| Alzhe mer's disease (AD) is chronic degenerative brain diseasecharacterized by progressive dementia and the main clinical manifestationsinclude memory loss, mental retardation and abnormal changes in behaviorand mood. According to the statistics, the incidence of AD is about10%incrowd of about65years old and rises up to47%in the population over85years of age. The survival time of patients with AD is expected to reach20years, but most of the cases after diagnosis can only survive8to10years, soAD has become the fourth killer to human's health.With rapid aging of thepopulation, the development of effective interventions and medications to cureAD has become a hot issure in AD research,and also an anxious socialproblem.The proposed pathogenic mechanisms of AD generally include loss ofcholinergic function, oxidative stress, amyloid cascade, inflammatorymediators and steroid hormone deficiencies. In addition, excitotoxicity,apoptosis and dysregulation of cell cycle are in close relation to AD.Neurobiochemistry revealed that central amino acid neurotransmitters are inclose relaiton to learning and memory. Asp and Glu are excitatory amino acids,Gly and GABA are inhibitory amino acids and they are very import in thebalance of exitation and repression of the nervous system.The level of aminoacids in the centre becomes abnormal when AD happens, especially thebalance between Glu and GABA, which is the key point of neuron injury.Apoptosis is a significant path of neuron retrograde death, so the interferenceto apoptosis could be a possible new method to treatment of AD. Apoptosis isregulated by many kinds of genes and Bcl-2gene family is very importantcontrolling gene containing numerous members in which Bax promotesapoptosis and Bcl-2restrains apoptosis. Bcl-2/Bax is a balanced system— while Bax is redundant the apoptosis process is promoted and while Bcl-2isredundant the process is restrained. In recent years, with the deep study of cellcycle and the regulation, people found that the mechanism of AD is in relationto the regulation of cell cycle and proposed the cell cycle hypothesis about themechanism of AD. The adult neurons of central nervous system can enter cellcycle again when some component elements become abnormal in theregulation of the cell cycle, but neurons in mitosis anaphase will appear thefailure of cell division and cell cycle arrest finally and form some pathologicalfeatures of AD or neuron apoptosis.Osthole (7-methoxy-8-isopentenoxy-coumarin) is an ingredient oftraditional Chinese medicine (TCM) from natural product Cnidium monnieri(L.) Cusson, possesses a variety of pharmacological properties, includingantiosteoporotic, antihepatitic, antiallergic, antiseizure and antiproliferativefunctions and so on, and has been clinically used in the treatment of skindisease and gynecopathy for many years.We have engaged in the research ofosthole on improvement of learning and memory and anti-aging for manyyears.The previous studies showed that osthole can improve learning andmemory impairment in dementia model mouse via inhibitingacetylcholinesterase (AChE) activity or by enhancing the activities ofglutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) andclearing oxygen free radical (OFR) in brain. Osthole can pass through theblood-brain barrier and its side effects are small, so it is expected to becomethe effective drug to prevent and treat AD.Based on these profiles,the study investigated the effects of osthole oncognitive impairment and central synaptic plasticity of AD rats induced byAβ25-35via behavioral and electrophysiological experiment method,observedthe effects of osthole on contents of amino acids in hippocampus of AD ratsinduced by Aβ25-35via pre-column derivatization high performance liquidchromatography and researched the effects of osthole on neuronal apoptosis,protein expression related to apoptosis and cell cycle in hippocampus of ADrats induced by Aβ25-35via HE, TUNEL and immunohistochemical staining method and flow cytometry. The specific contents as follows:1Effects of osthole on learning and memory of AD ratsObjective: We aimed to investigate the effects of osthole on cognitiveimpairment of AD rats induced by once intracerebroventricular (i.c.v.)injection of Aβ25-35.Methods: SD rats were randomly assigned to control, model, low-doseosthole (12.5mg·kg-1, Ost1), and high-dose osthole (25.0mg·kg-1, Ost2)group. On the first day of the experiment, an intracerebroventricular (i.c.v.)injection of Aβ25-35was administrated to establish AD rat model. After i.c.v.injection, control and model group rats were intraperitoneally injected withsolvent1mL·kg-1, and osthole treatment group rats were intraperitoneallyadministered with osthole. All animals were administered