莲房原花青素对老年认知障碍大鼠记忆功能的改善作用及其机制研究
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摘要
正常老化是一个独立于病理条件下的正常而复杂的生物学过程。在衰老过程中,机体的许多系统都出现各种进行性的结构和功能的改变。在神经系统,与年龄增加相关的最主要的衰退之一是认知功能障碍。认知功能是人类与感知、思维、记忆、学习、想象和推理等有关的信息处理能力,是人类正常生存的必需能力和智能发展和维持的基础。人类和动物的老化都导致认知障碍。学习记忆能力作为认知功能的一部分,大量研究已经证实人和动物学习和记忆能力都存在增龄性衰退现象。这种年龄相关性的记忆损害在从正常认知到轻度认知障碍(MCI, mild cognitive impairment)再到老年痴呆(AD, Alzheimer's disease)的整个过程中都可能发生。虽然这种年龄相关性的记忆损害对生活质量的影响没有AD那样严重,但其在老年人群中的发病率非常高。研究表明有38%的年龄在60-78岁的老年人符合年龄相关性记忆障碍(AAMI, age associated memory impairment)的标准,并且9%的AAMI病人将发展为痴呆,而诊断为MCI的个体中55%的人在4.5年内发展为痴呆。因此年龄相关性记忆损害是影响老年生活质量的和生活乐趣的一个关键因素,有必要寻找安全而又切实可行的防治措施。为了解释年龄相关性记忆障碍的发生机制,人们提出了各种各样的假说,这其中,老化的胆碱能假说、老化的自由基假说、老化的一氧化氮假说是被广泛接受的解释。另外,老化过程中脑CREB介导的转录受损也越来越引起人们的注意。
原花青素是一类多酚类物质,它们是由黄烷-3-醇单体组成的低聚物或多聚物,这些物质广泛分布于水果、蔬菜、种子、花以及树皮中。由于它们具有极强的抗氧化能力和极高的安全性,现在已经广受关注。原花青素清除自由基的能力明显优于维生素C、维生素E和β-胡萝卜素。除此之外,这些物质还具有抗菌、抗病毒、抗炎以及抗癌等多种功能。原花青素的代谢产物能够穿过血脑屏障而可被脑组织摄取。由于在脑中可以检测到这些代谢物的存在,因而其可能对脑功能发挥潜在的调节作用。原花青素在脑中的有益生物学作用一般认为是基于其优良的抗氧化能力。例如,以往的研究证实原花青素可抑制脑中的脂质过氧化和年龄相关性的DNA氧化损伤的蓄积。现在越来越多的证据表明原花青素所具有的其它一些调节机制可能也参与了对脑功能的调节作用。例如,蛋白组学研究表明原花青素能够系统地调节年轻大鼠脑中某些特定蛋白质的表达和活性；而(-)-表儿茶素则被发现可上调含有GluR2的AMPA受体,而上调此受体意味着对突触功能的潜在调节作用。
为了探讨莲房原花青素(LSPC, procyanidins from the lotus seedpod)对AAMI的改善作用以及作用机制,我们首先确定LSPC对东莨菪碱所致记忆障碍模型小鼠和老年认知障碍大鼠记忆能力是否具有改善作用,然后从自由基假说、胆碱能假说、一氧化氮假说和CREB改变几个方面探讨LSPC改善老年认知障碍大鼠记忆能力的可能机制。
第一部分行为学研究
第一节LSPC对东莨菪碱所致记忆障碍小鼠模型学习记忆能力的影响
目的确定LSPC对东莨菪碱所致记忆障碍小鼠学习记忆的改善作用。
方法2月龄雄性昆明种小鼠50只(18-22 g)被随机分为5组(每组10只)：对照组、低中高剂量组(L-、M-、-H-LSPC)和东莨菪碱组。L-、M-、H-LSPC组的小鼠分别给予50,100,150 mg/kg BW LSPC灌胃30 d,对照组和东莨菪碱组灌胃等量的蒸馏水。在进行行为学测试期间,L-、M-、H-LSPC组小鼠每天在第一次测试前1h灌胃LSPC, L-、M-、H-LSPC组和东莨菪碱组小鼠每天在第一次测试前10 min腹腔注射东莨菪碱(1mg/kg),对照组小鼠腹腔注射生理盐水。用Morris水迷宫和跳台试验评估小鼠学习记忆能力。在Morris水迷宫测试中,记录每次训练的潜伏期和游泳距离。跳台试验在Morris水迷宫结束7 d后进行,记录错误次数和潜伏期。跳台试验完成后,将小鼠处死并将脑匀浆进行AChE检测。
结果三个剂量的LSPC处理组,除了L-LSPC组第1 d的潜伏期明显高于对照组外,其它时间和剂量的LSPC组的潜伏期与对照组相当。所有LSPC剂量组在第4和第5 d的潜伏期均明显少于东莨菪碱组；所有LSPC剂量组在第4和第5 d的游泳距离均明显少于东莨菪碱组并与对照组没有显著性差异。在跳台试验中,所有LSPC剂量组的错误次数均明显低于东莨菪碱组,同时所有LSPC剂量组的潜伏期均明显低于对照组,但是又都显著高于东莨菪碱组。LSPC剂量依赖性地抑制AChE活性,所有LSPC剂量组的AChE活性均明显低于东莨菪碱组并与对照组相比无显著性差异。
结论通过Morris水迷宫和跳台试验评估,LSPC可改善东莨菪碱所致记忆障碍小鼠模型的学习记忆能力,其抑制AChE活性而增加胆碱能活性的作用可能参与了这一过程。
第二节LSPC对老年认知障碍大鼠学习记忆能力的影响
目的确定LSPC对老年认知障碍大鼠学习记忆能力的改善作用。
方法以40只年轻雌性大鼠在Morris水迷宫中的潜伏期为基础,从200只18月龄雌性SD大鼠中筛选出老年认知正常(AU)大鼠和老年认知障碍(AI)大鼠。从年轻大鼠中随机挑选14只大鼠组成年轻组；从AU大鼠中随机挑选16只大鼠组成AU组；同时将AI大鼠随机分为3组(每组16只)：AI组和低、高LSPC(L-、H-LSPC)剂量组。L-、H-LSPC组大鼠每日分别灌胃50和100 mg/kg BW LSPC,其它三组大鼠灌胃等量生理盐水。喂养周期为7 w。在其后进行的Morris水迷宫测试期间,所有动物在每日第一次训练前1 h继续进行相同处理。用与筛选时完全不同参照体系的Morris水迷宫试验评估各组大鼠的学习记忆能力。
结果与年轻组和AU组相比,AI组大鼠在Morris水迷宫中的潜伏期明显延长。除H-LSPC组在训练的第1 d与AI组差异无显著性外,两个LSPC剂量组在测试期间的潜伏期均明显少于AI组。与年轻组和AU组相比,H-LSPC组的潜伏期在整个测试期间没有显著性差异。在训练的第3-5 d,H-LSPC组的游泳距离明显少于AI组；在训练的第2和第4d, L-LSPC组的游泳距离也明显少于AI组。在整个训练期间,H-LSPC组的游泳距离与年轻组和AU组相比均未见显著差异。
结论LSPC可明显改善AI大鼠的空间学习记忆能力。
第二部分机制研究
第三节抗氧化和胆碱能系统的改变促进LSPC改善老年认知障碍大鼠学习记忆能力的机制研究
目的探讨LSPC对AI大鼠海马和皮质抗氧化和胆碱能系统的作用。
方法实验设计同第一部分第二节。将完成Morris水迷宫测试的动物处死后分离海马和皮质。每组随机挑选4只大鼠样本进行后续研究。对于剩下的样本,左侧海马和皮质匀浆后进行抗氧化和氧化损伤指标以及ChAT、AChE活性的检测。右侧海马和皮质用含有3 mg/ml毒扁豆碱的磷酸盐缓冲液匀浆后进行ACh检测。
结果所有实验组大鼠海马和皮质的SOD活性均未见明显改变。两个老年组的海马和皮质CAT和GPx活性均显著低于年轻组,然而这两种酶在AU和AI组之间没有发现明显差异。与AI组相比,CAT、GPx活性在H-LSPC海马和皮质中明显增加,在L-LSPC组海马中也显著增加。在AU和AI组,海马和皮质的CAT/SOD和GPx/SOD比值均明显降低；与AI组相比,LSPC,特别是H-LSPC可逆转CAT/SOD和GPx/SOD比值的降低。AI和AU大鼠海马和皮质中GSH水平明显低于年轻大鼠,H-LSPC明显上调这两个部分的GSH水平。AU组和AI组海马和皮质的总抗氧化能力(T-AOC)均显著下降,而两个LSPC剂量组的T-AOC水平均明显升高并与年轻组无显著性差异。AU组和AI组海马和皮质MDA水平和蛋白羰基含量均明显高于年轻组。AU组和AI组之间的海马和皮质MDA水平未见显著性差异,然而AI组海马和皮质的蛋白羰基含量明显高于AU组。与AI组相比,两个LSPC剂量组海马和皮质中MDA水平和蛋白羰基含量均明显降低。各个实验组海马和皮质中ChAT活性均未见明显改变。AU和AI组大鼠海马AChE活性均显著低于年轻大鼠；AI组大鼠皮质AChE活性明显低于年轻大鼠,而AU组大鼠皮质AChE活性与年轻大鼠没有显著性差异。H-LSPC组海马AChE活性以及L-、H-LSPC组皮质AChE活性均明显高于AI组。与年轻组和AU组大鼠相比,AI组大鼠的海马和皮质ACh含量显著下降。LSPC可剂量依赖性地增加ACh含量,并且两个LSPC剂量组大鼠的海马和皮质ACh含量均与年轻组大鼠相当。
结论LSPC改善AI大鼠海马和皮质抗氧化系统的功能。降低脑的氧化损伤和增加胆碱能系统功能参与了LSPC改善AI大鼠空间学习记忆能力的作用。
第四节一氧化氮系统改变促进LSPC改善老年认知障碍大鼠学习记忆能力的机制研究
目的探讨海马和皮质一氧化氮系统改变对LSPC改善AI大鼠学习记忆能力的影响。
