限制饮食对老年早期大鼠行为学和海马组织学及SirT1和FoxO3a信号途径的影响

发布时间：2018-08-31 13:15

【摘要】： 研究背景和目的衰老(Senescence)是随着年龄增长，机体各器官发生生物学功能退化的过程，表现为老年相关疾病的发病率增高，细胞更新的能力下降，死亡的可能性增加。热量限制(Calorie Restriction，CR)是一种目前公认的有效延缓衰老的方法。CR不仅能延缓衰老，还能延缓和预防一些与年龄相关疾病的发生发展。衰老的大脑在功能上主要表现为学习记忆能力减退，而CR能够减少与认知能力相关的疾病(如Alzheimer's病)的发病率，但是CR对自然衰老引起的学习记忆能力衰减的延缓作用仍缺乏行为学和组织学依据，并且多数的研究结果是基于对动物进行终生饮食限制获得的，研究周期较长。对于CR是否能够延缓老年早期的大鼠学习记忆能力的衰退，还存有争议，甚至有人对成年或老年早期的大鼠进行饮食限制是否能够提高大鼠的生存率提出质疑。在明确CR能否对认知能力的下降有延缓作用时，确定认知相关的脑区(如海马和额叶皮质)神经元的衰老状态是揭示CR延缓学习记忆能力下降的细胞学基础，衰老细胞的鉴定是确定细胞是否发生衰老的标志。尽管有报道，在培养的细胞，衰老细胞表现为细胞变大，变扁平，但在组织学，衰老细胞仅从形态学上难以与其它细胞区分。生物学标记为衰老细胞的鉴定提供了一个重要方法，衰老相关的β半乳糖苷酶(Senescence associatedβ-galactosidase，SA-β-GAL)是目前被广泛采用的一种生物学标记物，它在衰老细胞中表达升高，因此成为鉴定细胞衰老的一个标志。值得提出的是，有些衰老的生物学标记并不具有普遍性，SA-β-GAL作为一个衰老的生物学标记在体外检测复制衰老的细胞已经得到了广泛的认可，但其是否能够应用于脑组织，作为检测衰老神经元的生物学标记，目前还未得到证实，因此在应用上应加以验证。本研究选定老年早期的大鼠，对其进行饮食控制，即选在认知能力衰退的年龄段进行观察，旨在明确限制饮食对老年早期的大鼠的生存状态的影响和通过行为学检测观察学习记忆能力的改变，利用SA-β-GAL在学习记忆产生的主要部位海马检测衰老神经元。探讨认知能力的提高是否与限制饮食引起的延缓神经元衰老有关，同时验证SA-β-GAL作为衰老的生物标记在脑组织检测衰老神经元的可靠性，为进一步探讨CR延缓海马衰老的生物学机制提供行为学和组织学依据。材料与方法实验动物选用18月龄的健康雄性Sprague Dawley大鼠，根据进食情况分为两组，一组为CR组(n=29)给予对照组进食量的60％的食量喂养，对照组为ad libitum(AL)组(n=31)自由进食，观察6个月，在此期间，记录大鼠的生存状况，每周测定大鼠体重；6个月后，进行开放场实验(open-field test)观察自发活动能力；利用Morris水迷宫(Morris water maze，MWM)检测大鼠的空间学习能力和参考记忆能力。选用6月龄(n=10)，18月龄(n=6)和24月龄(n=7)的SD雄性大鼠，进行SA-β-GAL的组织化学检测；同时海马神经元原代培养，取培养6天，12天和20天的神经元进行SA-β-GAL的细胞化学检测。结果 CR组给予60％的饮食控制后1-10周内体重增长率下降明显，低于对照组的近30％，之后CR组的体重增长率有缓慢的升高，并维持在低于对照组约20％左右(p＜0.001)。CR组的生存率明显高于AL组(p=0.039)；在开放场实验中测定的大鼠白发活动路程明显长于AL组(p=0.021)，而自发活动速度也明显高于AL组(p=0.021)，并且CR组大鼠具有在中央区域的活动距离长(p=0.048)和在周边区域活动速度快(p=0.012)的特点。Morris水迷宫定向航行实验中，CR组的逃避潜伏期(Escape latency，EL)明显比AL组短(p=0.038)，而在空间探索实验中，比较两组的穿环次数无统计学差异(p=0.232)，两组大鼠在目的象限Ⅲ中的时间和路程差别无统计学意义(p=0.324和p=0.367)，但在象限Ⅱ中的时间和路程都有明显统计学差异(p=0.027和p=0.029)。与AL大鼠相比，CR组大鼠海马CA3区锥体细胞层细胞排列相对整齐、紧密，细胞计数明显增多(p=0.039)。SA-β-GAL在CR大鼠的神经元染色微弱(p＜0.001)。18月龄和24月龄的大鼠海马组织的SA-β-GAL染色较6月龄增加(P＜0.001)，培养12天和20天的海马神经元SA-β-GAL染色较培养6天的海马神经元明显增多(P＜0.001)。结论： 1．对老年早期的大鼠给予6个月的限制饮食(CR)可以提高大鼠的生存率。 2．CR能够提高大鼠的活动能力，反映了大鼠的健康状况改善。 3．CR延缓衰老引起的空间学习能力减弱，提高参考记忆力的准确性。 4．大鼠空间学习记忆能力的提高可能与CR延缓了大鼠海马CA3区神经元的衰老有关。 5．SA-β-GAL是可靠的检测海马CA3区海马衰老神经元的生物学标记物。限制饮食(calorie restriction，CR)能够不同程度的延长多种生物的寿命，有效延缓的衰老和减少老年相关疾病的发生。了解CR引起这些效应的作用机制有助于指导人类的饮食和生活方式，起到延缓衰老，预防疾病发生的作用。然而，CR是如何发挥抗衰老的作用，参与其调节的分子机制还远未了解清楚。沉默信息调节因子2(silence information regulator 2，Sir2)是一种烟酰胺腺嘌呤核苷酸(nicotinamide adenine dinucleotide，NAD)依赖性去乙酰化酶，研究发现，在CR能够引起Sir2的上调，并且这一现象从低等生物酵母到高等生物如哺乳类动物，灵长类动物等普遍存在，因此，Sir2被认为是介导CR发挥效应的一个关键调节因子。哺乳动物的同源基因(sirtuin 1，SirT1)能够与多种底物作用，如p53，NF-kappaB和叉头框转录因子(Forkhead transcription factors，FoxO3a)参与细胞对氧化应激的抵抗力和基因稳定性的调节。在哺乳动物生长激素和IGF-1也参与了寿命调节，对于信号通路中FOXO家族的一员，FoxO3a参与调节多种生物学功能，包括细胞周期停滞，修复受损的DNA和衰老。CR能够在多种生物中引起胰岛素／胰岛素样生长因子-1(insulin／insulin-like growth factor 1，insulin／IGF-1)信号的抑制，其中FOXO家族是这个信号通路下游的一个主要靶点。因此，FoxO3a在CR调节中可能参与了重要的介导作用，同时FoxO3a在大脑中大量表达，尤其在发育和成年的海马，这提示FoxO3a可能参与了学习记忆的过程和(或)调节神经元的衰老。