β-淀粉样蛋白(Aβ32-35)的血管活性及其机制探讨

目的 研究Ａβ３２－３５的血管活性机制。方法 建立鼠后爪垫皮肤微血管网模型，将新鲜制备的Ａβ３２－３５（１０μｍｏｌ）溶液、Ａｃｈ（１００μｍｏｌ）、ＳＮＰ（１００μｍｏｌ）、ＢＱ－１２３（１０μｍｏｌ）以及抗氧化剂ＳＯＤ（１００Ｕ／ｍｌ）、Ｃａｔａｌａｓｅ （１００Ｕ／ｍｌ）和ＮＡＣＥ（１００μｍｏｌ）溶液按一定顺序由微型泵驱动灌注微血管网、激光多普勒血流监测仪测量微血管 血液流量的变化。结果 灌注 Ａβ３２－３５引起血管收缩反应（ＶＣ）；灌注Ａｃｈ和 ＳＮＰ引起血管舒张反应（ＶＤ）。先灌 注Ａβ３２－３５，立即或２ｈ后再予Ａｃｈ均造成Ａｃｈ的ＶＤ消失：但立即予ＳＮＰ或２ｈ后再予ＳＮＰ则造成ＳＮＰ的ＶＤ减 弱或不受影响。先灌注ＢＱ－１２３或Ｃａｔａｌａｓｅ或ＮＡＣＥ后予Ａβ－３２－３５；再予Ａｃｈ，则Ａβ３２－３５引起的ＶＣ反应消失；但Ａｃｈ的ＶＤ反应在不同时间点得到部分恢复，ＳＯＤ对Ａβ３２－３５及Ａｃｈ的血管反应无影响。结论 提供活体鼠Ａβ３２－３５收缩外周微血管依据：其机制通过内皮细胞和平滑肌细胞的长短期作用实现，氧自由基和内皮素－１参与Ａβ３２－３５的缩血管作用。

Effect of amyloid peptides on serum withdrawal-induced cell differentiation and cell viability

Abnormal deposition of amyloid-p(Ap) peptides and formation of neuritic plaques are recognized as pathological processes in Alzheimer's disease (AD) brain. By using amyloid precursor protein (APP) transfected cells, this study aims to investigate the effect of overproduction of Aβ on cell differentiation and cell viability. It was shown that after serum withdrawal, untransfected cell (N2a/Wt) and vector transfected cells (N2a/vector) extended long and branched cell processes, whereas no neurites was induced in wild type APP (N2a/APP695) and Swedish mutant APP (N2a/ APPswe) transfected N2a cells. After differentiation by serum withdrawal, the localization of APP/AP and neurofilament was extended to neurites, whereas those of APP-transfected cells were stillrestricted within the cell body. Levels of both APP and Aβ were significantly higher in N2a/APP695 and N2a/APPswe than in N2a/Wt, as determined by Western blot and Sandwich ELISA, respectively. To further investigate the effect of A0 on the inhibition of cell differentiation, we added exogenously the similar level or about 10-times of the AP level produced by N2a/APP695 and N2a/APPswe to the culture medium and co-cultured with N2a/Wt for 12 h, and we found that the inhibition of serum withdrawal-induced differentiation observed in N2a/APP695 and N2a/APPswe could not be reproduced by exogenous administration of AP into N2a/Wt. We also observed that neither endogenous production nor exogenous addition of Aβ1-40 or Aβ1-42, even to hundreds fold of the physiological concentration, affected obviously the cell viability. These results suggest that the overproduction of AP could not arrest cell differentiation induced by serum deprivation and that, at least to a certain degree and in a limited time period, is not toxic to cell viability.

Pathway and mechanism of oxidative stress in Alzheimer's disease

Current hypotheses of pathogenesis of neuronal degeneration in Alzheimer's disease (AD) have been proposed, including formation of free radicals, oxidative stress, mitochondrial dysfunction, inflammatory processes, genetic factors, environmental impact factors, apoptosis, and so on. Especially, oxidative stress plays an essential role in AD pathogenesis by the function of linking agent. Oxidative stress in AD mainly includes lipid peroxidation, protein oxidation and DNA oxidation. Lipid peroxidation plays a key role in the development and progression of AD. Protein oxidation is an important mechanism in AD. Oxidative damage to DNA may plays an important role in aging and AD.