方法实验设计以及组织样品的准备同第二部分第三节。每组随机挑选4只大鼠进行实时定量PCR和western blot检测。其余样品的部分匀浆用于NO水平以及总NOS和分型NOS的检测。为了评估在蛋白酶酶抑制剂或磷酸酶抑制剂存在的情况下NOS活性,向匀浆中加入蛋白激酶抑制剂或磷酸酶抑制剂,然后用HEPES终止反应,再进行iNOS活性的检测。
结果AI大鼠海马和皮质NO水平明显高于年轻大鼠。所有实验组的海马和皮质tNOS活性改变均与其NO水平的改变相平行。AI大鼠海马iNOS活性明显高于AU组和年轻组,同时AU大鼠海马iNOS活性与年轻大鼠没有明显差异。另外,虽然AU和AI大鼠海马：iNOS活性均明显降低,但是AI大鼠又比AU大鼠进一步明显降低。虽然AU和AI大鼠皮质iNOS活性均大幅显著增加同时nNOS活性明显降低,但是AU和AI大鼠皮质iNOS以及nNOS活性并没有明显差异。iNOS和nNOS的基因和蛋白表达也有相似的变化。用蛋白激酶抑制剂或磷酸酶抑制剂处理后,年轻组nNOS活性改变率变化最大,AU组也有相当大幅度的改变；然而这些抑制剂对AI大鼠海马nNOS活性改变率的影响则很小。LSPC明显降低AI大鼠海马和皮质iNOS活性并因此导致tNOS活性和NO水平降低。虽然LSPC并没有明显改变AI大鼠海马和皮质nNOS活性,但是却有增加AI大鼠海马nNOS基因和蛋白表达的趋势。因此,LSPC剂量组大鼠海马的单位nNOS蛋白活性降低。另外,虽然LSPC在蛋白激酶抑制剂存在的情况下并没有明显改变AI大鼠的海马nNOS活性,但却在磷酸酶抑制剂存在的情况下明显改变AI大鼠海马nNOS活性。结论AI大鼠认知功能的降低主要是海马iNOS和nNOS的表达和活性改变所致,而iNOS和nNOS在皮质中的改变则不是主要因素。LSPC不但降低AI大鼠海马iNOS的表达和活性,而且也可使海马nNOS磷酸化调节更加有效,从而抑制年龄相关性的NO过表达、增加海马NO-sGC-cGMP信号转导。表明LSPC可能通过海马NO系统的上述改变而改善AI大鼠的认知功能。
第五节CREB磷酸化改变促进LSPC改善老年认知障碍大鼠学习记忆能力的机制研究
目的探讨海马和皮质中CREB磷酸化改变对LSPC改善AI大鼠学习记忆能力的影响。方法实验设计以及组织样品的准备同第二部分第三节。每组随机挑选4只大鼠样本进行BDNFmRNA的实时定量PCR检测和CREB、pCREB、BDNF、ERK 42/44、pERK 42/44、CaMKIV、pCaMKIV和PKA C-a的western blot检测。
结果AI大鼠海马CREB磷酸化程度明显低于年轻大鼠与AU大鼠；LSPC可剂量依赖性地明显增加AI大鼠海马CREB磷酸化程度。各个实验组的皮质CREB磷酸化程度没有明显差异。AI大鼠海马BDNF mRNA和蛋白表达水平明显低于年轻大鼠和AU大鼠；两个剂量的LSPC均明显增加AI大鼠海马BDNF mRNA和蛋白表达水平。各个实验组大鼠皮质BDNF mRNA和蛋白表达水平未见显著差异。AI大鼠海马ERK42、ERK44蛋白磷酸化程度显著低于年轻大鼠和AU大鼠。相对于AI组,L-LSPC组大鼠海马ERK42蛋白磷酸化程度有增加的趋势,H-LSPC组大鼠海马ERK42蛋白磷酸化程度在L-LSPC剂量组的基础上又明显增加,并与年轻组没有明显差异。两个LSPC剂量组海马ERK44蛋白磷酸化程度都明显高于AI组,并且与AU组相当。AI大鼠海马CaMKIV磷酸化状态显著低于年轻大鼠和AU大鼠；补充LSPC后没有改变海马的CaMKIV磷酸化状态。所有实验组动物海马PKA C-a水平均没有显著差异。
结论相对于年轻大鼠和AU大鼠,AI大鼠海马CREB磷酸化程度明显降低,然而其皮质CREB磷酸化程度没有明显改变。AI大鼠海马ERK 42／44和CaMKIV磷酸化程度降低都促进其CREB活性的降低。海马CREB活性降低导致CREB依赖性的转录降低,从而损害长期记忆的形成。例如,AI大鼠海马BDNF mRNA和蛋白水平明显降低。这些结果表明海马CREB活性降低明显参与了年龄相关性老年记忆障碍的形成和发展过程。LSPC可通过ERK介导的通路活化海马CREB,从而易化长期记忆的形成过程,并因此改善AI大鼠的学习记忆能力。
Aging is a normal but complicated, not pathological, process, which characterized by slowly progressing impairments in structure and function of most systems. In central nervous system (CNS), age-related cognitive dysfunction is one of the most prominent features. Cognitive function refers to a person's ability to process information, such as perception, thinking, recognition, sensing, learning, memory, imagining, and reasoning, and which is necessary for survival as well as intellectual development and maintenance. As the natural process of aging progresses, humans and animals experience a progressive decline in overall cognitive function. As an integral part of cognitive function, learning and memory abilities have been demonstrated to marked decline in the course of normal aging. The age associated memory impairment (AAMI) may range from normal to a mild cognitive impairment, or to Alzheimer's disease (AD). Although its passive impact on quality of life is not as severe as AD, the age associated memory impairment is very prevalent in elderly people. It has been shown that thirty-eight percent of 60-78 years old individuals fulfill the criteria for AAMI, and in the follow-up nine percent of AAMI patients became demented. In addition, during a follow-up period of four and a half years fifty-five percent of individuals with the diagnosis of mild cognitive impairment developed dementia. Therefore, AAMI is one of crucial determinants of the quality and enjoyment of life of elderly people, and it is necessary to seek for some measures for prevention and treatment AAMI with little or not damage to body. Various hypothesizes have been proposed to try to explain the mechanisms of geriatric memory dysfunction. Among them, the cholinergic hypothesis of geriatric memory dysfunction, the free radical hypothesis of aging and the nitric oxide hypothesis of aging have been most widely accepted. In addition, the impaired CREB-mediated transcription in brain during aging has also been paid more and more attention.
