Huanglian Jiedutang Regulates HIF-1α/VEGF Signaling Pathway to Improve Learning and Memory Abilities of APP/PS1 Transgenic Mice / 中国实验方剂学杂志

ObjectiveTo reveal the effects of Huanglian Jiedutang （HLJDT） on the learning and memory abilities of APP/PS1 transgenic mice via hypoxia-inducible factor-1α （HIF-1α）/vascular endothelial growth factor （VEGF） signaling pathway. MethodForty 5-month-old β-amyloid precursor protein （APP）/presenilin 1（PS1） mice were randomized into the model， donepezil （0.001 g·kg-1·d-1）， and low-， medium-， and high-dose （1.5， 3， 6 g·kg-1·d-1， respectively） HLJDT groups， and 8 C57BL/6 mice were taken as the normal group. After 45 days of continuous administration， Morris water maze test was conducted， and the organ indexes were calculated. The morphological structure of cerebral vascular endothelial cells in mice was observed under a transmission electron microscope. Western blot was employed to measure the protein levels of APP， HIF-1α， VEGF，VEGFA， and brain-derived neurotrophic factor （BDNF） in the hippocampus. The mRNA levels of APP， HIF-1α， and VEGF were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the normal group， the model group showed prolonged escape latency （P<0.05）， reduced distance and time around the target platform （P<0.05）， decrease brain and spleen indexes （P<0.05）， vascular endothelial cells with karyopyknosis and not abundant cytoplasm， up-regulated protein levels of APP， HIF-1α， VEGF， and VEGFA （P<0.05）， down-regulated protein level of BDNF （P<0.05）， and up-regulated mRNA levels of APP， HIF-1α， and VEGF （P<0.05） in the hippocampus. Compared with the model group， high-dose HLJDT shortened the escape latency （P<0.05）， increased the distance and time around the target platform （P<0.05）， raised the brain and spleen indexes （P<0.05）， repaired the organelles of vascular endothelial cells， down-regulated the protein levels of APP， HIF-1α， VEGF， and VEGFA （P<0.05）， up-regulated the protein level of BDNF （P<0.05）， and down-regulated the mRNA levels of APP， HIF-1α， and VEGF （P<0.05） in the hippocampus. ConclusionHLJDT can improve the learning and memory abilities of mice by reducing the expression of HIF-1α and VEGF， thus protecting the nerves.

Expression, purification and micelle reconstruction of the transmembrane domain of the human amyloid precursor protein for NMR studies / 生物工程学报

The multiple-step cleavage of amyloid precursor protein (APP) generates amyloid-β peptides (Aβ), highly toxic molecules causing Alzheimer's disease (AD). The nonspecific cleavage between the transmembrane region of APP (APPTM) and γ-secretase is the key step of Aβ generation. Reconstituting APPTM under physiologically-relevant conditions is crucial to investigate how it interacts with γ-secretase and for future AD drug discovery. Although producing recombinant APPTM was reported before, the large scale purification was hindered by the use of biological protease in the presence of membrane protein. Here, we expressed recombinant APPTM in Escherichia coli using the pMM-LR6 vector and recovered the fusion protein from inclusion bodies. By combining Ni-NTA chromatography, cyanogen bromide cleavage, and reverse phase high performance liquid chromatography (RP-HPLC), isotopically-labeled APPTM was obtained in high yield and high purity. The reconstitution of APPTM into dodecylphosphocholine (DPC) micelle generated mono dispersed 2D 15N-1H HSQC spectra in high quality. We successfully established an efficient and reliable method for the expression, purification and reconstruction of APPTM, which may facilitate future investigation of APPTM and its complex in more native like membrane mimetics such as bicelle and nanodiscs.

Abdominal acupoint thread embedding therapy based on "brain-intestinal connection" for mild-to-moderate Alzheimer's disease and its effects on serum levels of APP and Aβ / 中国针灸