Neuroprotective effect of fucoxanthin on β-amyloid-induced cell death

Alzheimer＇s disease （AD） is one of the most common cognitive disorders of the elderly. Fucoxanthin is a carotenoid that is found in common edible seaweed, and it is considered as a major active compound of marine algae with cancer-preventing, antioxidant and anti-inflammatory properties. In this study, we investigated the ability of fucoxanthin to protect against the β-amyloid protein （Aβ）-induced neurotoxicity in primary cortical cultured neurons and PC12 cells. Neuroprotective effects of fucoxanthin were determined by measuring cell viability and nuclei double-staining with Hoechst 33342 and propidium iodide following Aβ treatment with or without fucoxanthin. Moreover, we also evaluated its potential mechanism on antioxidation by detecting the total antioxidant capacity （T-AOC）, level of lipid peroxidation malondialdehyde （MDA） and activity of superoxide dismutase （SOD）. We found that exposure of cortical cultured neurons or PC12 cells to Aβ resulted in neuronal cell death, whereas pre-treatment with fucoxanthin reduced Aβ-induced cell death. The data on the T-AOC, MDA level and SOD activity showed that Aβ treatment resulted in decreases in T-AOC and SOD activity and an increase in MDA level. After fucoxanthin administration, the results of T-AOC, MDA level and SOD activity showed an opposite trend, indicating that T-AOC was increased and MDA level was reduced. These results suggested that fucoxanthin prevented Aβ-induced neurotoxicity through attenuating oxidative stress induced by Aβ. Therefore, fucoxanthin might be useful as a potential preventive or theraoeutic agent for AD.

Oxidative damage increased in presenilin1/presenilin2 conditional double knockout mice

Objective This report aims to describe the oxidative damage profile in brain ofpresenilinl andpresenilin2 conditional double knockout mice （dKO） at both early and late age stages, and to discuss the correlation between oxidative stress and the Alzheimer-like phenotypes of dKO mice. Methods The protein level of Aβ42 in dKO cortex and free 8-OHdG level in urine were measured by ELISA. Thiobarbituric acid method and spectrophotometric DNPH assay were used to determine the lipid peroxidation and protein oxidation in cortex, respectively. SOD and GSH-PX activities were assessed by SOD Assay Kit-WST and GSH-PX assay kit, separately. Results Significant decrease of Aβ42 was verified in dKO cortex at 6 months as compared to control mice. Although lipid peroxidation （assessed by MDA） was increased only in dKO cortex at 3 months and protein oxidation （assessed by carbonyl groups） was basically unchanged in dKO cortex, ELISA analysis revealed that free 8-OHdG, which was an indicator of DNA lesion, was significantly decreased in urine of dKO mice from 3 months to 1 2 months. Activities of SOD and GSH-PX in dKO and control cortices showed no statistical difference except a significant increase of GSH-PX activity in dKO mice at 9 months. Conclusion Oxidative damage, especially DNA lesion, was correlated with the neurodegenerative symptoms that appeared in dKO mice without the deposition of Aβ42. Triggers of oxidative damage could be the inflammatory mediators released by activated microglia and astrocytes.

Roles of NG2 glial cells in diseases of the central nervous system

NG2 cells are a novel distinct class of central nervous system（CNS） glial cells,characterized by the expression of the chondroitin sulfate proteoglycan NG2.They have been detected in a variety of human CNS diseases.As morphological,physiological and biomolecular studies of NG2 cells have been conducted,their roles have been gradually revealed.Research on cellular and molecular mechanisms in the pathophysiological state was built on the preliminary findings of their physiological functions;and in turn,this helps to clarify their physiological roles and leads to the identification of novel therapeutic targets.This review summarizes recent findings regarding the potential roles of NG2 cells in traumatic brain injury,multiple sclerosis,glioma,epilepsy,Alzheimer＇s disease and electroconvulsive therapy for depression.