但对于FoxO3a在CR的大鼠海马的表达变化及与SirT1变化的关系，仍是一个值得探讨的问题。为探讨限制饮食引起的延缓神经元衰老的调节机制，本研究选用结合观察SirT1及FoxO3a及相关信号因子，同时，利用SirT1的激动剂白藜芦醇作用于PC12细胞，观察对培养细胞的生物学和分子生物学改变，进一步探讨SirT1与FoxO3参与CR的调控机制。材料与方法：实验动物选用18月龄的健康雄性Sprague Dawley(SD)大鼠，根据进食情况分为两组，一组为CR组(n=29)给予60％的进食量，另一组为AL组(n=31)自由进食，采用免疫组织化学和Westem blot方法观察海马CA3区SirT1和FoxO3a蛋白水平的改变。在培养的PC12细胞中，给予不同浓度的白藜芦醇，观察细胞形态学改变，利用MTT试验观察细胞增殖情况，利用RT—PCR观察SirT1，FoxO3a及细胞周期素依赖性激酶(Cyclin-dependent Kinase Inhibitors，Cdk)抑制蛋白p27~(KIP1)的mRNA水平的改变，利用流式细胞术观察白藜芦醇对PC12细胞周期的影响。结果：免疫组织化学和Westem blot检测SirT1的表达结果显示，CR组明显升高(p=0.018)，免疫组织化学观察到FoxO3a在两组中呈现细胞定位的不同，，CR组FoxO3a在细胞核内的表达减少(p=0.002)而转移至胞浆(p=0.005)。在培养的PC12细胞中，随着白藜芦醇浓度增加(5-50μmol／L)出现明显的细胞生长抑制(p＜0.001)，在细胞周期的检测中，发现10μmol／L的白藜芦醇使G0／G1期的细胞比例增加(P=0.023)。RT-PCR结果显示，10μmol／L的白藜芦醇能够使SirT1表达升高(p=0.02)，而p27~(KIP1)在20μmol／L和50μmol／L白藜芦醇刺激下表达升高(p＜0.001)，而FoxO3a的改变不明显(p=0.09)。结论： 1．在SirT1的上调和FoxO3a的失活参与了限制饮食延缓海马神经元衰老的调节 2．SirT1的激活能够使PC12细胞G0／G1期停滞
[Abstract]:Research background and purpose
Senescence is a process in which the biological functions of various organs degenerate with age. It is characterized by an increased incidence of age-related diseases, a decreased ability to regenerate cells, and an increased likelihood of death. Old age also delays and prevents the development of age-related diseases. The aging brain is functionally characterized by impaired learning and memory, while CR can reduce the incidence of cognitive-related diseases such as Alzheimer's disease. However, CR's delaying effect on the impairment of learning and memory caused by natural aging is still lacking. It is controversial whether CR can delay the decline of learning and memory in rats in the early years of old age, and whether dietary restriction in adult or early old rats can improve the growth of rats. The survival rate of rats was questioned. In determining whether CR could delay cognitive decline, determining the aging state of neurons in cognitive-related brain areas (such as the hippocampus and frontal cortex) was the cytological basis for revealing that CR delayed the decline in learning and memory, and the identification of aging cells was a marker for determining whether cells were aging. It is reported that in cultured cells, senescent cells become larger and flatter, but histologically, senescent cells are difficult to distinguish from other cells only by morphology. Biological markers provide an important method for identifying senescent cells. Senescent associated beta-galactosidase (SA-beta-GAL) is an order. It is worth mentioning that some biological markers of aging are not universal, and SA-beta-GAL as a biological marker of aging has been widely used in vitro to detect replicative aging cells. This study selected early-aged rats for dietary control, i.e. at the age of cognitive decline, in order to clearly restrict diet to