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ObjectiveTo investigate the effect of soluble β-amyloid protein (Aβ) oligomers on the expression levels of insulin signaling transduction cascades-associated proteins including insulin receptor ( InsR),insulin receptor substrate-Ⅰ( IRS-Ⅰ) and protein kinase B (PKB) of rat hippocampal neurons,and the pathogenesis of Alzheimer's disease (AD) in depth.MethodsSoluble Aβ oligomers (5 μl) were injected into the lateral ventriculus of the AD group by a microinjector under the stereotaxic apparatus.Normal saline solution ( NS,5 μl) was injected into the NS group in the same way,and the control group received the puncture without injection. It was repeated after 1 week and the behavior of all rats was evaluatedbyY-mazetestafter2weeks.Thenhippocampuswasremovedandunderwent immunohistochemical staining to detect the expression of proteins associated.ResultsCompared with the other groups,learning and memory ability of the Aβ-treated rats were impaired.To be specific,the times of learning were increased and the times of memory were decreased. However,there was no significant difference between the NS group and the control group.Besides,the expression levels of InsR,IRS-Ⅰ,and PKB were decreased in AD group showing that a mean optical density of staining on these proteins ( InsR:0.12 ± 0.0l ; IRS-Ⅰ:0.14 ± 0.02; PKB:0.12 ± 0.03 ) was reduced in contrast with that in the NS group and the control group.Whereas there was no significant difference between the NS group (0.40 ± 0.02,0.39 ± 0.06,0.38 ± 0.03,mean difference:- 0.13,- 0.13,- 0.17,all P ＜ 0.05 ) and the control group (0.38 ± 0.07,0.35 ± 0.03,0.35 ± 0.06,mean difference:- 0.15,- 0.07,- 0.73,all P ＜ 0.05 ).ConclusionsSoluble Aβ1-42 induced learning and memory disability of the rats.The mechanism might be that Aβ can lead to disorders of the insulin signaling transduction pathway of hippocampal neurons and decrease the expression levels of the proteins in the pathway.
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Objective To develop urine AD7C-NTP diagnostic kit,analyze and evaluate its application value on AD.Methods Immunogenicity AD7C-NTP peptide fragments had synthesized by solidphase methods.The animals immunized to prepare antibodies.After matching screening.mouse antibody was uesed as coating antibody.biotin-labeled rabbit antibody wag used as testing antibody,and horseradish peroxidase was labeied with avidin.The urine AD7C-NTP ELISA detective method was established.The AD7C-NTP levels in morning urine samples of 121 AD patients and 118 age-matched controls were collected.Results AD7C-NTP antibodies were identified.Mouse anti-AD7C-NTP antibody titer in ELISAwas 1:8 000,and rabbit anti-AD7C-NTP antibody titer in ELISA was 1:32 000:WB was uesd to detect human brain specimens and there was a single band with molecular weight of 41 000.The lowst detection limit of ELISA methodology was 0.5μg/L The linear range was 0-10μg/L,normal reference value ≤1.5μg/J,the average recovery rate was 100.2%.The intra and inter of CV were 3.8%,4.5%,7.6%,6.8% respectively.The AD7C-NTP levels[2.25(0.43-8.62)μg/L]of urine in AD group was higher than those in contorl group[0.82(0.47-2.77)μg/L,P<0.01].The positive rates in AD group and control group were 89.3% and 15.3% respectively.The sensitivity Was 89.3%and specificity was 84.7%.Conclusions The animals are immunized with the self-designed synthetic peptide fragment to prepare AD7C-NTP antibodies successfully.The established ELISA method for detection of urine AD7C-NTP with high sensitivity,and precision can be used as an assistant examination in clinical diagnosis of AD.