Procyanidins chemically belong to polyphenols, they are oligomeric and polymeric flavonoids comprised of flavan-3-ol monomeric subunits. These compounds are widely distributed in fruits, vegetables, seeds, flowers and bark. They have received considerable attention in recent years due to their excellent antioxidant activity and a broad spectrum of safety. Procyanidins posses significantly better free radical scavenging capability than vitamin C, E orβ-carotene. In addition, these compounds have also been reported to exhibit a wide range of biological effects including antibacterial, antiviral, anti-inflammatory, and anticarcinogen actions. The metabolites of procyanidins are able to cross the blood brain barrier and be uptaken and detected in brain and thus exert potential effects on brain functions. Beneficial bioactivities of procyanidins in brain are often claimed to be based on their antioxidant properties. For example, previous experiments demonstrated that procyanidins had an inhibiting effect on the lipid peroxidation and accumulation of age-related oxidative DNA damage in brain. But increasing evidence suggests that some other mechanisms may involve the modulation of brain functions. A proteomics study indicated that procyanidins were able to systematically change specific brain proteins in expression or modification in young rats. (-)-Epicatechin has been found to up-regulate GluR2-containing AMPA receptors, which means it has the potential to modulate synaptic function.
In order to explore the effects of procyanidins extracted from the lotus seedpod (LSPC) on AAMI, the primary goal of this research is to find whether LSPC ameliorate memory impairment in scopolamine-induced mice and cognitively impaired aged rats. When the positive results were observed, we further explored the mechanisms which refer to cholinergic hypothesis, the free radical hypothesis, nitric oxide hypothesis and changes in CREB-mediated transcription.
PartⅠBehavioral study
Section one LSPC ameliorate scopolamine-induced memory impairment in mice
Objective To determine the ameliorating effect of LSPC on the learning and memory impairments induced by scopolamine in mice.
Methods Fifty two-month old male Kunming mice (18-22 g) were randomly divided into five groups with 10 mice in each group:the control group, the low, middle and high dose LSPC (L-, M-, and H-LSPC) groups and the scopolamine group. Mice in L-, M-, and H-LSPC groups were given 50,100,150 mg/kg BW LSPC orally by a gavage daily for 30 days respectively. The control group and scopolamine group were given an equivalent amount of distilled water daily by oral gavage. On each day of the behavioral tests period, mice in L-, M-, and H-LSPC groups were given LSPC 1 h before the first trial session. Animals in L-, M-, and H-LSPC groups and scopolamine group were treated with scopolamine (1 mg/kg, i.p) dissolved in normal saline at 10 min before the first trial session during the training phase of both behavioral tests. Mice in the control group received vehicle only. The capacities of memory and learning were evaluated by the Morris water maze and the step-down avoidance test. During each trial session in Morris water maze, escape latency and swimming distance were recorded. The step-down avoidance test was performed 7 days after the Morris water maze, and the numbers of errors and step-down latency were recorded. After completion of step-down avoidance test, the mice were killed and the brains were homogenized to determine the activity of AChE.
Results No difference in escape latency was observed among the LSPC groups and the control group during all the training days only except that L-LSPC group had significantly longer escape latency relative to the control groups on day 1. Escape latency of all the three LSPC groups on day 4 and 5 were significantly shorter compared to that of scopolamine group. Mice in three LSPC groups on day 4 and 5 exhibited significantly shorter swimming distances compared with that in scopolamine group, but no differences were observed when compared with that in control group. In step-down avoidance test, reduced the number of errors in all the three LSPC groups were observed compared with scopolamine group. In addition, all the three LSPC groups had significant shorter latencies than the control group and significantly longer latencies than scopolamine group. LSPC inhibited AChE activity in a dose-dependent manner, and no significant difference was found among all the three LSPC groups and the control group.
Conclusion LSPC has the ability to ameliorate the impairment of learning and memory induced by scopolamine in both Morris water maze test and step-down avoidance test, and increasing cholinergic activity by inhibition of AChE activity may involve in the ameliorative activities of LSPC on learning and memory dysfunction.
Section two LSPC ameliorate memory impairment in cognitively impaired aged rats
Objective To determine the ameliorating effect of LSPC on the learning and memory impairments in cognitively impaired aged rats.
Methods Based on Morris water maze performance compared with forty young female rats, aged-unimpaired (AU) and aged-impaired (AI) rats were chosen from two hundred 18-month-old female Sprague-Dawley rats. The rats in young control group (n=14) and AU group (n=16) were randomly selected from the young rats and aged unimpaired rats respectively. The animals categorized as AI were randomly subdivided into three groups consisting of 16 animals each:AI group, low and high dose LSPC (L-and H-LSPC) groups. Rats in L-and H-LSPC groups were given 50 and 100 mg/kg BW LSPC orally by a gavage daily for 7 weeks, respectively. The other three groups were given orally an equivalent volume of distilled water daily. On each day of the behavioral tests period in Morris water maze, animals continued to be administered intragastrically 1 h before the first trial session. The learning and memory abilities of rats in different groups were assessed by retraining the animals in Morris water maze with entirely different cues and testing the animals as before.
Results Rats in AI group exhibited a significant prolongation of escape latency in Morris water maze compared with those of young group and AU group. Both LSPC groups exhibited significantly shorter escape latencies than AI group during all sessions only except H-LSPC group on the first training day. Meanwhile, H-LSPC group did not exhibit significant difference in escape latency from young and AU groups. Significantly shorter swimming distance than that of AI group was observed in H-LSPC group on day 3-5 and L-LSPC group on day 2 and day 4. Compared with young and AU groups, H-LSPC group did not exhibit difference in swimming distance during all the test days.
Conclusion LSPC has the ability to ameliorate the impairment of learning and memory in AI animals in Morris water maze test.
