一：iNOS在高脂饮食诱导的学习记忆缺失中的作用及机制研究 二：白藜芦醇苷对鱼藤酮诱导帕金森病的保护作用及机制研究
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摘要
目的：认知功能障碍的发病率随年龄增长而升高,在当今老龄化日趋明显的社会,其已成为临床的常见症状。认知功能损伤严重者即为痴呆。据统计,目前全球痴呆患者约有2400万,其中逾半为阿尔茨海默病(Alzheimer's disease, AD),AD是老年期认知功能障碍最主要的病症。AD是一种以进行性认知障碍为主的中枢神经系统退行性疾病,其病理特征为老年斑(Senile plaque, SP)形成、神经原纤维缠结(Neurofibrillary tangle, NFT)和大量神经元丢失。理想的AD动物模型,对研究AD的发病机制及治疗具有重要意义。目前实验中常用的AD动物模型的建立途径有很多,但主要可归为以下三类：即以模拟AD症状学特征为主的动物模型,模拟AD组织病理特征为主的动物模型和多因素复合动物模型。AD动物模型的研究经过几十年的努力取得了很大的进展,每一种模型都在一定的程度或某些方面模拟了AD的症状和病理改变；但是至今尚无一种能够真实反映各个病例发展阶段特征并且广泛普及的动物模型。美国杜克大学医学院Colton CA.2006年首次提出APP单转基因小鼠再次敲除iNOS基因会导致病理样的Tau蛋白过度磷酸化,并再次分配到皮层和海马神经元的胞体树突室,聚集累积。缺乏iNOS基因的瑞典APP突变小鼠增加了不溶性淀粉样肽的水平、神经元变性、caspase-3的活化和Tau蛋白裂解；这表明NO是淀粉样肽、半胱天冬酶激活和Tau聚集的连接点。Colton CA.首次提出这一理论的时候,遭到很多人的质疑。iNOS基因定位于第17对常染色体上,只有在细胞受到刺激被激活后才表达,iNOS产生的NO能持续相对较长时间(几小时到几天)。NO是重要的细胞内和细胞间的信号调节分子,在人体的生理和病理过程中具有双重性。NO既是一个有细胞毒性效应器作用的分子,是组织损伤的诱发因子和各种病变的增强因子,有其病理上不好的一面；又是生物体许多部分的信号分子,是先天性免疫应答的调节性效应分子,作为一种信息传递物质维持正常的生理功能,有其生理性好的一面。在啮齿动物的研究中,提示iNOS活动在实验性自身免疫性或慢性炎症过程及其他一些疾病中可能起了有害作用。然而iNOS在很多疾病中的具体作用还不是很明确,尚需进一步深入研究。本课题目的在于研究iNOS跟学习记忆的关联；研究在长期高脂饮食诱导下iNOS在学习记忆中的作用及相关机制。
方法：3月龄的i(?)(?)OS基因敲除小鼠(iNOS knockout mice, iNOS-/-)和相同背景(背景鼠为C57BL/6小鼠)的同窝出生的野生型小鼠(wild type, WT)饲养一周以适应环境,转基因小鼠PCR鉴定基因型后筛选使用。iN(?)OS基因敲除小鼠和野生型小鼠分别喂养正常饲料(Chow)和高脂饲料(High fat cholesterol, HFC)。喂养20周后首先对各组小鼠进行焦虑行为的检测,包括旷场实验、高架十字迷宫实验和明暗箱实验。在焦虑行为学检测完之后,进行记忆行为的检测,包括Morris水迷宫实验和条件性恐惧实验。与此同时对各组小鼠进行了腹腔注射糖耐量实验(Intraperitoneal glucose tolerance test, IPGTT),所有小鼠禁食16小时后,称量体重,剪尾采血测定空腹血糖值。然后按2g/kg的剂量腹腔注射葡萄糖,分别在0、15、30、60、90、120min,用微血糖测量仪及配套的血糖试纸检测各时间段的血糖值,观察高脂饮食对糖耐量的影响。活体行为学和IPGTT测试结束后,各组部分小鼠,按50mg/kg的剂量腹腔注射伊文思蓝染料(Evan blue dye, EBD),三小时后,用抗凝管取血,迅速断头,粘去脑膜,分离出左、右脑半球和小脑,以及心脏,肝脏,肾脏,脾,和小鼠大腿肌肉组织,甲酰胺提取染料；并取血获得血浆。检测各组织和血浆中EBD的浓度,评价血脑屏障通透性以及EBD在血液组织中的分布情况。还有部分小鼠在行为学测试结束后：(1)心脏灌流后取出大脑做冰冻切片进行GFAP和Aβ40免疫荧光染色；(2)断头处死后,分离出半侧前额皮层和海马用组织裂解液提取蛋白,完成蛋白浓度测定后用Western blotting检测Akt信号分子和Tau蛋白的水平。另外半侧前额皮层和海马组织样品通过Real-time PCR检测炎症因子白细胞介素-1β(IL-1β)、肿瘤坏死因子α (TNF-α)基因表达水平的变化,以及星形胶质细胞中GFAP的表达情况。
结果：(1)焦虑行为：通过旷场实验、高架十字迷宫实验和明暗箱实验对各组小鼠进行焦虑行为的检测结果显示iNOS基因敲除和长达20周的高脂饮食对小鼠并不会产生焦虑行为,也不影响其自主活动性。
(2)记忆行为：在无焦虑行为的状态下对各组小鼠进行了记忆行为的检测。Morris水迷宫：1)5天习得性训练实验中各组小鼠差异不显著,这进一步说明iNOS基因敲除和长达20周的高脂饮食并不影响小鼠的视力和游泳运动能力。2)10天探索性实验中随着测试天数的增加,各组动物寻找到平台的时间都有不同程度地缩短。第1-5天里,各组间的差异不是很明显,但喂高脂饲料的iNOS基因敲除小鼠寻找平台潜伏期的趋势比其他各组稍微慢点。第6天之后,iNOS-/-HFC组的逃避潜伏期与WT-Chow正常组比,显著升高,这说明高脂饮食的iNOS基因敲除小鼠空间学习记忆能力出现一定程度的损伤。在第6天,iNOS组与iNOS-/-Chow组比较,其逃避潜伏期显著升高,说明高脂饮食会进一步加重iNOS基因敲除小鼠学习记忆的损伤。喂养高脂饮食的WT小鼠跟WT-Chow正常组比,尽管统计上没有显著差异,但喂养高脂饲料的WT小鼠的逃避潜伏期提示高于正常小鼠。
条件性恐惧实验：第一天对各组小鼠进行条件恐惧性训练,在受到电刺激后,各组小鼠恐惧不动的时间都有所增高,这表明各组小鼠对恐惧都有比较同步的反应能力。第二天对小鼠进行环境恐惧恢复测试的结果表明,与正常组小鼠相比,喂养高脂饮食小鼠的空间记忆能力显著下降；iNOS基因敲除后,其空间记忆能力也显著下降；其中喂高脂饮食的iNOS基因敲除小鼠空间记忆能力下降最为显著,且比喂高脂饮食的WT小鼠的空间记忆能力还要差。这一结果表明iNOS敲除后加剧了高脂饮食对学习记忆的损伤。声音恐惧恢复测试中各组间并未见显著性差异。
(3) IPGTT实验：结果表明各组小鼠在腹腔注射2g/kg葡糖糖后,血糖迅速升高,其中WT-Chow和iNO-/-Chow组小鼠血糖在15mmin达到峰值。而WT-HFC和iNOS-/-HFC组小鼠血糖在30mmin才达到峰值,且显著高于WT-Chow和iNOS-/-Chow组。2小时后,WT-Chow和iNOS-/-Chow组小鼠的血糖都趋于正常水平;WT-HFC和iNOS-/-HFC组小鼠的血糖仍呈现较高的水平(>150mg/dL),且峰时后延,这表明喂高脂饮食的WT和iNOS-/-小鼠都存在腹腔注射糖耐量异常。喂高脂饮食的iNOS-/-小鼠的血糖值总体都低于同喂高脂饮食的WT小鼠,特别是在第30min和90min;120min后血糖值基本趋于正常水平。由此推测,在类似糖尿病的病理条件下,iNOS基因的敲除有助于恢复糖耐量的异常。
基于IPGTT的检测结果,我们进一步检测了前额皮层和海马中磷酸化Akt和总的Akt水平分析其可能的机制。结果显示WT-Chow和WT-HFC组小鼠在前额皮层中p-Akt的基线水平都比较低。有趣的是,WT-HFC组小鼠p-Akt在海马的表达水平也比喂养正常饲料的WT小鼠低,这也许就可以解释为什么在IPGTT中WT-HFC组小鼠的糖耐量异常,且选择性地降低小鼠海马组织的p-Akt水平。iNOS基因敲除后的小鼠,无论是喂养正常饲料还是高脂饲料,其前额皮层中p-Akt的水平都比WT小鼠高,这说明iNOS基因敲除后有助于Akt信号分子的激活。更有趣的是,iNOS-/-小鼠在喂养高脂饲料后,其前额皮层和海马组织中的p-Akt水平跟WT-HFC组和iNOS-/-Chow组小鼠比都显著增高。因此,我们得出结论,iNOS基因敲除后有助于糖耐量异常的改善,且可能是通过激活Akt信号分子而使血糖下降,这一推测跟上面IPGTT的结果一致。
(4)腹腔注射EBD实验结果：喂养高脂饲料的WT小鼠其左右脑半球的EBD浓度与WT-Chow组比,显著升高,这说明高脂饮食会造成血脑屏障的损伤。EBD在左右脑半球的浓度差别不大,但是小脑的EBD浓度比较高。喂高脂饲料的iNOS基因敲除小鼠脑内EBD的浓度与WT-HFC组小鼠比,显著降低。这说明iNOS基因敲除后有助于高脂饮食诱导的血脑屏障障碍的恢复重建。与此同时,我们还观察到iNOS基因在肾脏具有同样类似的作用。但是不影响其他组织器官如脾、肝脏、心脏和肌肉的血管渗透性。为了确保各组小鼠对当量浓度的EBD都能吸收进血液循环,及腹腔注射的泄漏程度,我们检测了血浆中EBD的浓度。结果表明各组小鼠血浆中EBD的浓度没有显著差异。
(5)免疫炎症实验：检测结果显示长期高脂饮食会诱导炎症的发生,WT-HFC组小鼠与WT-Chow组小鼠比,其前额皮层和海马组织中IL-1p和TNF-α的基因水平都比较高,特别是在前额皮层组织。iNOS基因敲除后并不影响自身的免疫系统,因iNOS-/-Chow组小鼠与WT-Chow组小鼠比,其炎症因子IL-1β和TNF-α的基因水平变化不明显。喂高脂饲料的iNOS基因敲除小鼠,其前额皮层和海马组织中炎症因子IL-1β和TNF-α的基因水平与其他各组比都高,惊奇的是比WT-HFC组小鼠还高,尽管显著性差异不是很明显,仅TNF-α在前额皮层中的含量有差异。由此我们推测,在病理条件下(高脂饮食),iNOS基因敲除后会诱导增加其他炎症因子的水平,这可能是一种代偿反应。
(6)星形胶质细胞中GFAP表达情况：WT-HFC组小鼠与WT-Chow组小鼠比,其前额皮层和海马组织中GFAP的基因水平都显著增高。iNOS-/-Chow组小鼠与WT-Chow组小鼠比,GFAP的基因水平变化不明显；这表明iNOS基因敲除后并不影响自星型胶质细胞在前额皮层和海马中的数量。喂高脂饲料的iNOS基因敲除小鼠,其在前额皮层中GFAP的基因水平与其他各组比都高,且比WT-HFC组小鼠还高,尽管显著性差异不是很明显。由此我们推测,在病理条件下(高脂饮食),iNOS基因敲除后会进一步诱导GFAP表达的上调,从而加重AD样表型,损伤小鼠的记忆行为,这与前面的记忆行为学检测结果一致。在此基础上,我们对各组脑组织进行GFAP免疫荧光染色,结果与GFAP mRNA在前额皮层和海马中的表达情况一致。喂高脂饲料的iNOS基因敲除组小鼠其GFAP的荧光强度比其他各组高,星型胶质细胞的密度增加。
(7)AD样表型的检测(Tau和Aβ40)：检测各组小鼠前额皮层和海马中磷酸化Tau蛋白(p-Tau(AT8))和总Tau蛋白(Tau46)的蛋白表达水平。结果显示WT-HFC组小鼠与WT-Chow组小鼠比,其前额皮层和海马组织中磷酸化Tau与总Tau蛋白比的相对水平都显著增高。iNOS-/-Chow组小鼠与WT-Chow组小鼠比,p-Tau和Tau的比值变化不明显；但与WT-HFC组比显著降低,由此推测在正常生理状态下iNOS基因敲除后并不会诱导异常磷酸化的Tau蛋白的高表达。喂高脂饲料的iNOS基因敲除小鼠,其在前额皮层中p-Tau蛋白的相对水平与其他各组比都高,惊奇的是比WT-HFC组小鼠还高,呈显著性差异；在海马中的变化不是很显著。由此我们推测,在病理条件下(高脂饮食),iNOS基因敲除后会进一步诱导异常磷酸化的Tau蛋白主要在前额皮层表达的上调,从而加重AD样表型,损伤小鼠的记忆行为,这与前面的记忆行为学检测结果一致。免疫荧光法对各组小鼠前额皮层和海马中Aβ40的表达进行检测的结果显示高脂饮食和iNOS基因敲除都有诱导Aβ40表达增加的趋势。