continuously for14days. Morris water maze test was performed from day10to14after injectionof Aβ25-35. Rats were trained four days (d1-d4) and the escape latency wasrecorded. Twenty-four hours after the last training day (d5), a probe trial wasmade under the condition of removing the platform and the residence time inthe target quadrant and the number of crossing the platform site wererecorded.Results: With the increase of training day, the escape latenciescontinuously reduced in each experimental group, and there was no differencebetween the groups on the first day of training. Compared with control group,model group displayed longer escape latency from d2and lower percentage oftarget quadrant searching time and fewer number of crossing platform (inprobe trial). Compared with model group, osthole groups showed shorterescape latency fron d2and higher percentage of target quadrant searching timeand more number of crossing platform.Summary: The study indicated the establishment of AD rat model byonce i.c.v. injection of Aβ25-35is successful. The model rats showed learningand memory decline and osthole improved the learning and memoryimpairment of AD rats.2Effects of osthole on hippocampal LTP induced by HFS of AD rats Objective: We aimed to investigate the effects of osthole on centralsynaptic plasticity of AD rats induced by once intracerebroventricular (i.c.v.)injection of Aβ25-35.Methods: Electrophysiological test was conducted on day14after modelestablishment. According to Pellegrino stereotaxic atlas of rat brain,positioned stimulating and recording electrode. We adoptedelectrophysiological methods of extracellular recording in vivo to observe theeffects of osthole on hippocampal long-term potentiation (LTP) phenomenoninduced by high frequency stimulus (HFS) of AD rats induced by onceintracerebroventricular (i.c.v.) injection of Aβ25-35.Results: In all groups, the PS amplitude obviously increased during the60min observation and the LTP phenomenon was induced by HFS, but thegrowth extent of PS amplitude was different in each group. The PS amplitudein model group was lower than that in control group, while the PS amplitudein osthole groups was higher than that in model group on each time point afterHFS.Summary: Our experiments discovered that AD model rats appeared theinhibitory action of LTP induced by HFS, osthole could enhance neuronalsynaptic plasticity in the hippocampus and reversed the inhibitory action ofLTP induced by HFS of AD rats, which indicated osthole could improvelearning and memory impairment of AD rats from cell synaptic level.3Effects of osthole on level of hippocampal amino acids of AD ratsObjective: We aimed to observe the effects of osthole on level ofhippocampal amino acids of AD rats and investigate the mechanism of ostholeon cognitive impairment of AD rats from Glu/GABA learning and memoryregulatory system.Methods: Amino acids determination was conducted on day14afterestablishment of AD model.The levels of Asp, Glu, Gly and GABA weredetermined by pre-column derivatization high performance liquidchromatography (HPLC) and the wavelength of the ultraviolet detection was360nm. Rats were executed after anesthesia and the hippocampus tissues were separated on ice. Made10%homogenate with methanol and distilled water(1:1V/V) and centrifuged with10000rpm speed for10min. The supernatantswere stored at-80℃.The specimens were dissolved before determination andcentrifuged with10000rpm speed for10min at low temperature. Acetonitrile200μL and supernatant200μL were mixed and centrifuged with10000rpmspeed for10min at low temperature to precipitate protein. The supernatant,sodium hydrogen carbonate200μL and2,4-dinitrofluorobenzene100μL weremixed and derived for1h in60℃water bath. The whole process should payattention to avoid light. Finally the sample was filtered and taken out20μLliquid to determine the contents of amino acids.Results: Compared with control group, the contents of Asp, Glu, Gly,GABA and the ratio of Glu/GABA obviously increased in model group. Theincrease extent of Glu was more than those of other amino acids. Comparedwith model group, the contents of Glu in osthole groups decreased withsignificant difference, and the contents of other amino acids decreased withoutsignificant difference.The ratios of Glu/GABA in osthole groups decreasedcompared with that in model group, while higher than that in control group.Summary: HPLC results showed that the establishment of AD rat modelby i.c.v. injection of Aβ25-35could give rise to the metabolic disturbance ofamino acid transmitters in hippocampus and the increasion of Glu content andGlu/GABA ratio, which resulted in excitotoxicity. Osthole could inhibit theexcitotoxicity of Glu by regulate the