A possible role of myristoylated alanine-rich C kinase substrate in endocytic pathway of Alzheimer’s disease

It is believed that amyloid-βpeptide（Aβ）plays a central role in the pathogenesis of Alzheimer’s disease（AD）.Thus,the process of amyloid precursor protein（APP）cleavage is a key event and has raised much attention in the field of AD research.It is proposed that APP,β-andγ-secretases are all located on the lipid raft,and the meeting of them is an indispensable step for Aβgeneration.Endocytosis can lead to clustering of APP,β-andγ-secretases from separate smaller lipid rafts into a larger one.On the other hand,for myristoylated alanine-rich C kinase substrate（MARCKS）,phosphorylation by protein kinase C（PKC）or interaction with Ca2＋can lead to its release from membrane into cytoplasm.This process induces the release of actins and phosphatidylinositol 4,5-bisphosphate（PIP2）,which are important factors for endocytosis.Thus,the present review proposes that MARCKS may be implicated in Aβgeneration,by modulating free PIP 2 level and actin movement,causing endocytosis.

Ameliorating effect of Zhizi(Fructus Gardeniae) extract and its glycosides on scopolamine-induced memory impairment

OBJECTIVE： To study the chemical constituents of Zhizi （Fructus Gardeniae） and their antiamnesic ef- fect in a mouse model of Alzheimer＇s disease. METHODS： Ameliorating effects of the extracts, fractions and constituents on scopolamine-in- duced memory impairment in vivo using a passive avoidance task system and their inhibitory activi- ties on acetylcholinesterase （ACHE） in vitro were ex- amined. The isolation of components was per- formed by chromatographic techniques and their structures were identified on the basis of spectral analysis. RESULTS： Activity-guided fractionation of the total extracts resulted in the isolation of two glycosides, geniposide and cr0cin from the n-butanol fraction and genipin and crocetin from the ethylacetate fraction. Among the fractions tested, n-butanol fraction showed the strongest AChE inhibition （43.4% at a final dose of 0.03 mg/mL） and also ex- hibited outstanding efficacy （65.9% at a dose of2.50 mg/kg） in an experimental model of amnesia. Geniposide showed a 22.8% AChE inhibitory activi- ty and a potent ameliorating effect on scopol- amine-induced memory impairment in amnesic mice of 93.4% as compared to the control group. CONCLUSION： Geniposide, a main constituent of gardenia should be considered a candidate for fur- ther clinical study for the purpose of developing a cognition activator and its mechanism of action may be mediated, at least in part, by the acetylcho- line enhancing cholinergic nervous system.

Effect of Xixin decoction on O-linked N-acetylglucosamine Glycosylation of tau proteins in rat brain with sporadic Alzheimer disease

OBJECTIVE： To study the effects of Xixin decoction （XXD） on O-linked N-acetylglucosamine （O-GIcNAc） glycosylation of tau proteins in rat brain with spo- radic Alzheimer disease （SAD）, and discuss its possi- ble mechanism on prevention and treatment of SAD. METHODS： The rat model of SAD was established by intracerebroventricular injection of streptozoto- cin. The specific pathogen free male Sprague-Daw- ley rats were randomly divided into sham-opera- tion group （S）, model group （M）, donepezil group （D）, XXD at a low dose group （XL）, XXD at a medium dose group （XM） and XXD at a high dose group （XH）. After treatment and praxiology test, immuno- histochemistry and western blotting were used to detect O-GIcNAc glycosylation level of tau proteins in rat brain with SAD. O-GIcNAc glycosylation and expression of tau proteins were detected by O-GIcNAc-specific antibodies RL2 and CTD110.6. RESULTS： O-GIcNAc glycosylated proteins enriched by succinylated wheat germ agglutinin significant- ly improved in the hippocampus of SAD rats. Thedifferences were statistically significant among XXD groups （P〈0.05, P〈0.01）, while no obvious dif- ferences were observed between D group and M group （P〉0.05）. CONCLUSION： XXD can significantly improve O-GIcNAc glycosylation level of tau proteins in the hippocampus of SAD rats, which maybe inhibit hy- perphosphorylation of tau proteins on key sites and its toxicity, and prevent the pathological pro- cess of SAD.