结论：iNOS基因敲除和长达20周的高脂饮食不会使小鼠产生焦虑行为,也不影响其自主活动性,小鼠的视力和游泳运动能力也良好。在排除了焦虑行为干扰的情况下,高脂饮食的iNOS基因敲除小鼠其空间学习记忆能力仍有一定程度的损伤。iNOS敲除后加剧了高脂饮食对学习记忆的损伤,且依赖于海马组织,具有一定的组织选择性。高脂饮食是糖耐量异常的风险因素之一,IPGTT检测结果显示喂高脂饮食的WT和iNOS-/-小鼠都存在腹腔注射糖耐量异常。在类似糖尿病的病理条件下,iNOS基因的敲除有助于恢复糖耐量的异常。基于这一现象,我们探讨了其发生的可能机制,iNOS基因敲除后有助于Akt信号分子的激活。由此推测iNOS基因敲除后对糖耐量异常的改善,有可能是通过激活Akt信号分子而使血糖下降,这一推测跟上面IPGTT的结果一致。EBD实验结果证实高脂饮食会造成血脑屏障的损伤,iNOS基因敲除后有助于高脂饮食诱导的血脑屏障障碍的恢复重建。iNOS基因在肾脏具有同样类似的作用,但是不影响其他组织器官如脾、肝脏、心脏和肌肉的血管渗透性和血液的吸收和分布功能。长期的高脂饮食会诱导炎症的发生,其前额皮层和海马组织中IL-1β和TNF-α的基因水平都比较高,特别是在前额皮层组织。iNOS基因敲除后并不影响自身的免疫系统,但在病理条件下(高脂饮食),iNOS基因敲除后会诱导增加其他炎症因子的水平,这可能是一种代偿反应。iNOS基因敲除后并不影响自身星型胶质细胞在前额皮层和海马中的数量;在病理条件下(高脂饮食),iNOS基因敲除后会进一步诱导GFAP表达的上调,从而加重AD样表型,损伤小鼠的记忆行为。在正常生理状态下iNOS基因敲除后并不会诱导异常磷酸化的Tau蛋白的高表达；在病理条件下(高脂饮食),iNOS基因敲除后会进一步诱导异常磷酸化的Tau蛋白主要在前额皮层表达的上调,从而加重AD样表型,损伤小鼠的记忆行为,这与前面的记忆行为学检测结果一致。免疫荧光法对各组小鼠前额皮层和海马中Aβ40的表达进行检测的结果显示高脂饮食和iNOS基因敲除都有诱导Aβ40表达增加的趋势。本课题细致的探讨了iNOS在高脂饮食诱导的学习记忆缺失中的作用及可能的机制,这为建立一种新的比较理想的AD动物模型提供了可能。
目的：应用神经毒素鱼藤酮(Rotenone)建立稳定的帕金森病(Parkinson's disease, PD) SD大鼠模型,观察白藜芦醇苷对PD的神经保护作用,并在此基础上对其作用机制进行探讨。方法：采用大鼠背部皮下注射鱼藤酮(2.0mg·kg-1·d-1)持续5周以诱导其多巴胺神经元丢失为PD SD大鼠模型。SD大鼠随机分为6组：空白对照组,模型组(仅注射鱼藤酮),鱼藤酮+左旋多巴(10mg·kg-1·-1)组,鱼藤酮+白藜芦醇苷低、中、高剂量(20,40,80mg·kg-1·d-1)组,左旋多巴和不同剂量的白藜芦醇苷均通过口服给药,持续给药5周。通过Grid test、Bar test、 Rotarod test对大鼠进行僵直程度、运动障碍和肌肉活性的行为学检测;比色法测定大脑皮层、海马、纹状体中超氧物歧化酶(SOD)、谷胱甘肽(GSH)活力、丙二醛(MDA)含量及ATP水平；采用Western blot法测定大脑皮层、海马、纹状体中硫氧还蛋白(Trx)的表达。结果：低剂量长期背部皮下注射鱼藤酮可诱导SD大鼠出现类似PD的行为学表现,呈进行性僵直行为的增加,降低运动协调和肌活性。白藜芦醇苷剂量和时间依赖性的改善鱼藤酮诱导的行为障碍。在第五周,高剂量80mg/kg的白藜芦醇苷显著降低鱼藤酮诱导的僵直,并增加大鼠的运动协调性和肌活性。与模型组相比,各给药组纹状体中SOD、GSH活力和ATP含量均显著增加,MDA含量明显减少,Trx的免疫活性增加,并呈现一定的剂量依赖性。鱼藤酮对大鼠的损伤有一定的选择性,主要表现为黑质纹状体中多巴胺能神经元的丢失。结论：白藜芦醇苷对鱼藤酮诱导的帕金森病具有一定保护作用,此作用与其增加体内抗氧化酶的活性,减少过氧化脂质的生成,上调硫氧还蛋白系统中Trx的表达密切相关,并呈一定的剂量依赖性。
Objective:The incidence of cognitive dysfunction increases with age, it become a very common clinical symptoms in today's aging society. Severe cases of cognitive impairment were called dementia. According to statistics, the current global dementia patients is about24million, of which more than half are Alzheimer's disease (AD). AD is the most usual disease of old age cognitive dysfunction. AD is a progressive cognitive impairment of central neurodegenerative diseases, its pathology is characterized by the formation of senile plaques (SP), neurofibrillary tangles (NFT) and a large number of neurons lost. Ideal AD animal model is very important to study the pathogenesis of AD and the treatment. There are many ways to establish the AD animal models. They mainly can be grouped into the following three categories: simulate the characteristics of AD symptoms model, simulate histopathological features of AD model and multi-factor composite animal model. After decades of hard work AD animal model studies has made a lot of progress. Each animal model simulates a certain degree, or some aspects of the symptoms and pathological changes of AD. But as yet no one can truly reflect the various cases of stage of development characterized and widely popular animal model. Colton CA. in Duke University School of Medicine first proposed that genetic removal of iNOS in mice expressing mutated amyloid precursor protein results in pathological hyperphosphorylation of mouse tau, its redistribution to the somatodendritic compartment in cortical and hippocampal neurons, and aggregate formation. Lack of iNOS in the amyloid precursor protein Swedish mutant mouse increased insoluble β-amyloid peptide levels, neuronal degeneration, caspase-3activation, and tau cleavage, suggesting that NO acts at a junction point between β-amyloid peptides, caspase activation, and tau aggregation. Lot of people questioned this theory when Colton CA first proposed. iNOS gene is located on the17pairs of autosomes. It only can be expressed when the cells were stimulated and activated. NO, the production of iNOS can last a long time about several hours to days. NO is an important intracellular and intercellular signaling regulating molecular. It has a dual nature in human physiological and pathological processes. NO, a cytotoxic effector molecule is a tissue damage enhancement factor and predisposing factors of variety of lesions. This is the pathology bad side. In physiological good side, NO is also can be a signaling molecules in many organisms, innate immune response regulatory effector molecules and as an information material to maintain normal physiological function. In rodents experiments suggesting that iNOS activity play a harmful role in experimental autoimmune or chronic inflammatory processes, and several other diseases. But the detial of iNOS specific roles in many diseases are still unknown; it needs to be further in-depth study. The purpose of this project is to study the relationship of iNOS with learning and memory; study the effect and mechanism of iNOS on learning amnesia induced by high-fat diet.