Part II Mechanism study
Section three Rejuvenation of antioxidant and cholinergic systems contributes to the effect of LSPC ameliorating memory impairment in cognitively impaired aged rats
Objective To determine the effect of LSPC on the antioxidant and cholinergic systems in hippocampus and cerebral cortex in cognitively impaired aged rats.
Methods Study design was the same as part I, section two. After completion of water maze test, animals were killed and the hippocampus and cerebral cortex were dissected. Four animal samples in each group were randomly selected for next study. For the remaining samples, hippocampus and cerebral cortex in left hemisphere were homogenized to determine the antioxidant capacity, oxidative damage markers as well as activities of ChAT and AChE. The hippocampus and cerebral cortex in right hemisphere were homogenized in phosphate buffer containing 3 mg/ml eserine for ACh assay.
Results There were not significant changes in SOD activity in hippocampus and cerebral cortex in all the experimental groups. However, CAT and GPx activities of the both brain regions of old animals were significantly lower than those of young subjects, but no difference in activities was observed between AU and AI groups. Significant increase in CAT and GPx activities of both brain regions in H-LSPC group and in GPx activities of hippocampus in L-LSPC group were observed when compared with the AI group. The ratios of CAT/SOD and GPx/SOD were significantly lower in AU and AI groups. LSPC, especially H-LSPC, reversed the declines in these ratios when compared with AI. The levels of GSH in both brain regions of aged rats were significantly lower than those of young animals, and H-LSPC administration up-regulated significantly the GSH level in both regions. Hippocampal and cortical T-AOC in AU and AI groups markedly declined, and animals in both LSPC groups revealed higher T-AOC than that in AU and AI groups and were similar to their young counterparts. All AU and AI animals showed significant higher MDA levels and carbonyl content in hippocampus and cerebral cortex than young animals. There were not significant differences in MDA levels of both brain regions between AU and AI groups, whereas AI animals exhibited significantly higher carbonyl content in both brain regions than AU animals. Reduced MDA levels and carbonyl content in hippocampus and cerebral cortex in both LSPC groups were observed compared with AI group. There were not significant differences in ChAT activity in hippocampus and cerebral cortex in all the experimental groups. Animals in both AU and AI groups showed significantly lower AChE activities in hippocampus than their young counterparts. In cerebral cortex, animals in AI group also revealed significantly lower AChE activities than young animals, whereas AU animals were not significantly different from young animals. The AChE activities in hippocampus of H-LSPC group and in cerebral cortex of both LSPC groups were significantly higher than that in AI group. There was a substantial decrease in ACh content in both brain regions for AI group compared with young and AU groups. LSPC considerably increased ACh content in a dose-dependent manner, and no significant difference was found among both LSPC groups and the young group.
Conclusion LSPC attenuated functional decline in antioxidant system in hippocampus and cerebral cortex of AI rats. The decreased oxidative damage to brain and increasing cholinergic activity may involve in the ameliorative effects of LSPC on learning and memory dysfunction in AI animals.
Section four Changes in nitric oxide system contribute to the effect of LSPC ameliorating memory impairment in cognitively impaired aged rats
Objective To determine the effects of LSPC on the nitric oxide system in hippocampus and cerebral cortex in cognitively impaired aged rats.
Methods Study design and tissue preparation were the same as partⅡ, section three. Four animal samples in each group were randomly selected for real-time PCR and western blot analysis. For the remaining animal samples, part of the homogenate was used for NO level as well as total and NOS isoforms activities evaluation. In order to determination of the effect of phosphorylation/dephosphorylation on nNOS activity, homogenate were preincubated with protein kinase inhibitors cocktail or phosphatase inhibitor and terminated with HEPES, then nNOS activity were measured.
Results The hippocampal and cortical NO levels in AI animals were markedly higher than that in young animals. The alterations in tNOS activity of both brain regions closely paralleled the changes in NO level of that in all the experimental groups. AI rats exhibited significantly higher hippocampal iNOS activities than AU and young animals, and no differences in hippocampal iNOS activity were observed between young and AU animals. In addition, although marked decrease in hippocampal nNOS activity occurred in all AU and AI rats, AI animals exhibited further significant lower hippocampal nNOS activities than AU animals. However, although cortical iNOS activities were increased by a large margin and at the same time nNOS activities were reduced in all AU and AI animals, there was no differences in both iNOS and nNOS activities between AI and AU animals. Similar alteration trends were also observed in terms of the gene and protein expressions in both iNOS and nNOS. When treated with protein kinase inhibitors or phosphatase inhibitor, although young group revealed the most alteration in the rate of nNOS activity change, AU group also had considerable changes in that. However, the effects of these inhibitors on the rates of nNOS activity change were almost abrogated in hippocampus of AI animals. Both doses of LSPC significantly decreased iNOS activities and consequently resulted in tNOS activities and NO levels decrease in hippocampus and cerebral cortex of AI animals. LSPC supplementation did not lead to marked alterations for hippocampal and cortical nNOS activity in AI animals, whereas AI animals revealed a trend to increase nNOS gene and protein expressions in hippocampus with LSPC supplementation. As a result, animals in LSPC supplementation groups should have a decline in nNOS activity per unit protein in hippocampus. Moreover, although LSPC supplementation had not marked effect on the rates of hippocampal nNOS activity change in the presence of protein kinase inhibitors in AI animals, these compounds considerably altered these rates in these animals in the presence of phosphatase inhibitor.
Conclusion The alterations of expressions and activities in hippocampal but not cortical iNOS and nNOS accounted for the reduced cognitive behaviors in AI animals. LSPC supplementation not only decreased hippocampal iNOS activities and enzyme expression but also made hippocampal nNOS phosphorylation regulated effectively in AI animals, which led to the inhibition of age-related NO overproduction and increase of hippocampal NO-sGC-cGMP signal transduction in these animals. These changes in hippocampal NO system may involve, at least partly, in the ameliorative effects of LSPC on learning and memory dysfunction in AI animals.
Section five Memory impairment in cognitively impaired aged rats associated with decreased hippocampal CREB phosphorylation: reversal by LSPC
Objective To determine the effects of LSPC on CREB phosphorylation in hippocampus and cerebral cortex in cognitively impaired aged rats.
Methods Study design and tissue preparation were the same as part II, section three. Four animal samples in each group were randomly selected for real-time PCR of BDNF mRNA and western blot analysis of CREB, pCREB, BDNF, ERK 42/44, pERK 42/44, CaMKIV, pCaMKIV and PKA C-α.
Results The phosphorylation states of CREB in hippocampus of AI animals were markedly lower than that of young and AU animals. Supplementation of LSPC led to significant increase in hippocampal CREB phosphorylation with a dose-dependent manner. All groups were not significantly different from each other in cortical CREB phosphorylation. The BDNF mRNA and protein levels were markedly reduced in hippocampus of AI animals compared to that measured in young and AU animals. AI animals with LSPC supplementation uniformly exhibited significantly higher hippocampal BDNF mRNA and protein expression than AI animals. As for the BDNF mRNA and protein expression in cerebral cortex, no significant changes were detected in any of the groups. The significant lower states of phosphorylation in hippocampal ERK42 and ERK44 protein were observed in AI animals relative to young and AU animals. Animals in group with L-LSPC supplementation had a tendency to elevate the phosphorylation states of hippocampal ERK42 relative to AI rats, and rats in group with H-LSPC supplementation revealed further significant increase in that and were comparable with young animals. The phosphorylation states of hippocampal ERK44 in both LSPC groups were substantially increased than that in AI group and reach to the level of AU group. Substantial decrease in phosphorylation states of CaMKIV were detected in AI animals relative to young and AU counterparts. The phosphorylation states of this protein did not significantly increase in response to LSPC supplementation. No substantial alterations in the level of PKA C-a were detected among all the experimental groups.