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Objective To look for a small molecular neurotrophin which could promote production and/or differentiation of neural stem cells (NSCs). Methods 1.Embryo hippocampal NSCs were cultured in vitro . 2. The neurospheres were identified by antibodies of bromodeoxyuridine (BrdU), glial fibrillary acid protein (GFAP), microtubule associated protein 2 (MAP2) and Galactocerebroside (GalC). 3. The cells were divided into four groups, which were control group, FBS treated group, trans-sequence of APP 5-mer peptide treated group, APP 5-mer peptide treated group. The morphology of NSCs was observed in above four groups. 4. Cell counts, detection of clone information rate and diameter of clone were done to study the effect of APP 5-mer peptide on production of NSCs. 5. In addition, we also detected the MTT metablism rates in all groups. Results 1. The NSCs formed neurospheres and grew in floating. They were BrdU-positive. GFAP-positive, MAP2-positive and GalC-positive cells appeared after FBS were added into the medium. 2. The morphology of NSCs was not changed in APP 5-mer peptide treated group and trans-sequence group compared with the control group. 3. The cell number increased in APP 5-mer peptide treated group as compared with the control group. There were no apparent differences between the control group and the trans-sequence treated group. 4. The clone formation rate and diameters of neurospheres increased in APP 5-mer peptide treated group. 5. The MTT metabolism rate increased in APP 5-mer peptide treated group. Conclusion APP 5-mer peptide could promote the production of embryo hippocamal NSCs in vitro. APP 5-mer peptide doesn't promote the differentiation of embryo hippocamal NSCs in vitro.
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In order to provide the "guiding principles of clinical research on mild cognitive impairment (MCI) (protocol)" edited by Beijing United Study Group on MCI of the Capital Foundation of Medical Developments (CFMD) with evidence support, clinical criteria, subtypes, inclusion and exclusion of MCI, and use of rating scales were reviewed. The authors suggested that MCI clinical criteria and new diagnosis procedure from the MCI Working Group of the European Alzheimer's disease Consortium (EADC) may better reflect the heterogeneity of MCI syndrome. Diagnostic rating scales including Clinical Dementia Rating (CDR), Global Deterioration Scale (GDS), Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog) and Instrumental Activities of Daily Living (IADL) are very useful in definition of MCI but can not replace its clinical criteria. Absence of major repercussions on daily life in patients with MCI was emphasized, but the patients may have minimal impairment in complex IADL. According to their previous research, the authors concluded that highly recommendable neuropsychological scales with cut-off scores in the screening of MCI cases should include Mini-Mental State Examination (MMSE), logistic memory test such as Delayed Story Recall (DSR), executive function test such as Clock Draw Test (CDT), language test such as Verbal Category Fluency Test (VCFT), etc. And finally, the detection of biological and neuroimaging changes, including atrophy in hippocampus or medial temporal lobe in patients with MCI, was introduced.
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Mild cognitive impairment (MCI), as a nosological entity referring to elderly people with MCI but without dementia, was proposed as a warning signal of dementia occurrence and a novel therapeutic target. MCI clinical criteria and diagnostic procedure from the MCI Working Group of the European Alzheimer's Disease Consortium (EADC) may better reflect the heterogeneity of MCI syndrome. Beijing United Study Group on MCI funded by the Capital Foundation of Medical Developments (CFMD) proposed the guiding principles of clinical research on MCI. The diagnostic methods include clinical, neuropsychological, functional, neuroimaging and genetic measures. The diagnostic procedure includes three stages. Firstly, MCI syndrome must be defined, which should correspond to: (1) cognitive complaints coming from the patients or their families; (2) reporting of a relative decline in cognitive functioning during the past year by the patient or informant; (3) cognitive disorders evidenced by clinical evaluation; (4) activities of daily living preserved and complex instrumental functions either intact or minimally impaired; and (5) absence of dementia. Secondly, subtypes of MCI have to be recognized as amnestic MCI (aMCI), single non-memory MCI (snmMCI) and multiple-domains MCI (mdMCI). Finally, the subtype causes could be identified commonly as Alzheimer disease (AD), vascular dementia (VaD), and other degenerative diseases such as frontal-temporal dementia (FTD), Lewy body disease (LBD), semantic dementia (SM), as well as trauma, infection, toxicity and nutrition deficiency. The recommended special tests include serum vitamin B12 and folic acid, plasma insulin, insulin-degrading enzyme, Abeta40, Abeta42, inflammatory factors. Computed tomography (or preferentially magnetic resonance imaging, when available) is mandatory. As measurable therapeutic outcomes, the primary outcome should be the probability of progression to dementia, the secondary outcomes should be cognition and function, and the supplement outcome should be the syndrome defined by traditional Chinese medicine. And for APOE epsilon4 carrier, influence of the carrier status on progression rate to dementia and the effect of treatment should be evaluated.