Effect of reinforcing kidney-essence, removing phlegm, and promoting mental therapy on treating Alzheimer's disease

OBJECTIVE: To explore the mechanism of reinforcing kidney-essence, removing phlegm, and promoting mental therapy in treating Alzheimer's disease (AD). METHODS: Sixty patients with AD in Wuhan No.1 Hospital of Traditional Chinese Medicine for Geriatrics from May 2009 to April 2011 were randomly divided into two groups, with 30 in each group. Patients in Bushenhuatanyizhi group (BHY group) took BHY instant granules (6 g, twice per day). Patients in the control group took piracetam (0.8 g, 3 times per day). There were twelve weeks in a course. Changes in the mini-mental state examination (MMSE) score and Activity of Daily Living Scale (ADL) score were analyzed before and after treatment. RESULTS: MMSE scores in the two groups in-creased and ADL scores decreased after treatment, compared with those before treatment (both P< 0.05).The total effective rate was 86.67% in the BHY group (10 very effective, 13 effective, 7 non-responsive). The control group was 57.69% (5 very effective, 16 effective, 9 non-responsive) (P<0.05). Superoxide dismutase levels were increased, lipid peroxide and triglyceride levels decreased after treatment in the BHY group as compared with the control group. CONCLUSION: Reinforcing kidney-essence, removing phlegm, and promoting mental therapy can improve cognitive function and daily life of AD patients. The mechanism of the therapy might be related to improving blood fat, scavenging free radicals, and inhibiting lipid peroxides.

Nitric oxide in neurodegeneration:potential benefits of non-steroidal anti-inflammatories

The cellular messenger nitric oxide（NO） has been linked to neurodegenerative disorders due to the increased expression of the enzymes that catalyze its synthesis in postmortem tissues derived from sufferers of these diseases.Nitrated proteins have also been detected in these samples,revealing that NO is biologically active in regions damaged during neurodegeneration.Modulation of NO levels has been reported not only in the neurons of the central nervous system,but also in the glial cells（microglia and astroglia） activated during the neuroinflammatory response.Neuroinflammation has been found in some neurodegenerative conditions,and inhibition of these neuroinflammatory signals has been shown to delay the progress of such disorders.Thus NO and the pathways triggering its release are emerging as an important research focus in the search for strategies to prevent,halt or cure neurodegenerative diseases.

Effect of Bushenyisui Formula on brain tissue apoptosis and Bcl-2 in beta-amyloid protein-induced Alzheimer's disease rat models

OBJECTIVE： To investigate the effect of Bushenyisui Formula on cell apoptosis and positive B cell lym- phoma （Bcl-2） in the Brain of rat models of Alzheim- er＇s disease （AD） induced by beta-amyloid protein （All3） and the mechanism underlying the effect. METHODS： Total of 40 SD rats, 20 females and 20 males, were randomly assigned to 4 groups, con- trolled group （A）, model group （B）, conventional treatment group （C） and Bushenyisui Formula treat- ment （BYFT） group （D）, 10 rats in each group. AI3 1-42 was injected into the bilateral hippocampus of the rats in group B, C and D to create the models of AD. Sham operation was performed on the rats of group A in the same way by injecting equal volume of 0.9% sodium chloride solution into their bilateral hippocampus. 5 days after operation, Bushenyisui Formula was intraperitoneally administered at a dose of 450 mg/kg to the rats of group D （QD） for 20 days. Equal volume of 0.9% sodium chloride solution was intraperitoneally injected into the rats of group B with the same procedure. C suspension （20 mg/mL） was intraperitoneally injected into the rats of group B with the same procedure. The number of apoptot- ic cells in Brain and the positive Bcl-2 were count- ed. The changes of learning and memory abilities were evaluated using Y-maze. RESULTS： Right after the establishment of the mod- els, group B, C and D compared to group A respec- tively, the outcomes of Y-maze were significantly different from that of group A, which suggested ob- vious learning and memory disorder in those groups （P〈0.01）. After treatment, the times of elec- tronic shocks of group C and D were significantly less than that of group B （P〈0.05）, and the numbers of apoptotic cells and positive Bcl-2 were signifi- cantly different from those of group B, apoptotic sells＇ number of group C and D smaller than that of group B and the number of positive Bcl-2 greater than that of group B. CONCLUSION： Bushenyisui Formula could increase the number of Bcl-2 in brain, which improved the function of nervous system pertaining to learning and memory abilities.

