaques. Both in vivo and in vitro studies have shown thataccumulation of Aβ fibrils trigger neurodegeneration, supporting the view thatAβ aggregation is of importance in AD. Aβ causes oxidative damage,inflammatory responses, and memory impairment, all of which may lead tothe neuronal dysfunction and degeneration responsible for the cognitivedeficits observed in AD.
Steroids hormones and their metabolites within the central nervoussystem (CNS) are commonly defined as neuroactive steroids or neurosteroids.They can be either synthesized de novo in the CNS by glial cells and neuronsfrom cholesterol or synthesized in the periphery by the adrenals and gonads.The concentration of neurosteroid was higher in CNS than in periphery.Neurosteroids mainly include pregnenolone (PREG), dehydroepiandrosterone(DHEA), their sulfate derivatives pregnenolone sulfate (PREGS) anddehydroepiandrosterone sulfate (DHEAS), and progesterone (PROG),5α-dihydroprogesterone (5α-DHP),3α,5α-tetrahydroprogesterone(allopregnanolone/ALLO), deoxycorticosterone (DOC),tetrahydrodeoxycorticosterone (THDOC), estradiol (E2), etc. Neurosteroidsplay an important role as endogenous modulators in altering neuronalexcitability rapidly; however, no specific receptor has been reported to datefor neurosteroids. Most of their actions in the nervous tissue were reported tomodulate membrane neurotransmitter receptors, such as GABAA, NMDA, and sigma1receptors, and thus affect neuronal plasticity, anxiety, responses tostressful stimuli, and neuropsychiatric symptoms represented during AD.Some neurosteroids were reported to improve learning and memory abilityand protect against Aβ peptide-induced neurotoxicity, thus exerting neuronalprotection. However, changes in the metabolic pathway of neurosteroidsduring AD and their role in Aβ-mediated impairment remain relativelyelusive given the limitations in sample sizes and analysis methods.
Objective: We have reported previously that the concentration ofprogesterone decreased in the media of Aβ25-35-induced cortical neurons, andprogesterone treatment inhibited the declination of cell viability as well as theapoptosis induced by Aβ25-35. The present study was designed to investigatethe levels of neurosteroids in encephalic regions related to learning andmemory (the prefrontal cortex and hippocampus) of Sprague-Dawley (SD)rats injected with Aβ25-35as well as the expression of neurosteroidogenesisenzymes. Furthermore, the effect of progesterone administration againstAβ25-35induced impairment was investigated in vivo and in vitro. The presentstudy provided a potential therapeutic strategy for disorders with learning andmemory impairment, such as AD.
Material and Methods:
1Effect of Aβ micro-injection on the spatial learning and memory of ratsTo investigate the effect of Aβ25-35administration on spatial learning andmemory of rats, a total of thirty SD rats were divided into three groups (shamgroup, vehicle group, and Aβ group) randomly with ten in each. Afteranesthetized with2%pentobarbital sodium (40mg/kg weight, intraperitonealinjection), the animals were placed in stereotaxic apparatus (Jiangwai typeⅠ).Five microliters aggregated Aβ25-35or vehicle (sterile distilled water, forvehicle group) were injected slowly over10min into bilateral CA1hippocampus (anterior/posterior:-3.5mm, media/lateral:±2.0mm, anddorsal/ventral:-2.7mm ventral to the skull surface) with a10μLmicrosyringe, followed by10min remaining to allow the diffusion ofinjection content. The rectal temperature was maintained at36℃to37℃for all animals throughout the surgery. Six-day Morris water maze behavioraltask was employed to test the spatial learning and memory of the animalssince the seventh day after surgery. After the behavioral test, all animals wereanesthetized with pentobarbital sodium and perfused through the ascendingaorta with normal saline followed by4%paraformaldehyde. The brain tissueswere then dissected and fixed for histological examination.
Behavioral Tests: The Morris water maze (JLBehv-MWM, Shanghai,China) consisted of a black pool (180cm diameter,60cm high) filled withwater (40cm,23±1℃), and was divided into four quadrants with fourequidistant release points around the edge, with a circular black platform (10cm diameter) submerging1cm below the water surface and remaining thesame position of the target quadrant. The behavior of rats in the pool wastracked with a camera which allowing us to measure the swim distance andtime to find the platform. Rats were trained four trials each day, with aninterval of20min at least, for five consecutive days. The starting quadrantwas randomized every day with all animals according to the same order. Theanimals were faced towards the pool wall before being released, and allowedto stay on the platform for10s after finding the platform within60s, orguided to the platform to stay for10s if failed to reach the platform in60s.The time needed to reach the platform (escape latency) was analyzed asindices of spatial learning. The probe trial was conducted on the sixth day,when each subject allowed90s to search the water from which the platformhad been removed. The number of times the animal crossed the platform(number of platform crossings) and time ratio in platform quadrant (Ⅳ timeratio) were used as index of spatial memory retention. A camera was used totrack the time to find the platform as well as to measure the swim speed anddistance travelled.