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BACKGROUND: In brain insulin does its work through the insulin receptor substrate (IRS). Amyloid beta protein precursor 17 (APP17) peptide has the neurotrophic function, which may improve diabetic encephalopathy resulted from insulin deficiency by affecting insulin receptor substrate.OBJECTIVE: The mouse diabetic model was produced to observe the effect of APP17 peptide on the distribution of IRS-1 in brain tissues.DESIGN: Randomized control animal experiment.SETTING: Staff Room of Pathology, College of Basic Medical Sciences,Capital University of Medical Sciences; Beijing Research Laboratory for Brain Aging of Xuanwu Hospital.MATERIALS: The experiment was performed in Staff Room of Pathology,College of Basic Medical Sciences, Capital University of Medical Sciences and Beijing Research Laboratory for Brain Aging of Xuanwu Hospital from September to October 2003. Totally 18 male kunming mice were employed,and randomly assigned into control group, diabetic group and APP17 peptide treatment group with 6 mice in each group.METHODS: ①The mice were subjected to intraperitoneal injection of streptozotocin (STZ, Sigma) by 200 mg/kg, and 3 days later, the tail blood was sampled to examine non-fasting blood glucose, and the blood glucose over 15 mmol/L was set as the criteria for successful diabetic model establishment. ②In APP17 + diabetes mellitus group, the mice received subcutaneous injection of 0.35 μg APP17 peptide once daily for 2 weeks. The mice in the normal control group were not interfered. ③Then brain was removed and crystat sections were prepared. Immunohistochemical staining was done for IRS-1 at four weeks after giving streptozotocin.MAIN OUTCOME MEASURES: Pattern and distribution of IRS-1 positive cells of mice in each group.RESULTS: Totally 18 mice were involved in the result analysis. ①In the brains of diabetic mice the IRS-1 immunohistochemical positive cells distributed at cortex, hippocampus, thalamus, hypothalamus and so on, while the positive cells distributed only at cortex and hippocampus in the normal control group and APP17 peptide treatment group, lightly stained. ②Numbers of immunohistochemical positive cells of IRS-1 of cerebral hippocampus in the diabetic group, normal control group and APP17 peptide treatment group were (28.7±1.5), (9.2±1.5), (10.1±1.4) piece per 10 power object lens, and that in the diabetic group was higher than that in the other two groups (P ＜ 0. 001 ). CONCLUSION: Neurons in many regions of brains of diabetic mice have plenty of IRS-1 positive cells. APP17 peptide can make part and quantity of IRS-1 positive cells normality so as to ameliorate the degeneration of hippocampal neurons of diabetic mice.
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BACKGROUND: D-galactose-induced aging animal model is similar tohuman natural aging. Whether the expression of protein phosphatase-1(PP-1) in the brain of D-galactose-induced aging mice is related to cerebralaging process or not should be researched further. OBJECTIVE: To investigate the effect of the APP17 peptide and theliquid extract ofjiunao yizhi capsule on regulating the expression of PP-1 inhippocampal neurons of the aging mice induced by the D-galactose (D-gal). DESIGN: A random controlled study. SETTING: Department of Pathology, College of Basic Medical Sciences, Capital University of Medical Sciences and Beijing Research Laboratory forBrain Aging, Xuanwu Hospital of Capital University of Medical Sciences. MATERIALS: The study was completed between July 2003 and July2004 in the Experimental Center of Capital University of Medical Sciences. Forty male Kunming mice (SPF grade) with a body mass fron 28 g to 32 gwere purchased from Chinese Academy of Medical Sciences Institute ofMaterial Medical.METHODS: Kunming mice were randomly divided into 5 groups: control group, D-gal group, APP17 peptide treatment group, low dose herb treatment group, and high dose herb treatment group with 8 mice in each group. In Dgal group, APP17 peptide treatment group, low dose herb treatment group and high dose herb treatment group, galactose was injected subcutaneously (50 nmg/kg). Meanwhile, 0.1 mL normal saline containing 0.35 μg of APP17 peptide was injected subcutaneously into the mice in APP17 peptide treatment group, once a day for 3 months; liquid extract of jiunao yizhi capsule (provided by Beijing Chaoyangmen Hospital and Shanxi Quwo Traditional Medical Institute; the main component: dangshen, baizhu, guijia and chuanshanjia, etc.) was perfused by stomach (0.3 g/kg and 1.0 g/kg respectively) in low dose herb treatment group and high dose herb treatment group, once a day. And equivalent normal saline was injected and perfused in the two control groups. After 3 months of survival, the mice were killed and their brains were cut into sections. The immunohistochemical staining of these sections was then performed with PP-1 antibody.MAIN OUTCOME MEASURES: The results of immunohistochemical staining analysis of PP-1.RESULTS: Forty mice entered the final analysis without any loss. PP-1 positive cells in the hippocampus were poorly stained in the D-gal mice. In contrast, PP-1 positive neurons were widely distributed in the hippocampus of those normal mice, the APP17 peptide-treated D-gal mice and the high liquid extract of raw herb-treated D-gal mice. These cells were darkly stained in cytoplasm. The unexpected result was that in the low liquid extract of raw herb-treated D-gal mice the number of PP-1 positive neurons did not increase to normal.CONCLUSION: The results demonstrated that the expression of PP-1 decreased in the hippocampus of D-gal mice. The APP17 and low dose liquid extract of raw herbs can regulate the distribution of PP-1 in the brain of D-gal mice and make them recover to normal situation.
