The Study On The Effects And Mechanism Of Icariin On Learning And Memory In Senescence Accelerated Mouse Prone/8 (SAMP8)

Learning and memory deficits are the most common neurodegenerative disorders in the ageing process in humans, such as Alzheimer's disease (AD). A number of clinical studies have suggested that estrogen therapy may delay the onset or contribute to the prevention and/or attenuation of AD. However, estrogen therapy may lead to the severe side effects. Recently, phytoestrogen which has estrogen-like effects and can prevent the side effects of estrogen therapy has been attracted much attention. Traditional Chinese herb Epimedium has been used for the therapy of AD in Traditional Chinese Medicine prescription, and its estrogen-like activity has been found through animal experiments. Icariin (ICA) is a flavonoid extracted from Epimedium that is considered to be its major pharmacologically active component. More recently, icaritin (ICT), one of the metabolits of icariin, has been approved that it has estrogen-like effect and could be blocked by estrogen receptor antagonist in vitro. Senescence-accelerated mouse prone/8(SAMP8) exhibits many features that are known to occur early in the pathogenesis of AD, such as increased oxidative stress, amyloid-βalterations, and tau phosphorylation as well as age-related deterioration in learning and memory. The present studies of SAMP8 have focused on revealing mechanism of accelerated deficits in learning and memory, evaluating the efficiency of therapeutic drugs against AD. It has been reported that the level of estradial (E₂) in serum of female SAMP8 lost dramatically in the process of ageing. In this study, we used female SAMP8 as an animal model of AD to evaluate the effects of icariin orally administration for 12 weeks on learning and memory in SAMP8 and its mechanism related to estrogen-like activity. Donepezil (DON), a widely used acetylcholinesterase inhibitor, and diethylstilbestrol (DES), an estrogen replacement therapy drug, were also investigated as drug controls in female SAMP8.Part I. The Study on the estrogen-like activity of Epimedium and icariinObjective: To investigate the estrogen-like activity of Epimedium and icariin in mice. Methods: The female Kunming Mice in different groups were treated with the water extract of Epimedium 10, 20, 40 g/kg body weight for 2 weeks, and the female SAMP8 in different groups were treated with ICA 75, 150 mg/kg body weight for 12 weeks. (Groups including the normal control group, positive control group and treatment groups).Then the mice were killed and the uterus coefficient was calculated. The changes of mouse weight were observed before and after the drug administration. The serum levels of E₂ were detected by using radio-immunity method.Results: Orally administration of the water extract of Epimedium increased the uterus coefficient in mice and enhanced the level of E₂ in serum significantly; Orally administration of ICA could only enhance the level of E₂ in serum.Conclusions: The water extract of Epimedium has a powerful estrogen-like activity, and ICA has an estrogen-like activity, too.Part II. Effect of icariin on learning and memory in female SAMP8Objective: To investigate the effect of icariin on learning and memory in female SAMP8.Methods: SAMP8 divided in different groups were treated with ICA 75, 150 mg/kg body weight for 12 weeks, and the ability of learning and memory in female SAMP8 were studied by Morris water maze test and passive avoidance test. Normal aging control mouse senescence-accelerated resistant mouse I (SAMR1) was employed. DON 1.0 mg/kg body weight and DES 0.2 mg/kg body weight was used as positive drug controls.Results: Compare to SAMR1, in the Morris water maze test, the latency of SAMP8 in place navigation trail lasted longer, and the times of SAMP8 through the platform were more and the time of SAMP8 spent in the target quadrant lasted shorter in spatial probe trail; the latency of SAMP8 in passive avoidance test showed significantly longer, too. Orally administration of ICA in SAMP8 for 12 weeks could obviously shortened the latency in place navigation trail, increased the times through the platform and prolonged the time spent in the target quarter in spatial probe trail; ICA could also shorten the latency of SAMP8 in passive avoidance test. Positive drugs control DON and DES showed a significant cognitive-improving of SAMP8.Conclusions: Female SAMP8 showed learning and memory deficits at 10 months old. Orally administration of ICA could improve the learning and memory ability of SAMP8. It suggested that ICA could be an emerging candidate for AD. Part III. Effect of icariin on the changes of cholinergic system in the hippocampus of female SAMP8Objective: To investigate the effect of icariin on the changes of cholinergic system in the hippocampus of female SAMP8.Methods: The mice grouping and drug administration were the same with Part II. Activities of AChE in the hippocampus of SAMP8 were measured by chromatometry method; content of ACh and binding capacity of MCBC (M-cholinoceptor binding capacity) in the hippocampus of SAMP8 were detected by Elisa method. Results: Compare to SAMR1, the activity of AChE in the hippocampus of 10 months female SAMP8 had no obvious change, the level of ACh decreased, and the binding capacity of MCBC declined significantly. Orally administration of ICA in SAMP8 for 12 weeks could do no effect on the activity of AChE, while it could obviously increase the level of ACh and enhanced the binding capacity of MCBC in the hippocampus. Conclusions: The learning and memory deficits in SAMP8 were related to the cholinergic system disorder. Orally administration of ICA