Study On The Ameliorating Memory Impairment And The Mechanisms Of Procyanidins Extracted From The Lotus Seedpod In Cognitively Impaired Aged Rats

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 studySection one LSPC ameliorate scopolamine-induced memory impairment in miceObjective 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 ratsObjective 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 studySection three Rejuvenation of antioxidant and cholinergic systems contributes to the effect of LSPC ameliorating memory impairment in cognitively impaired aged ratsObjective 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 ratsObjective 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 LSPCObjective 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.