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BACKGROUND: Overexpression of phosphorylated Tau protein is a factor of dementia, and scholars abroad find that APP17 peptide may have effect on it.OBJECTIVE: To observe changes of phosphorylated Tau protein Ser202/Thr205 of mice with diabetes mellitus (DM) after injection of APP17 peptide.DESIGN: Randomized control study.SETTING: Department of Pathology, Capital University of Medical Sciences; Department of Brain Aging, Xuanwu Hospital, Capital University of Medical Sciences.MATERIALS: The experiment was carried out in the Pathological Department of Capital University of Medical Sciences and Brain Aging Department of Beijing Xuanwu Hospital. A total of 18 male Kunming mice of 8 weeks old and weighing 28-32 g were randomly divided into control group, DM group and APP17 peptide group with 6 in each group.METHODS: DM models were induced by streptozotocin (STZ) through selectively destroying β-islet cells; meanwhile, APP17 peptide was intraperitoneally injected into mice. Four weeks later, brain tissue underwentimmunohistochemical staining with AT-8 (Ser202/Thr205, a special monoclonal antibody).MAIN OUTCOME MEASURES: ① Morphological observation; ② AT-8 distribution; ③ quantitative analysis of immunohistochemical staining.RESULTS: Positive AT-8 cells in DM group were distributed in retrosplenial cortex, hippocampus, thalamus, hypothalamus, etc.; however, those incontrol and APP17 peptide groups were only distributed in retrosplenial cortex and hippocampus, and poorly stained.CONCLUSION: Positive AT-8 cells may be widely distributed in neurons of brains of DM mice; however, APP17 peptide may normalize the expression of positive AT-8 cells.
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Objective To analyze the expression of BDNF, NGF,GFAP in Wistar rat pretectel area and determine the relation between neuron damage and neurotrophin change of pretectal area and retinal neurodegeneration in the early stage of diabetes. Methods Male Wistar rats weighing about 200 g were used to create diabetic animal model by injecting STZ. Wistar rats were killed by heart perfusion with 4% FDP at 1-week, 2-week and 1-month after diabetes. The dissected pretectal area was used to determine the expression of BDNF, NGF and GFAP with immunohistochemistry. Results In pretectal area of diabetic rat, the expression of BDNF and NGF began to decrease from 1-week DM, the expression of GFAP began to increase from 2-week DM. Conclusions There are neuron damage and neurotrophin decrease in the pretectal area in the early stage of diabetes. Neurodegeneration and neurotrophin decrease in pretectal area, which is the target tissue of retina, might be one of the reasons resulting in retinal neurodegeneration at the early stage of diabetes.