Conclusion The alterations of expressions and activities in hippocampal but not cortical iNOS and nNOS accounted for the reduced cognitive behaviors in AI animals. LSPC supplementation not only decreased hippocampal iNOS activities and enzyme expression but also made hippocampal nNOS phosphorylation regulated effectively in AI animals, which led to the inhibition of age-related NO overproduction and increase of hippocampal NO-sGC-cGMP signal transduction in these animals. These changes in hippocampal NO system may involve, at least partly, in the ameliorative effects of LSPC on learning and memory dysfunction in AI animals.
原花青素是一类多酚类物质,它们是由黄烷-3-醇单体组成的低聚物或多聚物,这些物质广泛分布于水果、蔬菜、种子、花以及树皮中。由于它们具有极强的抗氧化能力和极高的安全性,现在已经广受关注。原花青素清除自由基的能力明显优于维生素C、维生素E和β-胡萝卜素。除此之外,这些物质还具有抗菌、抗病毒、抗炎以及抗癌等多种功能。原花青素的代谢产物能够穿过血脑屏障而可被脑组织摄取。由于在脑中可以检测到这些代谢物的存在,因而其可能对脑功能发挥潜在的调节作用。原花青素在脑中的有益生物学作用一般认为是基于其优良的抗氧化能力。例如,以往的研究证实原花青素可抑制脑中的脂质过氧化和年龄相关性的DNA氧化损伤的蓄积。现在越来越多的证据表明原花青素所具有的其它一些调节机制可能也参与了对脑功能的调节作用。例如,蛋白组学研究表明原花青素能够系统地调节年轻大鼠脑中某些特定蛋白质的表达和活性；而(-)-表儿茶素则被发现可上调含有GluR2的AMPA受体,而上调此受体意味着对突触功能的潜在调节作用。
为了探讨莲房原花青素(LSPC, procyanidins from the lotus seedpod)对AAMI的改善作用以及作用机制,我们首先确定LSPC对东莨菪碱所致记忆障碍模型小鼠和老年认知障碍大鼠记忆能力是否具有改善作用,然后从自由基假说、胆碱能假说、一氧化氮假说和CREB改变几个方面探讨LSPC改善老年认知障碍大鼠记忆能力的可能机制。
第一部分行为学研究
第一节LSPC对东莨菪碱所致记忆障碍小鼠模型学习记忆能力的影响
目的确定LSPC对东莨菪碱所致记忆障碍小鼠学习记忆的改善作用。
方法2月龄雄性昆明种小鼠50只(18-22 g)被随机分为5组(每组10只)：对照组、低中高剂量组(L-、M-、-H-LSPC)和东莨菪碱组。L-、M-、H-LSPC组的小鼠分别给予50,100,150 mg/kg BW LSPC灌胃30 d,对照组和东莨菪碱组灌胃等量的蒸馏水。在进行行为学测试期间,L-、M-、H-LSPC组小鼠每天在第一次测试前1h灌胃LSPC, L-、M-、H-LSPC组和东莨菪碱组小鼠每天在第一次测试前10 min腹腔注射东莨菪碱(1mg/kg),对照组小鼠腹腔注射生理盐水。用Morris水迷宫和跳台试验评估小鼠学习记忆能力。在Morris水迷宫测试中,记录每次训练的潜伏期和游泳距离。跳台试验在Morris水迷宫结束7 d后进行,记录错误次数和潜伏期。跳台试验完成后,将小鼠处死并将脑匀浆进行AChE检测。
结果三个剂量的LSPC处理组,除了L-LSPC组第1 d的潜伏期明显高于对照组外,其它时间和剂量的LSPC组的潜伏期与对照组相当。所有LSPC剂量组在第4和第5 d的潜伏期均明显少于东莨菪碱组；所有LSPC剂量组在第4和第5 d的游泳距离均明显少于东莨菪碱组并与对照组没有显著性差异。在跳台试验中,所有LSPC剂量组的错误次数均明显低于东莨菪碱组,同时所有LSPC剂量组的潜伏期均明显低于对照组,但是又都显著高于东莨菪碱组。LSPC剂量依赖性地抑制AChE活性,所有LSPC剂量组的AChE活性均明显低于东莨菪碱组并与对照组相比无显著性差异。
结论通过Morris水迷宫和跳台试验评估,LSPC可改善东莨菪碱所致记忆障碍小鼠模型的学习记忆能力,其抑制AChE活性而增加胆碱能活性的作用可能参与了这一过程。
第二节LSPC对老年认知障碍大鼠学习记忆能力的影响
目的确定LSPC对老年认知障碍大鼠学习记忆能力的改善作用。
方法以40只年轻雌性大鼠在Morris水迷宫中的潜伏期为基础,从200只18月龄雌性SD大鼠中筛选出老年认知正常(AU)大鼠和老年认知障碍(AI)大鼠。从年轻大鼠中随机挑选14只大鼠组成年轻组；从AU大鼠中随机挑选16只大鼠组成AU组；同时将AI大鼠随机分为3组(每组16只)：AI组和低、高LSPC(L-、H-LSPC)剂量组。L-、H-LSPC组大鼠每日分别灌胃50和100 mg/kg BW LSPC,其它三组大鼠灌胃等量生理盐水。喂养周期为7 w。在其后进行的Morris水迷宫测试期间,所有动物在每日第一次训练前1 h继续进行相同处理。用与筛选时完全不同参照体系的Morris水迷宫试验评估各组大鼠的学习记忆能力。
结果与年轻组和AU组相比,AI组大鼠在Morris水迷宫中的潜伏期明显延长。除H-LSPC组在训练的第1 d与AI组差异无显著性外,两个LSPC剂量组在测试期间的潜伏期均明显少于AI组。与年轻组和AU组相比,H-LSPC组的潜伏期在整个测试期间没有显著性差异。在训练的第3-5 d,H-LSPC组的游泳距离明显少于AI组；在训练的第2和第4d, L-LSPC组的游泳距离也明显少于AI组。在整个训练期间,H-LSPC组的游泳距离与年轻组和AU组相比均未见显著差异。
结论LSPC可明显改善AI大鼠的空间学习记忆能力。
第二部分机制研究
第三节抗氧化和胆碱能系统的改变促进LSPC改善老年认知障碍大鼠学习记忆能力的机制研究
目的探讨LSPC对AI大鼠海马和皮质抗氧化和胆碱能系统的作用。
方法实验设计同第一部分第二节。将完成Morris水迷宫测试的动物处死后分离海马和皮质。每组随机挑选4只大鼠样本进行后续研究。对于剩下的样本,左侧海马和皮质匀浆后进行抗氧化和氧化损伤指标以及ChAT、AChE活性的检测。右侧海马和皮质用含有3 mg/ml毒扁豆碱的磷酸盐缓冲液匀浆后进行ACh检测。
结果所有实验组大鼠海马和皮质的SOD活性均未见明显改变。两个老年组的海马和皮质CAT和GPx活性均显著低于年轻组,然而这两种酶在AU和AI组之间没有发现明显差异。与AI组相比,CAT、GPx活性在H-LSPC海马和皮质中明显增加,在L-LSPC组海马中也显著增加。在AU和AI组,海马和皮质的CAT/SOD和GPx/SOD比值均明显降低；与AI组相比,LSPC,特别是H-LSPC可逆转CAT/SOD和GPx/SOD比值的降低。AI和AU大鼠海马和皮质中GSH水平明显低于年轻大鼠,H-LSPC明显上调这两个部分的GSH水平。AU组和AI组海马和皮质的总抗氧化能力(T-AOC)均显著下降,而两个LSPC剂量组的T-AOC水平均明显升高并与年轻组无显著性差异。AU组和AI组海马和皮质MDA水平和蛋白羰基含量均明显高于年轻组。AU组和AI组之间的海马和皮质MDA水平未见显著性差异,然而AI组海马和皮质的蛋白羰基含量明显高于AU组。与AI组相比,两个LSPC剂量组海马和皮质中MDA水平和蛋白羰基含量均明显降低。各个实验组海马和皮质中ChAT活性均未见明显改变。AU和AI组大鼠海马AChE活性均显著低于年轻大鼠；AI组大鼠皮质AChE活性明显低于年轻大鼠,而AU组大鼠皮质AChE活性与年轻大鼠没有显著性差异。H-LSPC组海马AChE活性以及L-、H-LSPC组皮质AChE活性均明显高于AI组。与年轻组和AU组大鼠相比,AI组大鼠的海马和皮质ACh含量显著下降。LSPC可剂量依赖性地增加ACh含量,并且两个LSPC剂量组大鼠的海马和皮质ACh含量均与年轻组大鼠相当。
结论LSPC改善AI大鼠海马和皮质抗氧化系统的功能。降低脑的氧化损伤和增加胆碱能系统功能参与了LSPC改善AI大鼠空间学习记忆能力的作用。
第四节一氧化氮系统改变促进LSPC改善老年认知障碍大鼠学习记忆能力的机制研究
目的探讨海马和皮质一氧化氮系统改变对LSPC改善AI大鼠学习记忆能力的影响。