Methods:3-month-old iNOS gene knockout mice (iNOS-/-) and the same background (C57BL/6) with littermate wild-type mice (WT) were rearing to adapt to the environment one week. PCR was carried out to genotype the transgenic mice before the experiment. iNOS knockout mice and WT mice were fed with chow diet (Chow) and/or high-fat diet (HFC). After20weeks feeding mice in each group were did anxiety behavior test, including open field test, the elevated plus maze, and light dark box test. Memory behavior test were conducted after anxiety behavior, including the Morris water maze and fear condition test. At the same time for each group of mice did intraperitoneally tolerance test (IPGTT). All mice were fasted for ahout16hours and weighed, injected2g/kg dose of glucose. After0,15,30,60,90and120min, using micro-blood glucose meter and supporting blood glucose test strips to detect the blood glucose levels. After the end of the in vivo behavior and IPGTT test, some of mice were i.p. injected50mg/kg dose of Evans blue dye (EBD), three hours later, sacrificed mice and separated left and right brain hemispheres, cerebellum, as well as the heart, liver, kidney, spleen, and mouse thigh muscle tissue and plasma. Measure the EBD concentration in different tissues and plasma. For some other mice:(1) after heart perfusion remove the brain to do the GFAP and Aβ40immunofluorescence staining;(2) sacrificed mice, separated frontal cortex and hippocampus tissue; homogenated and measured the protein concentration. Using Western blotting method to analysis the Akt signaling molecule and Tau protein level. In addition, another frontal cortex and hippocampus tissue samples were added trisol to extracted RNA. Using real-time PCR to detected inflammatory cytokines, leukocyte interleukin-1β (IL-1β), tumor necrosis factor a (TNF-a) changes in gene expression levels, and GFAP expression in astrocytes.
Results:
(1) The results from open field test, elevated plus maze test, and light-dark box test showed that the mice in each group including iNOS knockout and up to20weeks of high-fat diet feeding, mice does not have anxiety behavior, nor does it affect the spontaneous motor activity.
(2) Memory behavior of each group of mice were detected in the state of non-anxiety behavior. Morris water maze:1)5days acquisition training experiments results showed that mice in each group have no significant difference, which further illustrates iNOS gene knock-out and up to20weeks of high-fat diet feeding did not affect the visual acuity and swimming ability.2)10days exploratory experiment: with the increasing of test days, the time of animals in each group to find the platform have different degrees of shortening. In the first1-5days, the differences between the groups is not very obvious, but iNOS knockout mice fed with high-fat diet the latency of finding the platform is slightly slower than the other groups. After6days, the escape latency of iNOS-/-HFC group was significantly higher than the WT-Chow normal group. It showed that iNOS gene knockout mice fed HFC had a certain degree of damage in spatial learning and memory. In the6th days, the escape latency of iNOS-/-HFC group was higher than the iNOS-/-Chow group, which indicated that high-fat diet will further aggravate the iNOS-/-mice learning and memory damage. WT mice fed a high-fat diet compared with the WT-Chow normal group have no significant difference in the statistics, but the escape latency of WT mice fed a high-fat diet is higher than in normal mice.
Fear conditioning experiment:The first day of each group of mice conditioned fear training by electrical stimulation. The fear freezing time has increased after time moving. It suggested that each group of mice to fear almost have the same degree of respond. At the2th days contextual fear test result showed that chronic fed high-fat diet will damage the spatial memory in mice, especially the iNOS gene knockout mice fed HFC. This result suggests that the iNOS knockout exacerbated the damage of the high-fat diet on learning and memory. The tone test results showed that there was no significant difference among the groups.
(3) The IPGTT experimental results show that the mice in each group after the intraperitoneal injection2g/kg glucose, the blood glucose concentration increased rapidly. WT-Chow and iNOS-/-Chow group mice reached the peaked blood glucose at15min. While the WT-HFC and iNOS-/-HFC group mice reached peaked in30min, and were significantly higher than the WT-Chow and iNOS-/-Chow group. After2hours, WT-Chow and iNOS-/-Chow mice blood glucose tends to normal levels; the WT-HFC and iNOS-/-HFC mice still showed a high level (>150mg/dl glucose), and the peaks after the extension, which indicates that fed with high fat diet of WT and iNOS-/-mice have abnormal glucose tolerance. INOS-/-mice blood glucose values overall were lower than with WT mice fed high-fat diet, especially in the first30min and90min; after120min the glucose values tends to normal levels. It inferred that iNOS gene knockout can help to recovery the abnormal glucose tolerance under the pathological conditions like diabetes.