Histological Examination: The brain tissues were fixation in4%paraformaldehyde in phosphate buffer (pH7.4), dehydration in a series ofethanol, embedded in wax and sectioned into5-micrometer-thick sections.Tissue sections were then deparaffinized, dehydrated, and stained with thionin, followed by differentiation, dehydration and clearance before they weremounted. The number of intact pyramidal cells (per1mm linear length of thesame section of CA1hippocampus) was then counted by three experimentersand analyzed.
2The metabolism alteration of neurosteroids in the prefrontal cortex andhippocampus of Aβ treated rats
To investigate the effect of Aβ25-35on neurosteroids level in brain, a totalof ninety SD rats were divided into three groups (sham group, vehicle group(sterile distilled water), and Aβ group) randomly with thirty in each group andtreated the same as above. Ten rats in each group were sacrificed (viadecapitation) during16:00-18:00on the seventh day and the twelfth dayafter Aβ injection respectively, with the prefrontal cortex and hippocampusremoving (on ice) and stored in-70℃until neurosteroids detectionthroughhigh performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. On the twelfth day after Aβ injection, five rats in eachgroup were sacrificed (via decapitation) with hippocampus removed (on ice)and stored at-70℃for steroidogenesis enzymes analysis through RT-PCR,Western blot, and immunohistochemisty. The rests were anesthetized withpentobarbital sodium and perfused through the ascending aorta with normalsaline followed by4%paraformaldehyde. The brain tissues were thendissected and fixed for histological examination.
Sample Preparation and HPLC-MS/MS Analysis: Samples (about100mg) were added with10μL30ng/mL MT (internal standards), homogenizedin1mL of phosphate buffered saline (PBS) using an ultrasonic homogenizer(Heidolph, Germany), mixed with2mL ethyl acetate/n-hexane (9:1, v/v)and centrifuged at12000rpm for10min. The pellet was extracted three timeswith2mL ethyl acetate/n-hexane (9:1, v/v), and the organic phases werecombined and dried with a gentle stream of nitrogen in a50℃water bath.The samples were derivatized with dansyl chloride in a60℃water bath for40min, concentrated at12000rpm for10min and then transferred inautosampler vials before the high performance liquid chromatography- tandem mass spectrometry (HPLC-MS/MS) analysis.
The HPLC-MS/MS system (Thermo Fisher Scientific, Waltham, MA,USA) consisted of Surveyor MS Pump Plus, Surveyor AS Plus, TSQQuantum Access triple quadrupole mass spectrometer and Xcalibur DataSystems (Thermo Fisher Scientific). Separation was achieved on a XDB C18analytical column (4.6mm×50mm, Agilent, Palo Alto, CA, USA) fittedwith a XDB C18guard column (4.6mm×12mm, Agilent). The HPLC mobilephases were (A) H2O/0.1%formic acid and (B) MeOH/0.1%formic acid,and the flow rate was0.5mL/min. The gradient was as follows:0-6.5min，36%A;6.5-6.6min,30%A;6.6-17min,10%A;17-18min,36%A.The column temperature was40℃and the injection volume was30μL. AMSD quadrupole mass spectrometer equipped with atmospheric pressurechemical ionization (APCI) source (Thermo Fisher Scientific) was used forthe detection of analytes in the positive ion mode. The optimized conditionswere as follows: the spray voltage,4500V; the vaporizer temperature,400℃; the sheath gas,30L/min; the aux gas,5L/min; the capillary temperature,270℃. The quantification was performed using multiple-reaction monitoring(MRM) method with the transitions of (m/z)299.03→(m/z)158.86and280.9for PREG,(m/z)315.03→(m/z)97and109.01for PROG,(m/z)301→(m/z)188.9and282.85for ALLO,(m/z)254.97→(m/z)132.9and158.9for17β-E2,(m/z)303.1→(m/z)97.04and109.06for MT, respectively.