Sirtuin 5:a review of structure,known inhibitors and clues for developing new inhibitors

Sirtuins(SIRTs) are nicotinamide adenine dinucleotide(NAD^+)-dependent protein deacetylases,which regulate important biological processes ranging from apoptosis,age-associated pathophysiologies,adipocyte and muscle differentiation,and energy expenditure to gluconeogenesis.Very recently,sirtuin 5(SIRT5) has received considerable attention due to that it was found to have weak deacetylase activity but strong desuccinylase,demalonylase and deglutarylase activities,and it was also found to be associated with several human diseases such as cancer,Alzheimer's disease,and Parkinson's disease.In this review,we for the first time summarized the structure characteristics,known peptide and small-molecule inhibitors of SIRT5,extracted some clues from current available information and introduced some feasible,practical in silico methods,which might be useful in further efforts to develop new SIRT5 inhibitors.

Effects of acupuncture plus medication on hippocampus SIRT1 and FOXO3a expression,MDA content,and SOD activity of rats with Alzheimer disease

Objective To observe the effect of the acupuncture plus medication on the expression of silent information regulator of transcription 1(SIRT1)and transcription factor forkhead box protein O3a(FOXO3a)in the hippocampus,the malondialdehyde(MDA)content,and the superoxide dismutase(SOD)activity of rats with Alzheimer disease(AD),and to explore the possible mechanism of combining acupuncture and medication in improving AD-related neurological symptoms.Methods Sixty male Sprague-Dawley rats were divided into a normal group,a model group,an electroacupuncture(EA)group,a drug group,and an acupuncture-medication combined group by the random number table method,with 12 rats in each group.The model was established by micro-injection of streptozotocin into the bilateral lateral ventricles.After successful modeling,rats in the EA group received EA at Zusanli(ST36)and Dazhui(GV14),those in the drug group received intragastric administration of resveratrol at a dose of 44 mg/(kg·bw),and those in the acupuncture-medication combined group received the combined intervention of EA and resveratrol.Rats in each group received intervention once a day for 4 consecutive weeks.Morris water maze was used to detect the rat behavioral changes.Nissl staining method was used to observe the cell morphology and changes in the number of rat hippocampal neurons.Western blotting and immunohistochemical staining methods were used to observe the expression changes of SIRT1 and FOXO3a.The thiobarbituric acid method was used to detect the MDA content.SOD activity was determined by the hydroxylamine method.Results Compared with the normal group,the escape latency was significantly prolonged(P<0.05);the percentage of stay in the target quadrant was reduced(P<0.05),the hippocampal neuronal cells were shrunken,nucleoli were unclear,and cell number was reduced(P<0.05);the SIRT1 expression and SIRT1 positive cell number were decreased,while the FOXO3a expression and FOXO3a positive cell number were increased significantly(P<0.05);the MDA content was increased significantly,and the SOD activity was decreased significantly(P<0.05)in the model group.Compared with the model group,the escape latency was shortened(P<0.05);the percentage of stay in the target quadrant was increased(P<0.05);the shape and number of hippocampal neurons tended to be normal(P<0.05);the SIRT1 protein expression and the SIRT1 positive cell numbers were increased,the FOXO3a protein expression and the FOXO3a positive cell number were decreased(P<0.05);the MDA content was significantly decreased,and the SOD activity was significantly increased(P<0.05)in the EA group,the drug group,and the acupuncture-medication combined group.The changes in the acupuncture-medication combined group were more obvious(P<0.05).Conclusion Both EA and resveratrol improve the learning and memory ability of AD rats by regulating the expression of SIRT1 and FOXO3a and improving the levels of MDA and SOD in the hippocampus and protect the hippocampal neurons,while the combined use of EA and medication is more effective than EA or resveratrol alone,suggesting that this combined treatment is more effective in AD treatment.