Based on the IPGTT results, we further examined the phosphorylated Akt and total Akt level in frontal cortex and hippocampus to analysis its possible mechanism. The results show that the WT-Chow and WT-HFC mice are relatively low baseline levels of p-Akt in the frontal cortex. Interestingly, p-Akt level in WT-HFC mice was low expression in the hippocampus than WT mice fed a normal diet, which perhaps can be explained why the IPGTT in WT-HFC mice of abnormal glucose tolerance, and the hippocampus of mice selectively reduce the level of p-Akt. iNOS knockout mice, both fed a normal diet or a high fat diet, their frontal cortex have high levels of p-Akt than WT mice, indicating that iNOS knockout contribute to the activation of Akt signaling molecules. More interesting the iNOS-/-mice fed the high fat diet, p-Akt levels in the frontal cortex and hippocampus was significantly increased compared with WT-HFC group and iNOS-/-Chow group of mice. Therefore, we concluded that iNOS knockout can help improve abnormal glucose tolerance by activating the Akt signaling molecules and decreased glucose level, this hypothesis is consistent with the above IPGTT results.
(4) Intraperitoneal injection of EBD experimental results:the left and right brain hemispheres of WT mice fed a high-fat diet EBD concentration was significantly increased than WT-Chow group, indicating that high-fat diet can cause damage to the blood-brain barrier. Cerebellum EBD concentration is relatively high than left and right brain hemispheres. Fed high-fat diet iNOS gene knockout mouse brain EBD concentration significantly reduced compared with WT-HFC mice. This shows that iNOS knockout contribute to the restoration and reconstruction of the BBB induced by high-fat diet. At the same time, we also observed the similar situation in the kidneys. But iNOS gene knockout does not affect other tissues or organs such as the spleen, liver, heart and muscle vascular permeability. In order to ensure that each group of mice can absorb the equivalent concentration of EBD into the blood circulation, and intraperitoneal injection technology, we examined plasma EBD concentration. The results showed the EBD's concentration in each group of mice plasma were no significant differences.
(5) Immune and inflammatory experiment:test results showed that long-term high fat diet induced inflammation. The frontal cortex and hippocampus IL-1β and TNF-a gene level were higher in WT-Chow group than WT-Chow group, especially in the prefrontal cortex tissue. iNOS knockout does not affect the immune system, as the inflammatory cytokines IL-1β and TNF-a gene levels did not change significantly in iNOS-/-Chow group compared with WT-Chow mice. iNOS knockout mice fed a high fat diet, the frontal cortex and hippocampus inflammatory cytokines IL-1β and TNF-a gene level were higher than other groups, than surprisingly compared with WT-HFC mice, despite the significant difference is not very obvious, only TNF-a in the frontal cortex has the significant. Thus, we speculate that under the pathological conditions (high fat diet), iNOS knockout induced increase in the levels of other inflammatory cytokines, which can also be interpreted as a compensatory response.
(6) Astrocyte GFAP expression:The GFAP gene levels in the frontal cortex and hippocampus significantly higher in WT-HFC group than WT-Chow group. GFAP gene level did not change significantly in iNOS-/-Chow mice than WT-Chow mice. This suggests that iNOS gene knockout does not affect the numbers of astrocyte in the frontal cortex and hippocampus. iNOS knockout mice fed high fat diet, the level of GFAP gene in the frontal cortex are higher than other groups, even higher than the WT-HFC mice, despite the significant difference is not very obvious. Thus, we speculate that the pathological conditions (high-fat diet), iNOS knockout further induced upregulation of GFAP expression, increasing the AD-like memory injury. These results were consistent with the memory behavior test results. Based on these, we tested the GFAP protein expression in brain by immunofluorescence staining method. The result was consistent with the results of GFAP mRNA expression in frontal cortex and hippocampus. iNOS gene knockout mice fed a high fat diet the GFAP fluorescence intensity is higher than the other groups, increasing the density of astrocytes.
(7) Detection of AD-like phenotype (Tau, Aβ40):detection the phosphorylation of Tau protein (p-Tau (AT8)) and total Tau protein (Tau46) expression levels in frontal cortex and hippocampus of each group of mice. The results showed that the ratio of phosphorylation tau with total Tau protein the relative level were significantly higher in the frontal cortex and hippocampus in WT-HFC mice than WT-Chow group. p-Tau and Tau ratio did not change significantly in iNOS-/-Chow group than WT-Chow group. This suggested that under normal physiological state iNOS knockout does not induce abnormal phosphorylation of Tau protein expression. iNOS knockout mice fed high fat diet, the relative level of p-Tau proteins is higher in the frontal cortex than other groups, eventualy higher than the WT-HFC mice. Thus, we speculate that in pathological conditions (high-fat diet) iNOS knockout further induced abnormally phosphorylated Tau protein upregulation in the frontal cortex, increasing the AD-like phenotype and memory injury. Detection the expression of Aβ40in frontal cortex and hippocampus of each group mice by immunofluorescence assay. The results show that the high-fat diet and iNOS gene knock can induce the Aβ40expression increasing trend.
Conclusion:iNOS knock-out addition to the high-fat diet for20weeks does not produce anxiety behavior, affect its own activities, visual acuity and swimming ability. Excluding the anxiety behavior interference, the high-fat diet iNOS knockout mice of spatial learning and memory ability still have a certain degree of injury. iNOS knockout exacerbated the damage of the high-fat diet on learning and memory, and is dependent on the hippocampus, has the tissue selectivity. High-fat diet is one of the risk factors to impaired glucose tolerance. IPGTT results showed that WT and iNOS-/-mice fed with high-fat diet existed abnormal glucose tolerance. In pathological conditions like diabetes, iNOS gene knockout help the recovery of abnormal glucose tolerance. Based on this phenomenon, we explore the possible mechanism of its occurrence. iNOS knockout contribute to the activation of Akt signaling molecules. It inferred that iNOS knockout on the improvement of abnormal glucose tolerance may through the activation of Akt signaling molecules and decline blood glucose level. This hypothesis is consistent with the results of the IPGTT. EBD experimental results confirmed that the high-fat diet can cause damage of the blood-brain barrier, and iNOS knockout contributed to the restoration and reconstruction of the blood-brain barrier disorder induced by high-fat diet. iNOS gene has the same similar role in the kidney, but does not affect other tissues and organs such as the spleen, liver, heart and muscle vascular permeability and blood absorption and distribution functions. The long-term high-fat diet can induce inflammation, IL-1β and TNF-α genes have higher levels in the frontal cortex and hippocampus, especially in the frontal cortex. iNOS knockout does not affect the immune system; but in pathological conditions (high-fat diet), iNOS knockout induced increase the levels of other inflammatory cytokines, which may be a compensatory response. iNOS knockout does not affect the number of astrocyte cells in the frontal cortex and hippocampus. While in pathological conditions (high-fat diet) iNOS knockout further induced upregulation of GFAP expression, increasing the AD-like phenotype, damage memory behavior of the mice. Normal physiological conditions, iNOS knockout does not induce abnormal phosphorylation of Tau protein expression; pathological conditions (high-fat diet), iNOS knockout will further induce abnormal phosphorylation of Tau protein upregulation in the frontal cortex, increasing the AD-like phenotype, damage memory behavior of the mice. These test results are consistent with previous memory behavior results. Detection the expression of A04O in frontal cortex and hippocampus of each group mice by immunofluorescence assay. The results showed that the high-fat diet and iNOS gene knockout can induced the Aβ40expression increasing trend. This project detail discussed the role of iNOS in learning amnesia induced by high-fat diet and the possible mechanisms, which may provide a possible to establish a new ideal AD animal model.
Objective:Oxidative stress has been implicated in the etiology of Parkinson's disease (PD) and may be an effective target of intervention. Peripherally and locally administered rotenone has been proposed as a well acknowledged preclinical model of PD as it induces selective nigrostriatal DA-ergic neuronal degeneration and produces motor dysfunction. Neuroprotective effects of piceid have been shown to be associated with its antioxidant properties, but the mechanisms remain unknown. Considering that enzymatic defense systems and the thioredoxin (Trx) systems, are the important cellular redox modulation systems, change under oxidative stress and could exert protective effects, the relationship between the antioxidant effects of piceid and these systems regulation were explored.