RT–PCR: Total RNA was isolated from cells using TRIzol Reagent(Invitrogen, CA, USA) and equal amounts were reverse transcribed intocDNA using the oligo dT primer, then the cDNAs were used as DNAtemplates for PCR. GAPDH was used to ensure equal loading. The PCRproducts were separated on1.5%agarose gel and visualized by ethidiumbromide.
Western Blot: Lysates from brain tissue were prepared with lysis buffer(1%Triton X-100,150mM NaCl,10mM Tris-HCl, pH7.4,1mM EDTA,pH8.0,0.2mM Na3VO4,0.2mM phenylmethylsulfonyl fluoride, and0.5%NP-40). Equal amounts of protein (40μg) were separated by10%SDS- PAGE, and electrotransferred to a PVDF membrane (Millipore, Billerica, MA,USA). Membranes were blocked with5%BSA for2h at room temperature,and incubated with the primary antibodies,1:1000mouse anti-TNF-α(Abcam),1:500rabbit anti-IL-1β (Abcam),1:500rabbit anti-CYP11A1(anbobio,),1:500rabbit anti-3β-HSD (Epitomics),1:500rabbit anti-aromatase (Abcam), and1:5000rabbit anti-β-actin (Santa Cruz) overnight,respectively, and then with the respective secondary antibody for2h. Proteinswere detected using the Chemiluminescence Plus Western blot analysis kit(Santa Cruz). The experiments were replicated three times.
Immunohistochemistry: Five-micrometer-thick sections weredeparaffinized, dehydrated, followed by Antigen retrieval (microwave). Thensections were in turn incubated with3%H2O2for10min, with primaryantibody overnight at4℃, with diluted biotinylated IgG secondary antibody,with DAB, followed by being differentiated, dehydrated, cleared, andmounted.
3The effect of progesterone on behavioral and cellular impairment in Aβtreated rats
To investigate the effect of PROG on cognition impairment induced byAβ25-35CA1hippocampus injection, a total of fifty SD rats were dividedrandomly into five groups: vehicle group (sterile distilled water), Aβ group, PLgroup (Aβ+progesterone4mg/kg weight), PMgroup (Aβ+progesterone8mg/kg weight), and PHgroup (Aβ+progesterone16mg/kg weight). During6-12days after Aβ injection described above, animals were injected withdifferent dosages of progesterone (PL,PM,and PHgroup) or vehicle (sesame oilfor vehicle group and Aβ group) daily at13:30. Behavioral tests wereconducted during7-12days after Aβ injection beginning at14:30.Immediately after behavior tests, five rats in each group were sacrificed (viadecapitation) with hippocampus removed (on ice) and stored at-70℃forinflammatory factors analysis through RT–PCR and Western blot. The restanimals were anesthetized with pentobarbital sodium and perfused through theascending aorta with normal saline followed by4%paraformaldehyde. The brain tissues were dissected and fixed for histological examination.
Behavioral tests, RT–PCR, Western blot and histological examinationwere conducted as mentioned above.
4The effect of PROG on Aβ25-35induced cell impairment in vitro
Hippocampus cells of newborn SD rat were cultured and treated asdescribed. In brief, bilateral hippocampus newborn rat were dissociated with0.125%trypsin/HBSS for15min at37℃, followed by trypsin quenchingwith DMEM-HG containing10%FBS. Cell suspensions were centrifuged(5min,200×g), resuspended, dissociated by repeated passage through a fire-polished Pasteur pipette, and then filtered through a sterile cell strainer. Thedissociated hippocampus cells plated on poly-L-lysine (Sigma,50μg/mL)coated96-well plates at a density1×106cells/mL were grown in Neurobasalmedium-A supplemented with5units/mL penicillin,5mg/mL streptomycin,1%GlutaMAX I and2%B27neuromix at37℃in humidified5%CO2/95%air atmosphere for7days to9days before experimentation.
The cultures were treated with or without Aβ (1μM,10μM, or50μM)for48h, or10μM Aβ for12h,24h, or48h. In the following experiment,cultures were pre-treated with or without PROG (0.1μM,0.5μM, or1μM)for60min followed by exposure to10μM aggregated Aβ for48h, or treatedsimultaneously with different dosages of PROG and Aβ for48h. Cellviability was assessed by MTT analysis. The results were presentedgraphically as a percentage of live cells in the control group.