方法实验设计以及组织样品的准备同第二部分第三节。每组随机挑选4只大鼠进行实时定量PCR和western blot检测。其余样品的部分匀浆用于NO水平以及总NOS和分型NOS的检测。为了评估在蛋白酶酶抑制剂或磷酸酶抑制剂存在的情况下NOS活性,向匀浆中加入蛋白激酶抑制剂或磷酸酶抑制剂,然后用HEPES终止反应,再进行iNOS活性的检测。
结果AI大鼠海马和皮质NO水平明显高于年轻大鼠。所有实验组的海马和皮质tNOS活性改变均与其NO水平的改变相平行。AI大鼠海马iNOS活性明显高于AU组和年轻组,同时AU大鼠海马iNOS活性与年轻大鼠没有明显差异。另外,虽然AU和AI大鼠海马：iNOS活性均明显降低,但是AI大鼠又比AU大鼠进一步明显降低。虽然AU和AI大鼠皮质iNOS活性均大幅显著增加同时nNOS活性明显降低,但是AU和AI大鼠皮质iNOS以及nNOS活性并没有明显差异。iNOS和nNOS的基因和蛋白表达也有相似的变化。用蛋白激酶抑制剂或磷酸酶抑制剂处理后,年轻组nNOS活性改变率变化最大,AU组也有相当大幅度的改变；然而这些抑制剂对AI大鼠海马nNOS活性改变率的影响则很小。LSPC明显降低AI大鼠海马和皮质iNOS活性并因此导致tNOS活性和NO水平降低。虽然LSPC并没有明显改变AI大鼠海马和皮质nNOS活性,但是却有增加AI大鼠海马nNOS基因和蛋白表达的趋势。因此,LSPC剂量组大鼠海马的单位nNOS蛋白活性降低。另外,虽然LSPC在蛋白激酶抑制剂存在的情况下并没有明显改变AI大鼠的海马nNOS活性,但却在磷酸酶抑制剂存在的情况下明显改变AI大鼠海马nNOS活性。结论AI大鼠认知功能的降低主要是海马iNOS和nNOS的表达和活性改变所致,而iNOS和nNOS在皮质中的改变则不是主要因素。LSPC不但降低AI大鼠海马iNOS的表达和活性,而且也可使海马nNOS磷酸化调节更加有效,从而抑制年龄相关性的NO过表达、增加海马NO-sGC-cGMP信号转导。表明LSPC可能通过海马NO系统的上述改变而改善AI大鼠的认知功能。
第五节CREB磷酸化改变促进LSPC改善老年认知障碍大鼠学习记忆能力的机制研究
目的探讨海马和皮质中CREB磷酸化改变对LSPC改善AI大鼠学习记忆能力的影响。方法实验设计以及组织样品的准备同第二部分第三节。每组随机挑选4只大鼠样本进行BDNFmRNA的实时定量PCR检测和CREB、pCREB、BDNF、ERK 42/44、pERK 42/44、CaMKIV、pCaMKIV和PKA C-a的western blot检测。
结果AI大鼠海马CREB磷酸化程度明显低于年轻大鼠与AU大鼠；LSPC可剂量依赖性地明显增加AI大鼠海马CREB磷酸化程度。各个实验组的皮质CREB磷酸化程度没有明显差异。AI大鼠海马BDNF mRNA和蛋白表达水平明显低于年轻大鼠和AU大鼠；两个剂量的LSPC均明显增加AI大鼠海马BDNF mRNA和蛋白表达水平。各个实验组大鼠皮质BDNF mRNA和蛋白表达水平未见显著差异。AI大鼠海马ERK42、ERK44蛋白磷酸化程度显著低于年轻大鼠和AU大鼠。相对于AI组,L-LSPC组大鼠海马ERK42蛋白磷酸化程度有增加的趋势,H-LSPC组大鼠海马ERK42蛋白磷酸化程度在L-LSPC剂量组的基础上又明显增加,并与年轻组没有明显差异。两个LSPC剂量组海马ERK44蛋白磷酸化程度都明显高于AI组,并且与AU组相当。AI大鼠海马CaMKIV磷酸化状态显著低于年轻大鼠和AU大鼠；补充LSPC后没有改变海马的CaMKIV磷酸化状态。所有实验组动物海马PKA C-a水平均没有显著差异。
结论相对于年轻大鼠和AU大鼠,AI大鼠海马CREB磷酸化程度明显降低,然而其皮质CREB磷酸化程度没有明显改变。AI大鼠海马ERK 42／44和CaMKIV磷酸化程度降低都促进其CREB活性的降低。海马CREB活性降低导致CREB依赖性的转录降低,从而损害长期记忆的形成。例如,AI大鼠海马BDNF mRNA和蛋白水平明显降低。这些结果表明海马CREB活性降低明显参与了年龄相关性老年记忆障碍的形成和发展过程。LSPC可通过ERK介导的通路活化海马CREB,从而易化长期记忆的形成过程,并因此改善AI大鼠的学习记忆能力。
Aging is a normal but complicated, not pathological, process, which characterized by slowly progressing impairments in structure and function of most systems. In central nervous system (CNS), age-related cognitive dysfunction is one of the most prominent features. Cognitive function refers to a person's ability to process information, such as perception, thinking, recognition, sensing, learning, memory, imagining, and reasoning, and which is necessary for survival as well as intellectual development and maintenance. As the natural process of aging progresses, humans and animals experience a progressive decline in overall cognitive function. As an integral part of cognitive function, learning and memory abilities have been demonstrated to marked decline in the course of normal aging. The age associated memory impairment (AAMI) may range from normal to a mild cognitive impairment, or to Alzheimer's disease (AD). Although its passive impact on quality of life is not as severe as AD, the age associated memory impairment is very prevalent in elderly people. It has been shown that thirty-eight percent of 60-78 years old individuals fulfill the criteria for AAMI, and in the follow-up nine percent of AAMI patients became demented. In addition, during a follow-up period of four and a half years fifty-five percent of individuals with the diagnosis of mild cognitive impairment developed dementia. Therefore, AAMI is one of crucial determinants of the quality and enjoyment of life of elderly people, and it is necessary to seek for some measures for prevention and treatment AAMI with little or not damage to body. Various hypothesizes have been proposed to try to explain the mechanisms of geriatric memory dysfunction. Among them, the cholinergic hypothesis of geriatric memory dysfunction, the free radical hypothesis of aging and the nitric oxide hypothesis of aging have been most widely accepted. In addition, the impaired CREB-mediated transcription in brain during aging has also been paid more and more attention.