Methods and Results:Male Sprague Dawley (SD) rats were treated with rotenone2.0mg/kg/day i.p. for5weeks. Behavioral data showed a strong increase in catalepsy score, a decrease in motor coordination activity from4-5weeks, and biochemical data showed profound deficient in the contents of reduced glutathione (GSH), the activities of superoxide dismutase (SOD) and the contents of adenosine triphosphate (ATP), and a significant increase the contents of malondialdehyde (MDA), and down-regulated the antioxidative enzyme thioredoxin (Trx) expression in the striatum in rotenone treated animals compared to vehicle. Chronic administration of rotenone to SD rats resulted in marked oxidative damage in the striatum region compared to other regions (cortex and hippocampus) of the brain. Oral administration of piceid (20,40,80mg/kg) or L-DOPA (10mg/kg) co-treatment is able to successfully attenuate these motor defects and most of biochemical changes in dose dependent manners. The administration with dose (40,80mg/kg) of piceid and L-DOPA (10mg/kg) caused the most marked symptomatic improvement in catalepsy score and motor coordination activity when compared to its administration with low dose of piceid (20mg/kg) or when compared to rotenone treated animals, respectively. Piceid in a dose40mg/kg especially in a high dose80mg/kg significantly attenuated rotenone-induced depletion in GSH and decreased the contents of MDA. In addition, the drug prevented the decrease in ATP level and also enhanced the activity of SOD. Western blot result showed piceid (80mg/kg) co-treatment could up-regulate Trx expression in the striatum region compared to cortex and hippocampus.
Conclusion:Taken together, these results strongly indicate the possible therapeutic potential of piceid as an antioxidant in PD and other movement disorders based on its up-regulating effects on enzymatic defense systems and neuroprotective protein thioredoxin, a substrate in the Trx redox system.
方法：3月龄的i(?)(?)OS基因敲除小鼠(iNOS knockout mice, iNOS-/-)和相同背景(背景鼠为C57BL/6小鼠)的同窝出生的野生型小鼠(wild type, WT)饲养一周以适应环境,转基因小鼠PCR鉴定基因型后筛选使用。iN(?)OS基因敲除小鼠和野生型小鼠分别喂养正常饲料(Chow)和高脂饲料(High fat cholesterol, HFC)。喂养20周后首先对各组小鼠进行焦虑行为的检测,包括旷场实验、高架十字迷宫实验和明暗箱实验。在焦虑行为学检测完之后,进行记忆行为的检测,包括Morris水迷宫实验和条件性恐惧实验。与此同时对各组小鼠进行了腹腔注射糖耐量实验(Intraperitoneal glucose tolerance test, IPGTT),所有小鼠禁食16小时后,称量体重,剪尾采血测定空腹血糖值。然后按2g/kg的剂量腹腔注射葡萄糖,分别在0、15、30、60、90、120min,用微血糖测量仪及配套的血糖试纸检测各时间段的血糖值,观察高脂饮食对糖耐量的影响。活体行为学和IPGTT测试结束后,各组部分小鼠,按50mg/kg的剂量腹腔注射伊文思蓝染料(Evan blue dye, EBD),三小时后,用抗凝管取血,迅速断头,粘去脑膜,分离出左、右脑半球和小脑,以及心脏,肝脏,肾脏,脾,和小鼠大腿肌肉组织,甲酰胺提取染料；并取血获得血浆。检测各组织和血浆中EBD的浓度,评价血脑屏障通透性以及EBD在血液组织中的分布情况。还有部分小鼠在行为学测试结束后：(1)心脏灌流后取出大脑做冰冻切片进行GFAP和Aβ40免疫荧光染色；(2)断头处死后,分离出半侧前额皮层和海马用组织裂解液提取蛋白,完成蛋白浓度测定后用Western blotting检测Akt信号分子和Tau蛋白的水平。另外半侧前额皮层和海马组织样品通过Real-time PCR检测炎症因子白细胞介素-1β(IL-1β)、肿瘤坏死因子α (TNF-α)基因表达水平的变化,以及星形胶质细胞中GFAP的表达情况。
结果：(1)焦虑行为：通过旷场实验、高架十字迷宫实验和明暗箱实验对各组小鼠进行焦虑行为的检测结果显示iNOS基因敲除和长达20周的高脂饮食对小鼠并不会产生焦虑行为,也不影响其自主活动性。
(2)记忆行为：在无焦虑行为的状态下对各组小鼠进行了记忆行为的检测。Morris水迷宫：1)5天习得性训练实验中各组小鼠差异不显著,这进一步说明iNOS基因敲除和长达20周的高脂饮食并不影响小鼠的视力和游泳运动能力。2)10天探索性实验中随着测试天数的增加,各组动物寻找到平台的时间都有不同程度地缩短。第1-5天里,各组间的差异不是很明显,但喂高脂饲料的iNOS基因敲除小鼠寻找平台潜伏期的趋势比其他各组稍微慢点。第6天之后,iNOS-/-HFC组的逃避潜伏期与WT-Chow正常组比,显著升高,这说明高脂饮食的iNOS基因敲除小鼠空间学习记忆能力出现一定程度的损伤。在第6天,iNOS组与iNOS-/-Chow组比较,其逃避潜伏期显著升高,说明高脂饮食会进一步加重iNOS基因敲除小鼠学习记忆的损伤。喂养高脂饮食的WT小鼠跟WT-Chow正常组比,尽管统计上没有显著差异,但喂养高脂饲料的WT小鼠的逃避潜伏期提示高于正常小鼠。
条件性恐惧实验：第一天对各组小鼠进行条件恐惧性训练,在受到电刺激后,各组小鼠恐惧不动的时间都有所增高,这表明各组小鼠对恐惧都有比较同步的反应能力。第二天对小鼠进行环境恐惧恢复测试的结果表明,与正常组小鼠相比,喂养高脂饮食小鼠的空间记忆能力显著下降；iNOS基因敲除后,其空间记忆能力也显著下降；其中喂高脂饮食的iNOS基因敲除小鼠空间记忆能力下降最为显著,且比喂高脂饮食的WT小鼠的空间记忆能力还要差。这一结果表明iNOS敲除后加剧了高脂饮食对学习记忆的损伤。声音恐惧恢复测试中各组间并未见显著性差异。
(3) IPGTT实验：结果表明各组小鼠在腹腔注射2g/kg葡糖糖后,血糖迅速升高,其中WT-Chow和iNO-/-Chow组小鼠血糖在15mmin达到峰值。而WT-HFC和iNOS-/-HFC组小鼠血糖在30mmin才达到峰值,且显著高于WT-Chow和iNOS-/-Chow组。2小时后,WT-Chow和iNOS-/-Chow组小鼠的血糖都趋于正常水平;WT-HFC和iNOS-/-HFC组小鼠的血糖仍呈现较高的水平(>150mg/dL),且峰时后延,这表明喂高脂饮食的WT和iNOS-/-小鼠都存在腹腔注射糖耐量异常。喂高脂饮食的iNOS-/-小鼠的血糖值总体都低于同喂高脂饮食的WT小鼠,特别是在第30min和90min;120min后血糖值基本趋于正常水平。由此推测,在类似糖尿病的病理条件下,iNOS基因的敲除有助于恢复糖耐量的异常。
基于IPGTT的检测结果,我们进一步检测了前额皮层和海马中磷酸化Akt和总的Akt水平分析其可能的机制。结果显示WT-Chow和WT-HFC组小鼠在前额皮层中p-Akt的基线水平都比较低。有趣的是,WT-HFC组小鼠p-Akt在海马的表达水平也比喂养正常饲料的WT小鼠低,这也许就可以解释为什么在IPGTT中WT-HFC组小鼠的糖耐量异常,且选择性地降低小鼠海马组织的p-Akt水平。iNOS基因敲除后的小鼠,无论是喂养正常饲料还是高脂饲料,其前额皮层中p-Akt的水平都比WT小鼠高,这说明iNOS基因敲除后有助于Akt信号分子的激活。更有趣的是,iNOS-/-小鼠在喂养高脂饲料后,其前额皮层和海马组织中的p-Akt水平跟WT-HFC组和iNOS-/-Chow组小鼠比都显著增高。因此,我们得出结论,iNOS基因敲除后有助于糖耐量异常的改善,且可能是通过激活Akt信号分子而使血糖下降,这一推测跟上面IPGTT的结果一致。
(4)腹腔注射EBD实验结果：喂养高脂饲料的WT小鼠其左右脑半球的EBD浓度与WT-Chow组比,显著升高,这说明高脂饮食会造成血脑屏障的损伤。EBD在左右脑半球的浓度差别不大,但是小脑的EBD浓度比较高。喂高脂饲料的iNOS基因敲除小鼠脑内EBD的浓度与WT-HFC组小鼠比,显著降低。这说明iNOS基因敲除后有助于高脂饮食诱导的血脑屏障障碍的恢复重建。与此同时,我们还观察到iNOS基因在肾脏具有同样类似的作用。但是不影响其他组织器官如脾、肝脏、心脏和肌肉的血管渗透性。为了确保各组小鼠对当量浓度的EBD都能吸收进血液循环,及腹腔注射的泄漏程度,我们检测了血浆中EBD的浓度。结果表明各组小鼠血浆中EBD的浓度没有显著差异。
(5)免疫炎症实验：检测结果显示长期高脂饮食会诱导炎症的发生,WT-HFC组小鼠与WT-Chow组小鼠比,其前额皮层和海马组织中IL-1p和TNF-α的基因水平都比较高,特别是在前额皮层组织。iNOS基因敲除后并不影响自身的免疫系统,因iNOS-/-Chow组小鼠与WT-Chow组小鼠比,其炎症因子IL-1β和TNF-α的基因水平变化不明显。喂高脂饲料的iNOS基因敲除小鼠,其前额皮层和海马组织中炎症因子IL-1β和TNF-α的基因水平与其他各组比都高,惊奇的是比WT-HFC组小鼠还高,尽管显著性差异不是很明显,仅TNF-α在前额皮层中的含量有差异。由此我们推测,在病理条件下(高脂饮食),iNOS基因敲除后会诱导增加其他炎症因子的水平,这可能是一种代偿反应。
(6)星形胶质细胞中GFAP表达情况：WT-HFC组小鼠与WT-Chow组小鼠比,其前额皮层和海马组织中GFAP的基因水平都显著增高。iNOS-/-Chow组小鼠与WT-Chow组小鼠比,GFAP的基因水平变化不明显；这表明iNOS基因敲除后并不影响自星型胶质细胞在前额皮层和海马中的数量。喂高脂饲料的iNOS基因敲除小鼠,其在前额皮层中GFAP的基因水平与其他各组比都高,且比WT-HFC组小鼠还高,尽管显著性差异不是很明显。由此我们推测,在病理条件下(高脂饮食),iNOS基因敲除后会进一步诱导GFAP表达的上调,从而加重AD样表型,损伤小鼠的记忆行为,这与前面的记忆行为学检测结果一致。在此基础上,我们对各组脑组织进行GFAP免疫荧光染色,结果与GFAP mRNA在前额皮层和海马中的表达情况一致。喂高脂饲料的iNOS基因敲除组小鼠其GFAP的荧光强度比其他各组高,星型胶质细胞的密度增加。