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@# ObjectiveTo observe the injured changes of brain myelin sheath structure and myelin basic protein (MBP) content induced by amyloid β peptide (Aβ) and effect of GETO on these changes.MethodsThe experimental rat model of Alzheimer's disease was established with Aβ1-42 injection into hippocampus. 4 weeks later, the myelin sheath structure of the CA1 area of the rat hippocampus was taken and observed by electromicroscope, and distribution and content of MBP were examined with immunohistochemical method.ResultsThe electromicroscope showed that the structure of myelin sheath became relaxing, disorder, homogenization and default of hippocampus CA1 in the model rats. In GETO treated group, the structure of myelin sheath was integrity and continuum. Immunohistochemical test showed that the staining and numbers of myelin sheath of model rats was thinner than that of normal rats and GETO treated rats. The numbers, mean area and mean density of positive staining axon in hippocampus CA1 of MBP in the model rats were significantly different from those in the normal group and GETO group (P<0.01).ConclusionAβ1-42 injection into hippocampus in rats can impair myelin sheath to make MBP release and GETO can ameliorate these changes.
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BACKGROUND: Amyloid β(Aβ) protein is the core of senile plaque.Being the toxic segment of Aβ, Aβ25-35 has been extensively applied in the experiments of recent years. The research in the past has verified that the self-prepared Chinese herb, fuzhisan can promote the survival of the cultured neural cells and probably acts on the treatment of Alzheimer disease (AD).OBJECTIVE: To study the resistance of fuzhisan to Aβ25-35 toxicity to cultured neural cells and the probable approaches.DESIGN: Repeated measurement based on the cells.SETTING: Department of neurology of a university hospital and key experimental room in brain aging in a university hospital.MATERIALS: The experiment was performed in Beijing Key Experimental Room in Brain Aging of Xuanwu Hospital from June 2002 to April 2003. Dopaminergic SH-SY5Y cell of neuroblastoma and Aβ25-35 were employed. Chinese herb, fuzhisan was decocted with mild fire and its upper clear solution was collected and prepared into storage solution at the concentration of 0. 5 g/mL. Antibody: Beijing Key Experimental Room in Brain Aging of Xuanwu Hospital prepared cAMP responsive element binding protein(CREB),Bcl-2 in B lymphatic leukaemia-2 genetic product and cytochrome C(CytC).METHODS: SH-SY5Y cell was incubated with Aβ25-35 of various doses alone or in combination with fuzhisan and was compared with blank control. MTF metabolic rate of cultured neural cells were determined under different incubation conditions. Western-blot method was used to measure the protein expression changes in incubation with fuzhisanalone, incubation with Aβ25-35 alone and the combination incubation, compared with the blank control.MAIN OUTCOME MEASURES: It was to study MTT metabolic rate in the comparison between each experimental group and the blank control and expressions of CREB, Bcl-2 and CytC relevant to survival/death of neural cells.RESULTS: Survival rate of SH-SY5Y cell was increased by 11.4% in incubation with fuzhisan alone. It was remarkably improved in incubation combining fu zhisan with Aβ25-35 as compared with Aβ25-35 alone. The expressions of CREB and Bcl-2 in Aβ25-35 group were decreased and were increased in fuzhisan group. CytC expression in cytoplasm was increased in Aβ25-35 group and was declined with fuzhisan incubation.CONCLUSION: Fuzhisan promotes the survival of cultured neural cells and its protection is still existed under Aβ25-35 injury. Fuzhisan brings such effects into play probably by the protein expressions relevant to survival/death of cells.