could recover the function of cholinergic system which could directly improve learning and memory ability of SAMP8.Part IV. Effect of icariin on the changes of neurotransmitters in the hippocampus of female SAMP8Objective: To investigate the effect of icariin on the changes of neurotransmitters in the hippocampus of female SAMP8.Methods: The mice grouping and drug administration was the same with Part II. The content of monoamines transmitters and amino acids transmitters in the hippocampus of 10 months female SAMP8 was detected by HPLC-electrochemical detection (HPLC-ECD).Results: Compare to SAMR1:â‘ the content of monoamines transmitters in the hippocampus of 10 months female SAMP8 declined obviously. The content of DA, 5-HT, 5-HIAA and NE decreased, respectively. Orally administration of ICA in SAMP8 for 12 weeks could significantly increase the content of these monoamines transmitters in the hippocampus.â‘¡the content of amino acids transmitters in the hippocampus of 10 months female SAMP8 showed metabolic disequilibrium, including the content of Asp and GABA decreased, the content of Glu and Gln increased obviously and the ratio of Glu/GABA went up significantly. There wasn't any marked change in the content of Tau and Gly. Orally administration of ICA in SAMP8 for 12 weeks could visibly increase the content of Asp and GABA, decrease the content of Glu and Gln, cut down the ratio of Glu/GABA and do no effect on the content of Tau and Gly in the hippocampus.Conclusions: The learning and memory deficits in SAMP8 were related to the decline of monoamines transmitters and the metabolic dysequilibrium of excitatory amino acids and inhibitory amino acids in the hippocampus. Orally administration of ICA could increase the content of monoamines transmitters, regulate the metabolic equilibrium of excitatory amino acids and inhibitory amino acids in the hippocampus of female SAMP8.Part V. Effect of icariin on the GFAP-positive horizontal cells in thebrain of female SAMP8Objective: To investigate the effect of icariin on the GFAP positive horizontal cells inthe brain of female SAMP8.Methods: The mice grouping and drug administration was the same with Part II. Theshape of GFAP positive horizontal cells in the brain of female SAMP8 was observedunder microscope by immunohistochemical staining method and the number of positivecells was calculated by the IPP5.1 image analysis software in the hippocampus.Results: The GFAP postive cells were displayed no staning of nucelus, dark brown ofcytoplasm and process, positive products distributed with cell process, and the shape ofcells seemed like the branch of spider. The GFAP postitive cells mainly distribted in thedentate gyrus (DG) region of hippocampus, while rarely distributed in the cerebralcortex.Compare to SAMR1, the number of GFAP positive cells in the hippocampus were themost within the brain of 10 months female SAMP8, especially in the DG region. Thecells body went bigger and the branch went thickening. Orally administration of ICA inSAMP8 for 12 weeks could partly recover the GFAP positive cells shape to normal anddecrease the number of positive cells significantly in the hippocampus.Conclusions: The horizontal cells in the hippocampus of female SAMP8 were activatedand proliferated. Orally administration of ICA could inhibit the hyperplasy of activatedcells in the hippocampus, and, in part, tranfer the cells shape to normal. Part VI. Effect of icariin on the content of lipid peroxidation and the activity of antioxidase in the brain of female SAMP8Objective: To investigate the effect of icariin on the content of lipid peroxidation and the activity of antioxidase in the brain of female SAMP8.Methods: The mice grouping and drug administration was the same with Part II. The content of MDA and the activity of SOD and GSH-Px in the cerebral cortex of female SAMP8 were detected by chromatometry method.Results: Compare to SAMR1, the content of MDA increased and the activity of SOD declined obviously while the activity of GSH-Px declined in some extent but no statistically significant in the cerebral cortex of 10 months female SAMP8. Orally administration of ICA in SAMP8 for 12 weeks could decrease the content of MDA and enhance the activity of SOD significantly, but do no effect on the activity of GSH-Px in the cerebral cortex.Conclusions: The lipid peroxidation in the cerebral cortex of female SAMP8 increased. The antioxidative defense systems of SAMP8, i.e., antioxidant enzyme activities and antioxidants, deteriorated, which were known as oxidative stress. Orally administration of ICA could inhibit the lipid peroxidation, enhance the antioxidative activities against oxidative stress.Part VII. Study of icariin and its metabolit icaritin on the antioxidativeactivities in vitroObjective: To investigate the antioxidative activities of icariin and its metabolit icaritin.Methods:â‘ DPPH (2,1-diphenyl-1-picrylhydrazyl) radical-scavenging activities of ICA and ICT were detected by chromatometry method.â‘¡ICA and ICT on the H₂O₂-induced injury PC12 cells viability was detected by 3-(4,5)-dimethylthiahiazo (-2-yl)-3,5-di-phenytetrazoliumromide test (MTT) and the cells shape was observed under microscope.Results:â‘ In the DPPH radical-scavenging test, ICT showed a better radical-scavenging activity (IC50= 76μg/ml) while ICA did not.â‘¡In the protective effect against H₂O₂-induced injury PC12 cells test, ICT could increase the cells viability significantly and protect the cells body against H₂O₂-induced injury while ICA could not. Conclusions: ICT exhibited a stronger antioxidative activity in vitro but ICA did not. The results suggest that ICT may be the pharmacologically active component of ICA in vivo.