Assessment of Cell Viability: Loss of cell viability was measured by theMTT (methyl thiazolyl tetrazolium) assay. In brief,2×105cells per well wereseeded in triplicate onto96-well plates in Neurobasal-A medium and allowedto grow for7days. The cells were treated according to the experimentaldesign. After drug treatment, cells were incubated with5mg/mL MTT for2h,and subsequently solubilized in dimethyl sulfoxide (DMSO). The absorbencyat570nm was then measured using ELIASA (BMG Labtech, Germany). Theresults were expressed as the mean of the absorbance relative to the controlgroup. The experiments were replicated three times.
5Statistical Analysis
The data were expressed as mean±standard error of the mean (SEM).The mean escape latency of the Morris water maze behavioral tests collectedduring the training days were analyzed by repeated-measure analysis ofvariance (ANOVA). Other data were statistically analyzed by one-wayANOVA, followed by between-group comparisons using LSD post hoc test.Statistical significance was concluded with a value of P <0.05for all analyses.Data analyses were performed with the SPSS software version13.0.
Results:
1Effect of Aβ micro-injection on the spatial learning and memory of rats
The Morris water maze behavioral task was employed to examinehippocampus-dependent spatial learning and memory. The mean swimspeeds did not differ across the groups. The mean escape latency in the Morriswater maze behavioral task was significantly (P <0.01) higher in Aβ group(47.9±2.3,39.1±3.0,37.4±2.9,33.3±2.6, and29.4±3.0; Day1to Day5,respectively) compared with those of the vehicle group (47.3±1.3,28.6±2.7,21.1±2.7,15.8±1.9, and12.8±1.4; Day1to Day5, respectively.).Moreover, the number of platform crossings (48.7%) and the staying timeratio in platform quadrant (32.7%) of Aβ group in the probe test wereconsiderably (P <0.01) lower compared with those of vehicle group, thusconfirming spatial learning and memory impairment in Aβ25-35treated rats.Overall, no difference was observed between sham and vehicle group.
Histological examination showed that Aβ25-35significantly (P <0.01)decreased (37.6%) the intact pyramidal cell number in the CA1hippocampuscompared with the vehicle group.
2The metabolism alteration of neurosteroids in the prefrontal cortex andhippocampus of Aβ treated rats
High performance liquid chromatography-tandem mass spectrometrywas employed to simultaneously quantify the level of neurosteroids indifferent encephalic region. Levels of neurosteroids were altered in theprefrontal cortex and hippocampus of Aβ treated rats7and12days after surgery. Levels of PREG in the hippocampus of Aβ group were significantly(P <0.05) lower (66.1%and70.8%)7days and12days after surgerycompared with those of vehicle group, while levels of PREG in the prefrontalcortex did not differ compared with those of vehicle group on either timepoint. Furthermore, levels of PROG were significantly (P <0.01) reduced inboth the prefrontal cortex and hippocampus (63.8%and61.8%for7daysand12days after surgery in the prefrontal cortex;44.3%and42.2%for7days and12days after surgery in hippocampus, respectively) compared withthose of vehicle group. However, the hippocampus of Aβ treated ratscontained significantly (P <0.05) elevated (146.2%and137.9%) levels of17β-E27days and12days after surgery compared with those of vehiclegroup, while the prefrontal cortex of Aβ treated rats contained significantly (P<0.05) higher (171.8%) levels of17β-E2only on7days after surgerycompared with those of vehicle group. The level of ALLO did not differ ineither brain regions on both time points. The level of PREG, PROG and17β-E2did not differ between sham and vehicle group in either brain region onboth time points.
The expression of neurosteroidogenesis enzymes was altered inhippocampus of Aβ treated rats. RT-PCR showed that gene expression ofCYP11A1,3β-HSD, and aromatase in hippocampus were significantly (P <0.01) higher in Aβ group (149.0%,247.6%, and159.5%) compared withthose of vehicle group. The up-regulation of CYP11A1,3β-HSD, andaromatase were confirmed when the protein level of CYP11A1,3β-HSD,and aromatase were significantly (P <0.01) increased in Aβ group (142.3%,219.9%, and145.0%) compared with those of vehicle group, showed byWestern blot. Results of immunohistochemisty analysis confirmed the up-regulation of aromatase in hippocampus of Aβ treated rats (113.6%, P <0.01,compared with vehicle group).