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BACKGROUND: It has been verified in the experiments over the past that the self-prepared Chinese herb, fuzhisan can retard natural aging in rats, suggesting that such drug acts on anti-aging.OBJECTIVE: To observe the effect of optimum effective concentration of nerve cell cultured with fuzhisan on morphological alternation of neuroblastoma SH-SY5Y cell.DESIGN: Repeated measurement.MATERIALS: The experiment was performed in Beijing Key Laboratory Room of Cerebral Aging of Xuanwu Hospital Affiliated to Capital University of Medical Sciences from June 2002 to April 2003. Self-prepared Chinese herb, fuzhisan [composed of 6 herbs, such as shichangbu (Rhizoma Acori Graminei), yuanzhi (Radix Polygalae), etc.] was co-developed by Prof.Wang De-shen from Department of Neurology of First Clinical Medical College of Harbin Medical University and Prof. Xu Xiao-yun from Department of Neurology of Shanghai Oriental Hospital. In addition, amyloid βprotein 25-35 segment and SH-SY5Y neuroblastoma were provided.METHODS: Fuzhisan of various concentrations were used for incubation of neuroblastoma SH-SY5Y cell. Thiazolyl blue (MTT), colorimetric method was used to determine the cell survival rate. Dose-effect relationship curve was drawn up to search optimum drug concentration. The cells cultured with 6-pore plate were divided into normal control, amyloid β-protein 25-35 25 μmol/L group, amyloid β-protein 25-35 25 μmol/L + fuzhisan 45×10-3 g/L groups and fuzhisan 45×10-3 g/L group. They were incubated for 24 hours to observe cell morphological alternation and determine neurosome area and axon length.ery group.length of every group: Those in amyloid β-protein 25-35 25 μmol/L group were decreased remarkably than the normal control [(505.5 ±122.36),(599.8 ±141.25) μm2; (26.0±13.97), (36.5 ±15.58) μm, (t =3.903,3.447, P=0.000)]. Those in fuzhisan 45×10-3g/L group and amyloid β-protein 25-35 25 μmol/L + fuzhisan 45×10-3 g/L group were increased remarkably than amyloid β-protein 25-35 25 μmol/L group [(918.3±178.34),(896.6 ±257.14), (505.5 ±122.36) μm2; (96.8 ±43.31), (88.3 ±30.23),(26.0±13.97) μm, (t=10.922, 14.172, P=-0.000)].CONCLUSION: With injury of amyloid β-protein 25-35, fuzhisan still enhances the survival of cultured nerve cell, manifested as promoting the increase of neurosome area and axonal extension.
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BACKGROUND: Learning and memory disorder exist in diabetic rats,which can be improved by APP 17-mer peptide. However, it is unclear whether learning and memory disorder in diabetes mellitus is caused by influencing neuronal mitochondrial transmembrane potentials and apoptosis in hippocampus or not and what is the related action mechanism of APP17-mer peptide.OBJECTIVE: To observe the effects of APP17-mer peptide on neuronal mitochondrial transmembrane potentials (△ψm) and apoptosis in hippocampal area of diabetic rats.DESIGN: A completely randomized, grouping and controlled trial.SETTING: Beijing Research Laboratory for Brain Aging, Beijing Xuanwu Hospital, Capital University of Medical Sciences; the Department of Endocrine, the First Central Hospital of Baoding.MATERIALS: The data measurement of the experiment was carried out in the Instrument Testing Center, the General Hospital of Chinese PLA between May 2002 and August 2002. The modeling and intervention of the experiment was carried out in the Animal Laboratory of Xuanwu Hospital, Capital University of Medical Sciences. Eighteen male Wistar rats were enrolled and randomized into control group, model group and APP17-mer peptide group with 6 rats in each group.METHODS: ① Diabetic models in the model and APP17-mer peptide groups were established by intraperitoneal injection of 60 mg/kg streptozotocin (pH=4.4) in fasted rats(fasting for 12 hours). Three days later, modeling was successful if blood sugar level in caudal vein was more than 15 mmol/L. Rats in the control group were not subjected to modeling.Then, the rats in the APP17-mer peptide group were subjected to the subcutaneous injection of APP17-mer peptide (3.4 μg for each rat once) three times a week and totally for ten weeks, whereas rats in the other groups were given saline of the same volume. ② After ten weeks, rats were anesthetized and decapitated to take out brain tissues, and then hippocampal tissues were isolated in ice bath for preparation of single cell suspension.JC-1 labeled mitochondrial transmembrane potentials and cell apoptosis in hippocampal area were measured by means of flow cytometry. ③ One-way analysis of variance was adopted in the comparison among groups.RESULTS: Eighteen rats were involved in the results analysis. ①Neuronal mitochondrial transmembrane potential was lower in the model group as compared with the control group [(551.91±53.36) vs (809.88±82.41) △ψm,P＜0.01] while it was higher in the APP17-mer peptide group as compared with the model group [(705.99±89.92) vs (551.91±53.36) △ψm, P ＜ 0.05].There was no difference between the APP17-mer peptide group and control group (P=0.146). ②) Apoptotic percentage of single cell in hippocampus was significantly higher in the model group than in the control and APP17-mer peptide groups [(5.32±1.37)%, (1.03±0.55)%, (2.80±0.92)%, P＜0.01, 0.05].CONCLUSION: Neuronal mitochondrial transmembrane potential and cell apoptosis in hippocampus may be involved in the occurrence and development of diabetes mellitus, and APP17-mer peptide plays an improved role in the process.