Procyanidins chemically belong to polyphenols, they are oligomeric and polymeric flavonoids comprised of flavan-3-ol monomeric subunits. These compounds are widely distributed in fruits, vegetables, seeds, flowers and bark. They have received considerable attention in recent years due to their excellent antioxidant activity and a broad spectrum of safety. Procyanidins posses significantly better free radical scavenging capability than vitamin C, E orβ-carotene. In addition, these compounds have also been reported to exhibit a wide range of biological effects including antibacterial, antiviral, anti-inflammatory, and anticarcinogen actions. The metabolites of procyanidins are able to cross the blood brain barrier and be uptaken and detected in brain and thus exert potential effects on brain functions. Beneficial bioactivities of procyanidins in brain are often claimed to be based on their antioxidant properties. For example, previous experiments demonstrated that procyanidins had an inhibiting effect on the lipid peroxidation and accumulation of age-related oxidative DNA damage in brain. But increasing evidence suggests that some other mechanisms may involve the modulation of brain functions. A proteomics study indicated that procyanidins were able to systematically change specific brain proteins in expression or modification in young rats. (-)-Epicatechin has been found to up-regulate GluR2-containing AMPA receptors, which means it has the potential to modulate synaptic function.
In order to explore the effects of procyanidins extracted from the lotus seedpod (LSPC) on AAMI, the primary goal of this research is to find whether LSPC ameliorate memory impairment in scopolamine-induced mice and cognitively impaired aged rats. When the positive results were observed, we further explored the mechanisms which refer to cholinergic hypothesis, the free radical hypothesis, nitric oxide hypothesis and changes in CREB-mediated transcription.
PartⅠBehavioral study
Section one LSPC ameliorate scopolamine-induced memory impairment in mice
Objective To determine the ameliorating effect of LSPC on the learning and memory impairments induced by scopolamine in mice.
Methods Fifty two-month old male Kunming mice (18-22 g) were randomly divided into five groups with 10 mice in each group:the control group, the low, middle and high dose LSPC (L-, M-, and H-LSPC) groups and the scopolamine group. Mice in L-, M-, and H-LSPC groups were given 50,100,150 mg/kg BW LSPC orally by a gavage daily for 30 days respectively. The control group and scopolamine group were given an equivalent amount of distilled water daily by oral gavage. On each day of the behavioral tests period, mice in L-, M-, and H-LSPC groups were given LSPC 1 h before the first trial session. Animals in L-, M-, and H-LSPC groups and scopolamine group were treated with scopolamine (1 mg/kg, i.p) dissolved in normal saline at 10 min before the first trial session during the training phase of both behavioral tests. Mice in the control group received vehicle only. The capacities of memory and learning were evaluated by the Morris water maze and the step-down avoidance test. During each trial session in Morris water maze, escape latency and swimming distance were recorded. The step-down avoidance test was performed 7 days after the Morris water maze, and the numbers of errors and step-down latency were recorded. After completion of step-down avoidance test, the mice were killed and the brains were homogenized to determine the activity of AChE.
Results No difference in escape latency was observed among the LSPC groups and the control group during all the training days only except that L-LSPC group had significantly longer escape latency relative to the control groups on day 1. Escape latency of all the three LSPC groups on day 4 and 5 were significantly shorter compared to that of scopolamine group. Mice in three LSPC groups on day 4 and 5 exhibited significantly shorter swimming distances compared with that in scopolamine group, but no differences were observed when compared with that in control group. In step-down avoidance test, reduced the number of errors in all the three LSPC groups were observed compared with scopolamine group. In addition, all the three LSPC groups had significant shorter latencies than the control group and significantly longer latencies than scopolamine group. LSPC inhibited AChE activity in a dose-dependent manner, and no significant difference was found among all the three LSPC groups and the control group.
Conclusion LSPC has the ability to ameliorate the impairment of learning and memory induced by scopolamine in both Morris water maze test and step-down avoidance test, and increasing cholinergic activity by inhibition of AChE activity may involve in the ameliorative activities of LSPC on learning and memory dysfunction.
Section two LSPC ameliorate memory impairment in cognitively impaired aged rats
Objective To determine the ameliorating effect of LSPC on the learning and memory impairments in cognitively impaired aged rats.
Methods Based on Morris water maze performance compared with forty young female rats, aged-unimpaired (AU) and aged-impaired (AI) rats were chosen from two hundred 18-month-old female Sprague-Dawley rats. The rats in young control group (n=14) and AU group (n=16) were randomly selected from the young rats and aged unimpaired rats respectively. The animals categorized as AI were randomly subdivided into three groups consisting of 16 animals each:AI group, low and high dose LSPC (L-and H-LSPC) groups. Rats in L-and H-LSPC groups were given 50 and 100 mg/kg BW LSPC orally by a gavage daily for 7 weeks, respectively. The other three groups were given orally an equivalent volume of distilled water daily. On each day of the behavioral tests period in Morris water maze, animals continued to be administered intragastrically 1 h before the first trial session. The learning and memory abilities of rats in different groups were assessed by retraining the animals in Morris water maze with entirely different cues and testing the animals as before.
Results Rats in AI group exhibited a significant prolongation of escape latency in Morris water maze compared with those of young group and AU group. Both LSPC groups exhibited significantly shorter escape latencies than AI group during all sessions only except H-LSPC group on the first training day. Meanwhile, H-LSPC group did not exhibit significant difference in escape latency from young and AU groups. Significantly shorter swimming distance than that of AI group was observed in H-LSPC group on day 3-5 and L-LSPC group on day 2 and day 4. Compared with young and AU groups, H-LSPC group did not exhibit difference in swimming distance during all the test days.
Conclusion LSPC has the ability to ameliorate the impairment of learning and memory in AI animals in Morris water maze test.
Part II Mechanism study
Section three Rejuvenation of antioxidant and cholinergic systems contributes to the effect of LSPC ameliorating memory impairment in cognitively impaired aged rats
Objective To determine the effect of LSPC on the antioxidant and cholinergic systems in hippocampus and cerebral cortex in cognitively impaired aged rats.
Methods Study design was the same as part I, section two. After completion of water maze test, animals were killed and the hippocampus and cerebral cortex were dissected. Four animal samples in each group were randomly selected for next study. For the remaining samples, hippocampus and cerebral cortex in left hemisphere were homogenized to determine the antioxidant capacity, oxidative damage markers as well as activities of ChAT and AChE. The hippocampus and cerebral cortex in right hemisphere were homogenized in phosphate buffer containing 3 mg/ml eserine for ACh assay.