(7)AD样表型的检测(Tau和Aβ40)：检测各组小鼠前额皮层和海马中磷酸化Tau蛋白(p-Tau(AT8))和总Tau蛋白(Tau46)的蛋白表达水平。结果显示WT-HFC组小鼠与WT-Chow组小鼠比,其前额皮层和海马组织中磷酸化Tau与总Tau蛋白比的相对水平都显著增高。iNOS-/-Chow组小鼠与WT-Chow组小鼠比,p-Tau和Tau的比值变化不明显；但与WT-HFC组比显著降低,由此推测在正常生理状态下iNOS基因敲除后并不会诱导异常磷酸化的Tau蛋白的高表达。喂高脂饲料的iNOS基因敲除小鼠,其在前额皮层中p-Tau蛋白的相对水平与其他各组比都高,惊奇的是比WT-HFC组小鼠还高,呈显著性差异；在海马中的变化不是很显著。由此我们推测,在病理条件下(高脂饮食),iNOS基因敲除后会进一步诱导异常磷酸化的Tau蛋白主要在前额皮层表达的上调,从而加重AD样表型,损伤小鼠的记忆行为,这与前面的记忆行为学检测结果一致。免疫荧光法对各组小鼠前额皮层和海马中Aβ40的表达进行检测的结果显示高脂饮食和iNOS基因敲除都有诱导Aβ40表达增加的趋势。
结论：iNOS基因敲除和长达20周的高脂饮食不会使小鼠产生焦虑行为,也不影响其自主活动性,小鼠的视力和游泳运动能力也良好。在排除了焦虑行为干扰的情况下,高脂饮食的iNOS基因敲除小鼠其空间学习记忆能力仍有一定程度的损伤。iNOS敲除后加剧了高脂饮食对学习记忆的损伤,且依赖于海马组织,具有一定的组织选择性。高脂饮食是糖耐量异常的风险因素之一,IPGTT检测结果显示喂高脂饮食的WT和iNOS-/-小鼠都存在腹腔注射糖耐量异常。在类似糖尿病的病理条件下,iNOS基因的敲除有助于恢复糖耐量的异常。基于这一现象,我们探讨了其发生的可能机制,iNOS基因敲除后有助于Akt信号分子的激活。由此推测iNOS基因敲除后对糖耐量异常的改善,有可能是通过激活Akt信号分子而使血糖下降,这一推测跟上面IPGTT的结果一致。EBD实验结果证实高脂饮食会造成血脑屏障的损伤,iNOS基因敲除后有助于高脂饮食诱导的血脑屏障障碍的恢复重建。iNOS基因在肾脏具有同样类似的作用,但是不影响其他组织器官如脾、肝脏、心脏和肌肉的血管渗透性和血液的吸收和分布功能。长期的高脂饮食会诱导炎症的发生,其前额皮层和海马组织中IL-1β和TNF-α的基因水平都比较高,特别是在前额皮层组织。iNOS基因敲除后并不影响自身的免疫系统,但在病理条件下(高脂饮食),iNOS基因敲除后会诱导增加其他炎症因子的水平,这可能是一种代偿反应。iNOS基因敲除后并不影响自身星型胶质细胞在前额皮层和海马中的数量;在病理条件下(高脂饮食),iNOS基因敲除后会进一步诱导GFAP表达的上调,从而加重AD样表型,损伤小鼠的记忆行为。在正常生理状态下iNOS基因敲除后并不会诱导异常磷酸化的Tau蛋白的高表达；在病理条件下(高脂饮食),iNOS基因敲除后会进一步诱导异常磷酸化的Tau蛋白主要在前额皮层表达的上调,从而加重AD样表型,损伤小鼠的记忆行为,这与前面的记忆行为学检测结果一致。免疫荧光法对各组小鼠前额皮层和海马中Aβ40的表达进行检测的结果显示高脂饮食和iNOS基因敲除都有诱导Aβ40表达增加的趋势。本课题细致的探讨了iNOS在高脂饮食诱导的学习记忆缺失中的作用及可能的机制,这为建立一种新的比较理想的AD动物模型提供了可能。
目的：应用神经毒素鱼藤酮(Rotenone)建立稳定的帕金森病(Parkinson's disease, PD) SD大鼠模型,观察白藜芦醇苷对PD的神经保护作用,并在此基础上对其作用机制进行探讨。方法：采用大鼠背部皮下注射鱼藤酮(2.0mg·kg-1·d-1)持续5周以诱导其多巴胺神经元丢失为PD SD大鼠模型。SD大鼠随机分为6组：空白对照组,模型组(仅注射鱼藤酮),鱼藤酮+左旋多巴(10mg·kg-1·-1)组,鱼藤酮+白藜芦醇苷低、中、高剂量(20,40,80mg·kg-1·d-1)组,左旋多巴和不同剂量的白藜芦醇苷均通过口服给药,持续给药5周。通过Grid test、Bar test、 Rotarod test对大鼠进行僵直程度、运动障碍和肌肉活性的行为学检测;比色法测定大脑皮层、海马、纹状体中超氧物歧化酶(SOD)、谷胱甘肽(GSH)活力、丙二醛(MDA)含量及ATP水平；采用Western blot法测定大脑皮层、海马、纹状体中硫氧还蛋白(Trx)的表达。结果：低剂量长期背部皮下注射鱼藤酮可诱导SD大鼠出现类似PD的行为学表现,呈进行性僵直行为的增加,降低运动协调和肌活性。白藜芦醇苷剂量和时间依赖性的改善鱼藤酮诱导的行为障碍。在第五周,高剂量80mg/kg的白藜芦醇苷显著降低鱼藤酮诱导的僵直,并增加大鼠的运动协调性和肌活性。与模型组相比,各给药组纹状体中SOD、GSH活力和ATP含量均显著增加,MDA含量明显减少,Trx的免疫活性增加,并呈现一定的剂量依赖性。鱼藤酮对大鼠的损伤有一定的选择性,主要表现为黑质纹状体中多巴胺能神经元的丢失。结论：白藜芦醇苷对鱼藤酮诱导的帕金森病具有一定保护作用,此作用与其增加体内抗氧化酶的活性,减少过氧化脂质的生成,上调硫氧还蛋白系统中Trx的表达密切相关,并呈一定的剂量依赖性。
Objective:The incidence of cognitive dysfunction increases with age, it become a very common clinical symptoms in today's aging society. Severe cases of cognitive impairment were called dementia. According to statistics, the current global dementia patients is about24million, of which more than half are Alzheimer's disease (AD). AD is the most usual disease of old age cognitive dysfunction. AD is a progressive cognitive impairment of central neurodegenerative diseases, its pathology is characterized by the formation of senile plaques (SP), neurofibrillary tangles (NFT) and a large number of neurons lost. Ideal AD animal model is very important to study the pathogenesis of AD and the treatment. There are many ways to establish the AD animal models. They mainly can be grouped into the following three categories: simulate the characteristics of AD symptoms model, simulate histopathological features of AD model and multi-factor composite animal model. After decades of hard work AD animal model studies has made a lot of progress. Each animal model simulates a certain degree, or some aspects of the symptoms and pathological changes of AD. But as yet no one can truly reflect the various cases of stage of development characterized and widely popular animal model. Colton CA. in Duke University School of Medicine first proposed that genetic removal of iNOS in mice expressing mutated amyloid precursor protein results in pathological hyperphosphorylation of mouse tau, its redistribution to the somatodendritic compartment in cortical and hippocampal neurons, and aggregate formation. Lack of iNOS in the amyloid precursor protein Swedish mutant mouse increased insoluble β-amyloid peptide levels, neuronal degeneration, caspase-3activation, and tau cleavage, suggesting that NO acts at a junction point between β-amyloid peptides, caspase activation, and tau aggregation. Lot of people questioned this theory when Colton CA first proposed. iNOS gene is located on the17pairs of autosomes. It only can be expressed when the cells were stimulated and activated. NO, the production of iNOS can last a long time about several hours to days. NO is an important intracellular and intercellular signaling regulating molecular. It has a dual nature in human physiological and pathological processes. NO, a cytotoxic effector molecule is a tissue damage enhancement factor and predisposing factors of variety of lesions. This is the pathology bad side. In physiological good side, NO is also can be a signaling molecules in many organisms, innate immune response regulatory effector molecules and as an information material to maintain normal physiological function. In rodents experiments suggesting that iNOS activity play a harmful role in experimental autoimmune or chronic inflammatory processes, and several other diseases. But the detial of iNOS specific roles in many diseases are still unknown; it needs to be further in-depth study. The purpose of this project is to study the relationship of iNOS with learning and memory; study the effect and mechanism of iNOS on learning amnesia induced by high-fat diet.