the elderly. The effect of early survival state on rats and the change of learning and memory ability were observed by behavioral tests. Aging neurons were detected by SA-beta-GAL in the hippocampus, the main part of learning and memory production. The reliability of biomarkers in detecting senescent neurons in brain tissue provides behavioral and histological evidence for further study of the biological mechanism of CR delaying senescence in hippocampus.
Materials and methods
Eighteen-month-old healthy male Sprague Dawley rats were divided into two groups according to their dietary intake. One group was CR group (n=29) fed 60% of the control group, and the other group was ad libitum (AL) group (n=31) fed freely. The rats were observed for six months. During this period, the survival status of the rats was recorded and the weight of the rats was measured weekly for six months. Then, open-field test was used to observe the spontaneous activity, Morris water maze (MWM) was used to detect the spatial learning ability and reference memory ability of rats, and 6-month-old (n = 10), 18-month-old (n = 6) and 24-month-old (n = 7) SD male rats were selected to carry out SA-beta-GAL histochemical detection; meanwhile, the hippocampal neurons were detected by histochemistry. Cultured neurons were cultured for 6 days, 12 days and 20 days to carry out cytochemical detection of SA- beta -GAL.
Result
The body weight growth rate of CR group decreased significantly within 1-10 weeks after 60% dietary control, which was lower than 30% of the control group. After that, the body weight growth rate of CR group increased slowly and remained about 20% lower than that of the control group (p < 0.001). The survival rate of CR group was significantly higher than that of AL group (p = 0.039). The escape latency (EL) of the CR group was significantly shorter than that of the AL group (p = 0.021), and the spontaneous activity speed was significantly higher than that of the AL group (p = 0.021), and the CR group had longer distance in the central region (p = 0.048) and faster speed in the peripheral region (p = 0.012). In the space exploration experiment, there was no significant difference in the number of perforations between the two groups (p = 0.232). There was no significant difference in time and distance between the two groups (p = 0.324 and P = 0.367), but there was significant difference in time and distance between the two groups (p = 0.027 and P = 0.029). SA-beta-GAL staining was weaker in CR rats (p < 0.001). The SA-beta-GAL staining in hippocampus of 18-month-old and 24-month-old rats was higher than that of 6-month-old rats (P < 0.001). The SA-beta-GAL staining in hippocampus of 12-day-old and 20-day-old rats was higher than that of 6-day-old rats (P < 0.001). Hippocampal neurons increased significantly (P < 0.001).