Glu/GABA learning and memoryregulatory system and possessed the protective action to central neurons.4Effects of osthole on neuronal apoptosis of AD ratsObjective: We aimed to investigate the effects of osthole onhippocampal neuronal apoptosis, protein expression related to apoptosis andcell cycle of AD rats.Methods: Rats were anesthetized on day14after establishment of ADmodel and perfused heart with heparinized normal saline and4%paraformaldehyde for30minutes. Decapitation and brain extraction wereperformed quickly.Cut the brain into pieces of3mm and fixed the tissues with 4%paraformaldehyde for24hours. Dehydration, lucidification, steeping wax,embedding, slicing and coating were performed in order andhematoxylin-eosine staining, Bcl-2and Bax immunohistochemistry stainingand TUNEL staining were also performed respectively. Slides were observedwith light microscope and results were recorded. Another part of rats wereexecuted to obtain hippocampus tissues on day14after model establishment.Made the monoplast suspension and fixed them with70%precooling alcoholfor more than24hours at4℃. Derived with propidium iodide of50μg·mL-1for30minutes at4℃and analyzed the cell cycle with flow cytometry.Results:①HE staining: No remarkable neuronal abnormalities in thehippocampus of the control group rats were observed, while the slides ofmodel group showed the disorder of neurons array, some neurons appearedapoptosis signs and some apoptotic cells was observed in vision. While theneurons in the osthole group were close to those of control group and thenumber of apoptotic cells was decreased compared to the number in modelgroup.②Apoptosis detected with TUNEL: There were few apoptotic cells inthe control group and much more apoptotic cells in the model group than thosein the control group. There were fewer apoptotic cells in the osthole groupthan those in the model group.③Bcl-2and Bax detected withimmunohistochemistry: There were an extremely small quantitiy of Bcl-2andBax positive cells in the control group. Compared with the control group, BothBcl-2and Bax expression increased, the Bax expression increased much morethan Bcl-2expression and so Bcl-2/Bax ratio decreased in the model group.Compared with the model group, Bcl-2expression increased, Bax expressiondecreased and Bcl-2/Bax ratio increased in the osthole group.④Cell cycledetected with flow cytometry: Compared with the control group,the G0/G1stage percentage increased, the S stage percentage decreased, the G2/M stagepercentage increased and proliferation index decreased in the modelgroup.Compared with the model group, both the S stage percentage and PIincreased and the G2/M stage percentage decreased in the osthole groups.Summary: Osthole could increase the Bcl-2/Bax ratio by regulating Bcl-2and Bax, proteins related to apoptosis, inhibit apoptosis, and protecthippocampus neurons. Moreover, osthole could increase the percentage of Sphase cells, promote the G2/M phase cells to divide further, strengthen theproliferation activity of cells, regulate cell cycle and benefit to themaintenance of normal physiological function of hippocampus.Conclusions:1The study constructed the AD rat model establiahment via once i.c.v.injection of Aβ25-35is successful.The model rats showed AD signs: learningand memory impairment, decrease of the PS amplitude of hippocampalLTP induced by HFS, increase of hippocampal apoptosis cells, decrease ofthe proliferation activity of hippocampus cells, cell retardation at G2/Mphase, increase of Glu and Glu/GABA ratio leading to excitotoxicity.2Osthole can improve the learning and memory impairment and strenghenthe synaptic plasticity in hippocampus of AD rats, which indicated thatosthole has the effect of improving the cognitive disorder of AD ratsimproved from the level of whole animal and cell synapsis.3Osthole can cut down the level of Glu and the Glu/GABA ratio inhippocampus of AD rats, regulate the Glu/GABA learning and memoryregulatory system, and decrease the excitotoxicity of Glu, which may beits one of the mechanisms of improving learning and memory impairmentof AD rats.4Osthole can increase the Bcl-2/Bax ratio by regulating Bcl-2and Bax,proteins related to apoptosis, inhibit apoptosis and protect hippocampusneurons, which may be its one of the mechanisms of improving learningand memory disorder of AD rats.5Osthole can regulate cell cycle and benefit to the maintenance of normalphysiological function of hippocampus by increasing the percentage of Sphase cells, promoting the G2/M phase cells to divide further andstrengthening the proliferation activity of cells, which may be its one ofthe mechanisms of improving learning and memory disturbance of ADrats. |
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