Methods:3-month-old iNOS gene knockout mice (iNOS-/-) and the same background (C57BL/6) with littermate wild-type mice (WT) were rearing to adapt to the environment one week. PCR was carried out to genotype the transgenic mice before the experiment. iNOS knockout mice and WT mice were fed with chow diet (Chow) and/or high-fat diet (HFC). After20weeks feeding mice in each group were did anxiety behavior test, including open field test, the elevated plus maze, and light dark box test. Memory behavior test were conducted after anxiety behavior, including the Morris water maze and fear condition test. At the same time for each group of mice did intraperitoneally tolerance test (IPGTT). All mice were fasted for ahout16hours and weighed, injected2g/kg dose of glucose. After0,15,30,60,90and120min, using micro-blood glucose meter and supporting blood glucose test strips to detect the blood glucose levels. After the end of the in vivo behavior and IPGTT test, some of mice were i.p. injected50mg/kg dose of Evans blue dye (EBD), three hours later, sacrificed mice and separated left and right brain hemispheres, cerebellum, as well as the heart, liver, kidney, spleen, and mouse thigh muscle tissue and plasma. Measure the EBD concentration in different tissues and plasma. For some other mice:(1) after heart perfusion remove the brain to do the GFAP and Aβ40immunofluorescence staining;(2) sacrificed mice, separated frontal cortex and hippocampus tissue; homogenated and measured the protein concentration. Using Western blotting method to analysis the Akt signaling molecule and Tau protein level. In addition, another frontal cortex and hippocampus tissue samples were added trisol to extracted RNA. Using real-time PCR to detected inflammatory cytokines, leukocyte interleukin-1β (IL-1β), tumor necrosis factor a (TNF-a) changes in gene expression levels, and GFAP expression in astrocytes.
Results:
(1) The results from open field test, elevated plus maze test, and light-dark box test showed that the mice in each group including iNOS knockout and up to20weeks of high-fat diet feeding, mice does not have anxiety behavior, nor does it affect the spontaneous motor activity.
(2) Memory behavior of each group of mice were detected in the state of non-anxiety behavior. Morris water maze:1)5days acquisition training experiments results showed that mice in each group have no significant difference, which further illustrates iNOS gene knock-out and up to20weeks of high-fat diet feeding did not affect the visual acuity and swimming ability.2)10days exploratory experiment: with the increasing of test days, the time of animals in each group to find the platform have different degrees of shortening. In the first1-5days, the differences between the groups is not very obvious, but iNOS knockout mice fed with high-fat diet the latency of finding the platform is slightly slower than the other groups. After6days, the escape latency of iNOS-/-HFC group was significantly higher than the WT-Chow normal group. It showed that iNOS gene knockout mice fed HFC had a certain degree of damage in spatial learning and memory. In the6th days, the escape latency of iNOS-/-HFC group was higher than the iNOS-/-Chow group, which indicated that high-fat diet will further aggravate the iNOS-/-mice learning and memory damage. WT mice fed a high-fat diet compared with the WT-Chow normal group have no significant difference in the statistics, but the escape latency of WT mice fed a high-fat diet is higher than in normal mice.
Fear conditioning experiment:The first day of each group of mice conditioned fear training by electrical stimulation. The fear freezing time has increased after time moving. It suggested that each group of mice to fear almost have the same degree of respond. At the2th days contextual fear test result showed that chronic fed high-fat diet will damage the spatial memory in mice, especially the iNOS gene knockout mice fed HFC. This result suggests that the iNOS knockout exacerbated the damage of the high-fat diet on learning and memory. The tone test results showed that there was no significant difference among the groups.
(3) The IPGTT experimental results show that the mice in each group after the intraperitoneal injection2g/kg glucose, the blood glucose concentration increased rapidly. WT-Chow and iNOS-/-Chow group mice reached the peaked blood glucose at15min. While the WT-HFC and iNOS-/-HFC group mice reached peaked in30min, and were significantly higher than the WT-Chow and iNOS-/-Chow group. After2hours, WT-Chow and iNOS-/-Chow mice blood glucose tends to normal levels; the WT-HFC and iNOS-/-HFC mice still showed a high level (>150mg/dl glucose), and the peaks after the extension, which indicates that fed with high fat diet of WT and iNOS-/-mice have abnormal glucose tolerance. INOS-/-mice blood glucose values overall were lower than with WT mice fed high-fat diet, especially in the first30min and90min; after120min the glucose values tends to normal levels. It inferred that iNOS gene knockout can help to recovery the abnormal glucose tolerance under the pathological conditions like diabetes.
Based on the IPGTT results, we further examined the phosphorylated Akt and total Akt level in frontal cortex and hippocampus to analysis its possible mechanism. The results show that the WT-Chow and WT-HFC mice are relatively low baseline levels of p-Akt in the frontal cortex. Interestingly, p-Akt level in WT-HFC mice was low expression in the hippocampus than WT mice fed a normal diet, which perhaps can be explained why the IPGTT in WT-HFC mice of abnormal glucose tolerance, and the hippocampus of mice selectively reduce the level of p-Akt. iNOS knockout mice, both fed a normal diet or a high fat diet, their frontal cortex have high levels of p-Akt than WT mice, indicating that iNOS knockout contribute to the activation of Akt signaling molecules. More interesting the iNOS-/-mice fed the high fat diet, p-Akt levels in the frontal cortex and hippocampus was significantly increased compared with WT-HFC group and iNOS-/-Chow group of mice. Therefore, we concluded that iNOS knockout can help improve abnormal glucose tolerance by activating the Akt signaling molecules and decreased glucose level, this hypothesis is consistent with the above IPGTT results.