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@# ObjectiveTo observe the influence of mitochondrial cytochrome c oxidase(COX) decrease on cholinergic and dopaminergic system in brain of model rats.MethodsRats were administrated with 1mg/kg/h or 2mg/kg/h subcutaneously via an Alzet minipump for 30 days. Choline-acetyl-transfertase(ChAT) and acetylcholinesterase(AChE) activity in hippocampus and cortex of rats were measured by radiochemical method and hydroxylamine colorimetry separately. The contents of Norepinephrine(NE), dopamine, 5-hydroxytryptamine(5-HT) and their metabolic products in striatum were measured by HPLC.ResultsChAT activity was significantly inhibited in hippocampus and cortex of model rats, however, the activity of AChE increased in hippocampus and was not affected at the cortex. Therefore, the ratio of ChAT/AChE decreased in model rats. The content of NE, dopamine, 5-HT and their metabolic products in striatum were not different among all groups.ConclusionsCOX deficiency can induce abnormality of ChAT and AChE activity in model rats. Dysfunction of neurotransmitter-acetylcholine would be account for learning-memory deficiency. Model rats indicated cholinergic system deficit without any dopaminergic system abnormality, so chronic infusion of sodium azide via minipump may specially serve as a tool for developing the experimental model of Alzheimer's disease.
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<p><b>OBJECTIVES</b>To explore the mechanisms of neuronal loss and apoptosis in the brains of Alzheimer's disease (AD) patients, through studying the expression of proteins related to signal transduction pathways, which are important for neuron survival.</p><p><b>METHODS</b>(1) Immunohistochemistry: Sections were double stained with Tunel and NSE antibodies. (2) The hippocampal tissue taken from 6 cases of AD and 6 cases of non-AD brains was homogenized. Protein estimation was done by Lowry method. Equal amounts of protein were taken from each specimen and immunoprecipitation was performed and analyzed by Western blot; color development was done by alkaline phosphatase method or luminol reagent.</p><p><b>RESULTS</b>(1) Tunel positive neurons were found in both AD and non-AD brains, but the number in the former was more than the latter. (2) The AD hippocampal tissue showed diminished expression of Akt/PKB, CREB, P-CREB, increased expression of apoptosis-related protein apoptosis-inducing factor, and diminished expression of apoptosis-related protein bcl-2. The expression of bax did not change.</p><p><b>CONCLUSIONS</b>Diminished expression of CREB, P-CREB, bcl-2 in AD hippocampus indicates that the neuron survival signal transduction pathway in AD brains is impaired. Neurons are in apoptotic or pro-apoptotic state. In addition, increased expression of apoptosis-inducing factor, diminished expression of bcl-2, which is an anti-apoptotic factor, promotes further neuron apoptosis.</p>
Subject(s)
Aged , Aged, 80 and over , Humans , Male , Alzheimer Disease , Metabolism , Pathology , Apoptosis , Cyclic AMP Response Element-Binding Protein , Metabolism , Neurons , Metabolism , Pathology , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Signal Transduction , PhysiologyABSTRACT
Objective:To analyze a novel epitope of Homo sapiens synapse associated protein and synthesize polyclonal antibody.Methods:FRG4 full-length sequence was obtained by PCR from human fetal liver library;by bioinformatics to detect the second structure of amino acids encoded by FRG4 and its epitope and motifs;by solid-phase peptide synthesis method to synthesize FRG4 peptides,then peptides were immunized to rabbits;by immunohistory to detect the expression of FRG4 in HepG_2 cells.Results:Select 13-peptides PKLVKEEVFWRNY by bioinformatics to synthesize rabbit anti-human FRG4 polyclonal antibody.Antibody purity was 82.79% and antibody dilution was 1∶16 000 detected by ELISA.The antibody had a good reaction and speciality in Western blot,it was mainly expressed in cytoplasm of HepG_2 cells.Conclusion:A novel Homo sapiens synapse associated protein(FRG4) antibody was synthesized successfully.