3The protective effect of progesterone against the behavioral and cellularimpairment in Aβ treated rats
Administration of PROG enhanced the cognitive performance of Aβ treated rats in a dose-dependent manner (4mg/kg,8mg/kg,16mg/kg for PL,PM, PHgroups, respectively). The mean swim speeds did not differ across thegroups. The mean escape latency of PL, PMand PHgroups were significantly(P <0.05for PL; P <0.01for PMand PH) lower compared with Aβ group. Therestoration of cognitive impairment was confirmed when the number ofplatform crossings of the PMand PHgroup was significantly increased by170.6%(P <0.05) and217.7%(P <0.01), compared with those of Aβ group.Meanwhile, the Ⅳ time ratio of PL, PM, and PHgroup was significantly (P <0.01) increased by170.6%,190.13%, and217.7%, compared with Aβgroup.
PROG reversed Aβ25-35-induced cell loss in CA1hippocampus dosedependently. Compared with vehicle group, the intact pyramidal cell numberof Aβ group significantly (P <0.01) decreased by35.9%. Compared with Aβgroup, the intact pyramidal cell number of PL, PM, and PHgroups weresignificantly (P <0.01) increased by130.9%,190.1%, and241.3%,respectively.
On the cellular level, PROG reversed Aβ25-35injection induced up-regulation of TNF-α and IL-1β. RT-PCR showed that that gene expressionof TNF-α and IL-1β in hippocampus were significantly (P <0.01) higher inAβ group (176.4%and176.4%) compared with those of vehicle group. Theup-regulation of TNF-α and IL-1β were confirmed when the protein levelof TNF-α and IL-1β were significantly (P
p (162.7%and158.3%) compared with those of vehicle group, showedby Western blot. The gene expression of TNF–α (83.6%,71.9%, and55.5%for PL, PM, and PHgroups, P <0.01, respectively) and IL-1β (90.9%,80.2%, and67.5%for PL(P <0.05), PM(P <0.01), and PHgroups (P <0.01),respectively) in hippocampus were significantly lower in the PROG-treatedgroup compared with Aβ group. The down-regulation of TNF-α and IL-1βby PROG were confirmed when the protein level of TNF–α (86.4%,73.8%,and58.1%for PL, PM, and PHgroups, P <0.01, respectively) and IL-1β(91.9%,81.8%, and69.6%for PL(P <0.05), PM(P <0.01), and PHgroups (P <0.01), respectively) were significantly decreased in the PROG-treatedgroup compared with Aβ group, showed by Western blot.
4The protective effect of progesterone against Aβ25-35-induced culturedhippocampus cells viability decrease in vitro
The MTT assay was used to evaluate the viability of culturedhippocampus cells. Aβ25-35decreased cell viability dose and time dependently.Compared with vehicle group, the cell viability of Aβ treated group (48h)significantly (P <0.05for Aβ10μM, and P <0.01for Aβ50μM) decreasedby66.6%and55.4%. Compared with vehicle group, the cell viability of Aβtreated group (10μM) significantly (P <0.05for Aβ24h, and P <0.01forAβ48h) decreased by68.2%and66.9%. Meanwhile, PROG protectedagainst Aβ25-35-induced impairment dose dependently. PROG (1μM)treatment simultaneously with Aβ25-35significantly (P <0.05) reversed Aβ25-35-induced cell viability decrease. Moreover, PROG treatment1h prior toAβ25-35incubation significantly (P <0.05for PROG0.5μΜ, and P <0.01forPROG1μM) reversed Aβ25-35-induced cell viability decrease, confirming theprotection of PROG against Aβ25-35-induced cultured hippocampus cellsviability decrease in vitro.
Conclusions:
1Injection of Aβ25-35into the CA1hippocampus sub-region impairedthe spatial learning and memory of rats. Aβ-induced learning and memeoryimpairment model was successfully established.
2The present study showed, for the first time, that injection of Aβ25-35into the CA1hippocampus sub-region of rats decreased levels of PREG andPROG and increased the level of17β-E2in the prefrontal cortex andhippocampus simultaneously quantified by HPLC-MS/MS. Moreover, theexpression of neurosteroidogenesis enzymes CYP11A1,3β-HSD, andaromatase was up-regulated in Aβ treated rats. Increased expression of3β-HSD and aromatase accelerated the metabolism of PREG and PROG thusreduced the levels of PREG and PROG and increased the level of E2.Meanwhile, the expression of CYP11A1was up-regulated to compensate the decreased level of PREG and PROG.
3Administration of PROG reversed the up-regulation of pro-inflammatory cytokines TNF-α and IL-1β, as well as the behavioral andcellular impairment of Aβ treated rats, thus exerting neuronal protection.
4PROG protectived against Aβ25-35-induced cells viability decrease ofcultured hippocampus neurons in vitro, comfirming the protection of PROGagainst Aβ25-35-induced neural impaiment.
引文
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