Conclusion:
1. 6 months restriction diet (CR) for early aged rats can improve the survival rate of rats.
2.CR can improve the activity of rats and reflect the improvement of the health status of rats.
3.CR slowed down the spatial learning ability caused by aging and improved the accuracy of reference memory.
4. The improvement of spatial learning and memory in rats may be related to CR delaying the senescence of neurons in hippocampal CA3 area.
5.SA- beta -GAL is a reliable biomarker for detecting hippocampal aging neurons in hippocampal CA3 region.
Calorie restriction (CR) can prolong the life span of various organisms, effectively delay aging and reduce the incidence of diseases related to aging. Understanding the mechanism of these effects can help guide human diet and lifestyle, delay aging and prevent disease. The molecular mechanism underlying the role of anti aging and its involvement in regulation is far from clear.
Silence information regulator 2 (Sir2) is a nicotinamide adenine dinucleotide (NAD) dependent deacetylase. It has been found that CR can induce the up-regulation of Sir2, and this phenomenon can be seen from lower yeast to higher organisms such as mammals, primates and so on. Sir2 is considered to be a key regulator of CR response. Mammalian homologous genes (sirtuin 1, SirT1) can interact with a variety of substrates, such as p53, NF-kappaB and Forkhead transcription factors (FoxO3a) involved in cell resistance to oxidative stress and regulation of gene stability. Festival.
In mammals, growth hormone and IGF-1 are also involved in life-span regulation. For FOXO family members in the signaling pathway, FoxO3a is involved in regulating a variety of biological functions, including cell cycle arrest, repair of damaged DNA and aging. CR can cause insulin/insulin-like growth factor-1 (insulin/insulin-like growth facts) in a variety of organisms. Therefore, FoxO3a may play an important role in CR regulation. At the same time, FoxO3a is highly expressed in the brain, especially in the developing and adult hippocampus, suggesting that FoxO3a may be involved in the learning and memory process and/or modulation. Aging of ganglion neurons. However, the expression of FoxO3a in the hippocampus of CR rats and the relationship between FoxO3a and SirT1 is still a problem worth discussing.
To investigate the mechanism of dietary restriction in delaying neuronal senescence, SirT1 and FoxO3a and related signaling factors were observed. At the same time, resveratrol, an agonist of SirT1, was used to treat PC12 cells to observe the biological and molecular biological changes of cultured cells, and to further explore the role of SirT1 and FoxO3 in the regulation of CR. System.
Materials and methods:
Eighteen-month-old male Sprague Dawley (SD) rats were divided into two groups according to their dietary status. One group was given 60% of the food intake in CR group (n=29) and the other group was given free food in AL group (n=31). Immunohistochemistry and Western blot were used to observe the changes of SirT1 and FoxO3a protein levels in hippocampal CA3 region. Resveratrol was given at different concentrations to observe the morphological changes of cells. MTT assay was used to observe the proliferation of cells. RT-PCR was used to observe the mRNA levels of SirT1, FoxO3a and Cyclin-dependent Kinase Inhibitors (Cdk) inhibitor protein p27~ (KIP1) and flow cytometry was used to observe the effect of resveratrol on P C12 cell cycle.
Result:
Immunohistochemistry and Western blot showed that the expression of SirT1 in CR group was significantly increased (p=0.018). Immunohistochemistry showed that FoxO3a was differentially localized in the two groups. The expression of FoxO3a in the nucleus of CR group was decreased (p=0.002) and transferred to the cytoplasm (p=0.005).
In cultured PC12 cells, significant cell growth inhibition (p < 0.001) was observed with the increase of resveratrol concentration (5-50 micromol/L). 10 micromol/L of resveratrol increased the proportion of G0/G1 phase cells (P = 0.023). RT-PCR results showed that 10 micromol/L of resveratrol could increase the expression of SirT1 (p = 0.02). However, the expression of p27~ (KIP1) was increased (p < 0.001) when stimulated by 20 and 50 micromol/L resveratrol, while the change of FoxO3a was not significant (p = 0.09).
Conclusion:
1. up regulation of SirT1 and inactivation of FoxO3a are involved in regulating the aging of hippocampal neurons by restricting diet.
Activation of 2.SirT1 can arrest the G0 / G1 phase of PC12 cells.
【学位授予单位】：汕头大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363

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