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The purpose of the present work is to observe whether Tau protein Ser202/Thr205 is hyperphosphorylated in braintissues of diabetic mice and to study the effect of App17 peptide. Mouse diabetic model was produced with streptozotocin, andApp1 7 peptide as a treatment was injected subcutaneously into diabetic mice. Four weeks later, fixative was injected intravascu-larly into the mice, the brain was removed and crystat sections prepared. Immunohistochemical staining was done with AT-8. Inthe brains of diabetic mice positive AT-8 reacting neurons were numerous, darkly stained, and widely distributed in retrosplenialgranular cortex, hippocampus, thalamus et al. , while in normal mice and App17 peptide-treated diabetic mice positive cells werescarce and poorly stained. Tau protein is hyperphosphorylated at Scr202/Thr205 site and widely distributed in the brains of dia-betic mice, while App17 peptide can normalize the expression of AT-8 positive cells.
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AIM: To observe the expression of apoptosis-related proteins in hippocampal neurons of (ovariectomized) (OVX) rats and explore the neuroprotective mechanism of the App17-mer peptide. METHODS: Female Wistar rats were randomly divided into three groups. Bilaterally ovariectomized rats with injection of App 17P peptide (3.5 ?g in 0.1 mL/per rat, three times a week) formed the experimental group (17P+ OVX group). Anti-AIF, Bcl-2 and Bax antibodies were applied in the immunohistochemistry experiment. TUNEL was employed to detect apoptosis. RESULTS: The number of apoptotic neurons was clearly higher in hippocampal and cortex in OVX group than that in OVX+17P group. Immunohistochemistry demonstrated the increased expression of AIF, Bax in hippocampal neurons of OVX group. OVX group showed a significantly reduced expression of Bcl-2 in hippocampal neurons. Hippocampal tissue from OVX group showed the increased expression of AIF, Bax, and showed diminished expression of Bcl-2, treating with App17-mer peptide normalized the expression of these proteins. CONCLUSIONS: The expression of apoptosis-related proteins were abnormal in the OVX rats, App17-mer peptide normalized these changes. Estrogen deficiency induced neuronal apoptosis, and App17-mer peptide diminished apoptosis.
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AIM:To observe the influence of beta-amyloid precursor protein (APP17) on the study ability, memory and the expression of neurotrophin-3 (NT-3), nerve growth factor(NGF)in the hippocampus neuron of the model mice. METHODS: Mice brain aging model were produced with D-galactose(D-gal), the model mice were given hypodermic injection of APP17 peptide. APP17 peptide is the 319-335 peptide sequence of beta-amyloid precursor protein. Eight weeks later, the animals were observed by water labyrinth test and immunohistochemistry assay. RESULT:(1) The whole time needed and total times of wrong response for the D-gal group mice to complete the whole course of the water labyrinth test is significantly higher than the normal control group. (2) The expression of NT-3, NGF in the hippocampus neurons of the mice in APP17 peptide group is significantly higher than that of the normal control group and D-gal mice group, P
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AIM To examine the effects of the APP17-mer peptide against A? 25-35 -induced apoptosis and gain some insight into the neuroprotective mechanism of the APP17-mer peptide. METHODS Protective effects of APP17-mer peptide against A? 25-35 -induced apoptosis in SH-SY5Y cell was proved by cell morphology, LM-PCR DNA ladder assay and FCM assay. The antiapoptotic mechanism of APP17-mer peptide was investigated using the MTT assay to measure mitochondrial energy redox state, using the fluorescent probe DCF-DA?Rhodamine 123 to measure relative levels of cellular peroxides and mitochondrial membrane potential and using Western blot for AIF and NF-?B to detect the expression of AIF and NF-?B. RESULTS Damage of cell morphology was ameliorated by pretreating with APP17-mer peptide. The apoptotic rate of the SH-SY5Y cells exposed to A? 25-35 in the presence of APP17-mer peptide decreased from 63.75% to 28.25%. Exposure of SH-SY5Y to A? 25-35 for 48 h resulted in an increase in DCF-DA fluorescence,a decrease in Rhodamine 123 fluorescence and MTT reduction, the results were weakened by pre-incubating with APP17-mer peptide for 30 minutes. Treatment of cells with APP17-mer peptide resulted in a significant attenuation in the expression of AIF and a strong increase in the expression of NF-?B. CONCLUSION APP17-mer is protective against cell apoptosis induced by A? 25-35 by provoking and sustaining upregulation of a key antiapoptotic transcription factor NF-?B, by suppressing oxyradical production and by preserving mitochondrial function and inhibiting the release of apoptotic protein from mitochondria.