Results There were not significant changes in SOD activity in hippocampus and cerebral cortex in all the experimental groups. However, CAT and GPx activities of the both brain regions of old animals were significantly lower than those of young subjects, but no difference in activities was observed between AU and AI groups. Significant increase in CAT and GPx activities of both brain regions in H-LSPC group and in GPx activities of hippocampus in L-LSPC group were observed when compared with the AI group. The ratios of CAT/SOD and GPx/SOD were significantly lower in AU and AI groups. LSPC, especially H-LSPC, reversed the declines in these ratios when compared with AI. The levels of GSH in both brain regions of aged rats were significantly lower than those of young animals, and H-LSPC administration up-regulated significantly the GSH level in both regions. Hippocampal and cortical T-AOC in AU and AI groups markedly declined, and animals in both LSPC groups revealed higher T-AOC than that in AU and AI groups and were similar to their young counterparts. All AU and AI animals showed significant higher MDA levels and carbonyl content in hippocampus and cerebral cortex than young animals. There were not significant differences in MDA levels of both brain regions between AU and AI groups, whereas AI animals exhibited significantly higher carbonyl content in both brain regions than AU animals. Reduced MDA levels and carbonyl content in hippocampus and cerebral cortex in both LSPC groups were observed compared with AI group. There were not significant differences in ChAT activity in hippocampus and cerebral cortex in all the experimental groups. Animals in both AU and AI groups showed significantly lower AChE activities in hippocampus than their young counterparts. In cerebral cortex, animals in AI group also revealed significantly lower AChE activities than young animals, whereas AU animals were not significantly different from young animals. The AChE activities in hippocampus of H-LSPC group and in cerebral cortex of both LSPC groups were significantly higher than that in AI group. There was a substantial decrease in ACh content in both brain regions for AI group compared with young and AU groups. LSPC considerably increased ACh content in a dose-dependent manner, and no significant difference was found among both LSPC groups and the young group.
Conclusion LSPC attenuated functional decline in antioxidant system in hippocampus and cerebral cortex of AI rats. The decreased oxidative damage to brain and increasing cholinergic activity may involve in the ameliorative effects of LSPC on learning and memory dysfunction in AI animals.
Section four Changes in nitric oxide system contribute to the effect of LSPC ameliorating memory impairment in cognitively impaired aged rats
Objective To determine the effects of LSPC on the nitric oxide system in hippocampus and cerebral cortex in cognitively impaired aged rats.
Methods Study design and tissue preparation were the same as partⅡ, section three. Four animal samples in each group were randomly selected for real-time PCR and western blot analysis. For the remaining animal samples, part of the homogenate was used for NO level as well as total and NOS isoforms activities evaluation. In order to determination of the effect of phosphorylation/dephosphorylation on nNOS activity, homogenate were preincubated with protein kinase inhibitors cocktail or phosphatase inhibitor and terminated with HEPES, then nNOS activity were measured.
Results The hippocampal and cortical NO levels in AI animals were markedly higher than that in young animals. The alterations in tNOS activity of both brain regions closely paralleled the changes in NO level of that in all the experimental groups. AI rats exhibited significantly higher hippocampal iNOS activities than AU and young animals, and no differences in hippocampal iNOS activity were observed between young and AU animals. In addition, although marked decrease in hippocampal nNOS activity occurred in all AU and AI rats, AI animals exhibited further significant lower hippocampal nNOS activities than AU animals. However, although cortical iNOS activities were increased by a large margin and at the same time nNOS activities were reduced in all AU and AI animals, there was no differences in both iNOS and nNOS activities between AI and AU animals. Similar alteration trends were also observed in terms of the gene and protein expressions in both iNOS and nNOS. When treated with protein kinase inhibitors or phosphatase inhibitor, although young group revealed the most alteration in the rate of nNOS activity change, AU group also had considerable changes in that. However, the effects of these inhibitors on the rates of nNOS activity change were almost abrogated in hippocampus of AI animals. Both doses of LSPC significantly decreased iNOS activities and consequently resulted in tNOS activities and NO levels decrease in hippocampus and cerebral cortex of AI animals. LSPC supplementation did not lead to marked alterations for hippocampal and cortical nNOS activity in AI animals, whereas AI animals revealed a trend to increase nNOS gene and protein expressions in hippocampus with LSPC supplementation. As a result, animals in LSPC supplementation groups should have a decline in nNOS activity per unit protein in hippocampus. Moreover, although LSPC supplementation had not marked effect on the rates of hippocampal nNOS activity change in the presence of protein kinase inhibitors in AI animals, these compounds considerably altered these rates in these animals in the presence of phosphatase inhibitor.
Conclusion The alterations of expressions and activities in hippocampal but not cortical iNOS and nNOS accounted for the reduced cognitive behaviors in AI animals. LSPC supplementation not only decreased hippocampal iNOS activities and enzyme expression but also made hippocampal nNOS phosphorylation regulated effectively in AI animals, which led to the inhibition of age-related NO overproduction and increase of hippocampal NO-sGC-cGMP signal transduction in these animals. These changes in hippocampal NO system may involve, at least partly, in the ameliorative effects of LSPC on learning and memory dysfunction in AI animals.
Section five Memory impairment in cognitively impaired aged rats associated with decreased hippocampal CREB phosphorylation: reversal by LSPC
Objective To determine the effects of LSPC on CREB phosphorylation in hippocampus and cerebral cortex in cognitively impaired aged rats.
Methods Study design and tissue preparation were the same as part II, section three. Four animal samples in each group were randomly selected for real-time PCR of BDNF mRNA and western blot analysis of CREB, pCREB, BDNF, ERK 42/44, pERK 42/44, CaMKIV, pCaMKIV and PKA C-α.
Results The phosphorylation states of CREB in hippocampus of AI animals were markedly lower than that of young and AU animals. Supplementation of LSPC led to significant increase in hippocampal CREB phosphorylation with a dose-dependent manner. All groups were not significantly different from each other in cortical CREB phosphorylation. The BDNF mRNA and protein levels were markedly reduced in hippocampus of AI animals compared to that measured in young and AU animals. AI animals with LSPC supplementation uniformly exhibited significantly higher hippocampal BDNF mRNA and protein expression than AI animals. As for the BDNF mRNA and protein expression in cerebral cortex, no significant changes were detected in any of the groups. The significant lower states of phosphorylation in hippocampal ERK42 and ERK44 protein were observed in AI animals relative to young and AU animals. Animals in group with L-LSPC supplementation had a tendency to elevate the phosphorylation states of hippocampal ERK42 relative to AI rats, and rats in group with H-LSPC supplementation revealed further significant increase in that and were comparable with young animals. The phosphorylation states of hippocampal ERK44 in both LSPC groups were substantially increased than that in AI group and reach to the level of AU group. Substantial decrease in phosphorylation states of CaMKIV were detected in AI animals relative to young and AU counterparts. The phosphorylation states of this protein did not significantly increase in response to LSPC supplementation. No substantial alterations in the level of PKA C-a were detected among all the experimental groups.
Conclusion The alterations of expressions and activities in hippocampal but not cortical iNOS and nNOS accounted for the reduced cognitive behaviors in AI animals. LSPC supplementation not only decreased hippocampal iNOS activities and enzyme expression but also made hippocampal nNOS phosphorylation regulated effectively in AI animals, which led to the inhibition of age-related NO overproduction and increase of hippocampal NO-sGC-cGMP signal transduction in these animals. These changes in hippocampal NO system may involve, at least partly, in the ameliorative effects of LSPC on learning and memory dysfunction in AI animals.
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