(4) Intraperitoneal injection of EBD experimental results:the left and right brain hemispheres of WT mice fed a high-fat diet EBD concentration was significantly increased than WT-Chow group, indicating that high-fat diet can cause damage to the blood-brain barrier. Cerebellum EBD concentration is relatively high than left and right brain hemispheres. Fed high-fat diet iNOS gene knockout mouse brain EBD concentration significantly reduced compared with WT-HFC mice. This shows that iNOS knockout contribute to the restoration and reconstruction of the BBB induced by high-fat diet. At the same time, we also observed the similar situation in the kidneys. But iNOS gene knockout does not affect other tissues or organs such as the spleen, liver, heart and muscle vascular permeability. In order to ensure that each group of mice can absorb the equivalent concentration of EBD into the blood circulation, and intraperitoneal injection technology, we examined plasma EBD concentration. The results showed the EBD's concentration in each group of mice plasma were no significant differences.
(5) Immune and inflammatory experiment:test results showed that long-term high fat diet induced inflammation. The frontal cortex and hippocampus IL-1β and TNF-a gene level were higher in WT-Chow group than WT-Chow group, especially in the prefrontal cortex tissue. iNOS knockout does not affect the immune system, as the inflammatory cytokines IL-1β and TNF-a gene levels did not change significantly in iNOS-/-Chow group compared with WT-Chow mice. iNOS knockout mice fed a high fat diet, the frontal cortex and hippocampus inflammatory cytokines IL-1β and TNF-a gene level were higher than other groups, than surprisingly compared with WT-HFC mice, despite the significant difference is not very obvious, only TNF-a in the frontal cortex has the significant. Thus, we speculate that under the pathological conditions (high fat diet), iNOS knockout induced increase in the levels of other inflammatory cytokines, which can also be interpreted as a compensatory response.
(6) Astrocyte GFAP expression:The GFAP gene levels in the frontal cortex and hippocampus significantly higher in WT-HFC group than WT-Chow group. GFAP gene level did not change significantly in iNOS-/-Chow mice than WT-Chow mice. This suggests that iNOS gene knockout does not affect the numbers of astrocyte in the frontal cortex and hippocampus. iNOS knockout mice fed high fat diet, the level of GFAP gene in the frontal cortex are higher than other groups, even higher than the WT-HFC mice, despite the significant difference is not very obvious. Thus, we speculate that the pathological conditions (high-fat diet), iNOS knockout further induced upregulation of GFAP expression, increasing the AD-like memory injury. These results were consistent with the memory behavior test results. Based on these, we tested the GFAP protein expression in brain by immunofluorescence staining method. The result was consistent with the results of GFAP mRNA expression in frontal cortex and hippocampus. iNOS gene knockout mice fed a high fat diet the GFAP fluorescence intensity is higher than the other groups, increasing the density of astrocytes.
(7) Detection of AD-like phenotype (Tau, Aβ40):detection the phosphorylation of Tau protein (p-Tau (AT8)) and total Tau protein (Tau46) expression levels in frontal cortex and hippocampus of each group of mice. The results showed that the ratio of phosphorylation tau with total Tau protein the relative level were significantly higher in the frontal cortex and hippocampus in WT-HFC mice than WT-Chow group. p-Tau and Tau ratio did not change significantly in iNOS-/-Chow group than WT-Chow group. This suggested that under normal physiological state iNOS knockout does not induce abnormal phosphorylation of Tau protein expression. iNOS knockout mice fed high fat diet, the relative level of p-Tau proteins is higher in the frontal cortex than other groups, eventualy higher than the WT-HFC mice. Thus, we speculate that in pathological conditions (high-fat diet) iNOS knockout further induced abnormally phosphorylated Tau protein upregulation in the frontal cortex, increasing the AD-like phenotype and memory injury. Detection the expression of Aβ40in frontal cortex and hippocampus of each group mice by immunofluorescence assay. The results show that the high-fat diet and iNOS gene knock can induce the Aβ40expression increasing trend.
Conclusion:iNOS knock-out addition to the high-fat diet for20weeks does not produce anxiety behavior, affect its own activities, visual acuity and swimming ability. Excluding the anxiety behavior interference, the high-fat diet iNOS knockout mice of spatial learning and memory ability still have a certain degree of injury. iNOS knockout exacerbated the damage of the high-fat diet on learning and memory, and is dependent on the hippocampus, has the tissue selectivity. High-fat diet is one of the risk factors to impaired glucose tolerance. IPGTT results showed that WT and iNOS-/-mice fed with high-fat diet existed abnormal glucose tolerance. In pathological conditions like diabetes, iNOS gene knockout help the recovery of abnormal glucose tolerance. Based on this phenomenon, we explore the possible mechanism of its occurrence. iNOS knockout contribute to the activation of Akt signaling molecules. It inferred that iNOS knockout on the improvement of abnormal glucose tolerance may through the activation of Akt signaling molecules and decline blood glucose level. This hypothesis is consistent with the results of the IPGTT. EBD experimental results confirmed that the high-fat diet can cause damage of the blood-brain barrier, and iNOS knockout contributed to the restoration and reconstruction of the blood-brain barrier disorder induced by high-fat diet. iNOS gene has the same similar role in the kidney, but does not affect other tissues and organs such as the spleen, liver, heart and muscle vascular permeability and blood absorption and distribution functions. The long-term high-fat diet can induce inflammation, IL-1β and TNF-α genes have higher levels in the frontal cortex and hippocampus, especially in the frontal cortex. iNOS knockout does not affect the immune system; but in pathological conditions (high-fat diet), iNOS knockout induced increase the levels of other inflammatory cytokines, which may be a compensatory response. iNOS knockout does not affect the number of astrocyte cells in the frontal cortex and hippocampus. While in pathological conditions (high-fat diet) iNOS knockout further induced upregulation of GFAP expression, increasing the AD-like phenotype, damage memory behavior of the mice. Normal physiological conditions, iNOS knockout does not induce abnormal phosphorylation of Tau protein expression; pathological conditions (high-fat diet), iNOS knockout will further induce abnormal phosphorylation of Tau protein upregulation in the frontal cortex, increasing the AD-like phenotype, damage memory behavior of the mice. These test results are consistent with previous memory behavior results. Detection the expression of A04O in frontal cortex and hippocampus of each group mice by immunofluorescence assay. The results showed that the high-fat diet and iNOS gene knockout can induced the Aβ40expression increasing trend. This project detail discussed the role of iNOS in learning amnesia induced by high-fat diet and the possible mechanisms, which may provide a possible to establish a new ideal AD animal model.
Objective:Oxidative stress has been implicated in the etiology of Parkinson's disease (PD) and may be an effective target of intervention. Peripherally and locally administered rotenone has been proposed as a well acknowledged preclinical model of PD as it induces selective nigrostriatal DA-ergic neuronal degeneration and produces motor dysfunction. Neuroprotective effects of piceid have been shown to be associated with its antioxidant properties, but the mechanisms remain unknown. Considering that enzymatic defense systems and the thioredoxin (Trx) systems, are the important cellular redox modulation systems, change under oxidative stress and could exert protective effects, the relationship between the antioxidant effects of piceid and these systems regulation were explored.
Methods and Results:Male Sprague Dawley (SD) rats were treated with rotenone2.0mg/kg/day i.p. for5weeks. Behavioral data showed a strong increase in catalepsy score, a decrease in motor coordination activity from4-5weeks, and biochemical data showed profound deficient in the contents of reduced glutathione (GSH), the activities of superoxide dismutase (SOD) and the contents of adenosine triphosphate (ATP), and a significant increase the contents of malondialdehyde (MDA), and down-regulated the antioxidative enzyme thioredoxin (Trx) expression in the striatum in rotenone treated animals compared to vehicle. Chronic administration of rotenone to SD rats resulted in marked oxidative damage in the striatum region compared to other regions (cortex and hippocampus) of the brain. Oral administration of piceid (20,40,80mg/kg) or L-DOPA (10mg/kg) co-treatment is able to successfully attenuate these motor defects and most of biochemical changes in dose dependent manners. The administration with dose (40,80mg/kg) of piceid and L-DOPA (10mg/kg) caused the most marked symptomatic improvement in catalepsy score and motor coordination activity when compared to its administration with low dose of piceid (20mg/kg) or when compared to rotenone treated animals, respectively. Piceid in a dose40mg/kg especially in a high dose80mg/kg significantly attenuated rotenone-induced depletion in GSH and decreased the contents of MDA. In addition, the drug prevented the decrease in ATP level and also enhanced the activity of SOD. Western blot result showed piceid (80mg/kg) co-treatment could up-regulate Trx expression in the striatum region compared to cortex and hippocampus.
Conclusion:Taken together, these results strongly indicate the possible therapeutic potential of piceid as an antioxidant in PD and other movement disorders based on its up-regulating effects on enzymatic defense systems and neuroprotective protein thioredoxin, a substrate in the Trx redox system.
引文
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