Brain natriuretic peptide consists of 26 amino acids and has diuretic, natriuretic and hypotensive activities. We have synthesized BNP and its analog (BNP-A) (Mpr4, D-Ala613)-BNP(4-26)-NH2 using solid phase method and examined their biological activities. Our results showed that 1) BNP (10-7mol/L) dilated human basal cerebral artery which was pre-constricted with endothelin, 5-HT and noradrenaline in vitro; 2) both BNP (5 ?g/ rat) and BNP-A (5 ?g/ rat) increased regional cerebral blood flow following i.v. injection in rats; 3) intracerebroventricular injection of BNP (3 ?g/ rat) induced an obvious natriuretic effect when blood volume was extremely expanded with hypoosmotic glucose solution in rats; 4) BNP (5 ?g/ rat) infusion for 60 minutes caused a decline of plasma concentration of angiotensin II and aldosterone at the end of infusion in spontaneous hypertensive rats (SHR), and the bolus injection of BNP (10 ?g/ rat) led to a fall in blood pressure about 1.33kPa. We conclude that BNP is an important peptide in regulating water and sodium metabolism and plays an important role in regulating blood flow to various organs.

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.

This paper briefly describes the mechanism of neurochemical changes in AD,the roles of the epigenetics,the regulation of gene expression and the mechanism of epigenetics in SAD.On the basis of the basic study on the AD during the decades,researchers initially revealed the mystery of the unfathomable neurochemical changes attack on AD.Especially in recentyears,researchers summarized the abnormal protein expression on SAD into epigenetics disease.This achievement helps researchers point out the new and strategic direction on AD pathogenesis,prevention and control mechanism.With the understanding of biological characteristics of epigenetics,AD needs to be studied in depth.

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.

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.

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.

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.

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.

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.

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.