OBJECTIVE@#To compare the clinical efficacy of abdominal acupoint thread embedding therapy based on "brain-intestinal connection" combined with donepezil hydrochloride tablets and oral donepezil hydrochloride tablets alone for mild-to-moderate Alzheimer's disease (AD) and observe its effects on amyloid precursor protein (APP) and β-amyloid protein@*METHODS@#Sixty patients with AD were randomly divided into an observation group (30 cases, 3 cases dropped off) and a control group (30 cases, 3 cases dropped off). The patients in the control group were treated with donepezil hydrochloride tablets (5 mg per day); based on the treatment in the control group, the patients in the observation group were treated with abdominal acupoint thread embedding therapy at Zhongwan (CV 12), Xiawan (CV 10), Huaroumen (ST 24), Wailing (ST 26), Daheng (SP 15), etc., once every 10 days. Both groups were treated for 2 months. The mini-mental state examination (MMSE), Alzheimer's disease assessment scale-cognitive subscale (ADAS-Cog), activity of daily living scale (ADL), neuropsychiatric inventory questionnaire (NPI) as well as the serum levels of APP and Aβ@*RESULTS@#After treatment, the MMSE scores in the two groups were higher than those before treatment (@*CONCLUSION@#The abdominal acupoint thread embedding therapy based on the theory of "brain-intestinal connection" combined with donepezil hydrochloride tablets can improve cognitive function, self-care ability of daily life and mental behavior, and reduce the serum levels of APP and Aβ

The Essentials of Biochemistry of the Proteins as Related to Alzheimer’s Disease: A Review

Amyloid plaques and Tau tangles, constitute the pathological hallmarks of the brains of the patients suffering from Alzheimer’s disease. They are identified as far back as 1996 by Alois Alzheimer, a German psychiatrist and neuropathologist, but till this date, how they produce neuronal death remained an enigma. The amyloid cascade theory held its sway until recent times until the emphasis is shifted to the metabolites of amyloid Beta precursor protein (APP). Several metabolites of APP are formed depending on by which pathway, the APP is metabolized, either by the non - amyloidogenic pathway (forming ? - C terminal fragment - CTF? / C83 and the N - terminal fragment sAPP? / P3 and the APP intracellular domain AICD). Or amyloidogenic pathways. ( Forming extracellular A? and APP intracellular domain - AICD). The hyperphosphorylation is held responsible for the tau protein tangles. The over activity of the tau kinases or the failure of inhibition by the tau phosphatases i s implicated, in tau tangle deposits. These biochemical aspects of AD assumed importance in connection with the interventional therapeutic strategies that are developed in the years bygone, as well as those still are in the developing stage. In keeping with this fact, it is attempted to review the essentials of the biochemical aspects of the involved proteins, as related to AD, in this article

Effect of Huangjingwan on Antioxidant Functions and Aβ1-42 and APP Protein Expressions in Brain of Alzheimer' s Disease Rats / 中国实验方剂学杂志

Objective::To observe the effect of Huangjingwan (HW) on antioxidant functions and β-amyloid 1-42 (Aβ1-42) and amyloid precursor protein (APP) expressions in the brain of Alzheimer' s disease (AD) rats. Method::SD rats were randomly divided into normal control group, sham model control group, AD model group, and low, medium, high-dose (equivalent raw drug dose 1, 3, 9 g·kg-1·d-1) HW groups.The AD models were established through intraperitoneal injection with 1.25% D-galactose (120 mg·kg-1·d-1, 6 consecutive weeks) and then one-time right ventricular injection with Aβ1-42 (10 μg). Two weeks after modeling, the rats in each HW group received corresponding drugs through intragastric administration, once a day, while the rats in sham model control group, AD model group were given normal saline 1 mL through intragastric administration, once a day.Gastric perfusion lasted for 8 weeks.At the end of the experiment, learning and memory abilities of the rats were assessed by Platform Jumping Test.The changes of physical endurance in rats were tested by 10% weight swimming under load.The activities of superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GSH-Px) antioxidant enzymes and the contents of glutathione (GSH) and malondialdehyde (MDA) in rat brain tissue were detected by colorimetry.The changes of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), Aβ1-42 and APP protein in rat brain tissues were determined by enzyme linked immunosorbent assay (ELISA). Western blot analysis was used to measure the expression of APP protein in rat brain. Result::Compared with the normal control group, rats in AD model group showed an obvious dementia state, that is more lying and less movement, longer learning response time, significant increase in the number of learning and memory errors, significant attenuation in physical fitness, significant decrease in the activities of antioxidant enzymes (SOD, GR, GSH-Px) and anti-inflammatory factors GSH in brain, significant rise in the levels of inflammatory factors MDA, IL-1β and TNF-α and the content of Aβ1-42 protein, and significant reduction in the content of APP protein in brain (P<0.01). Low, medium and high-dose HW could ameliorate dementia symptoms in AD rats, improve the achievement of learning and memory, antagonize body weakness and increase physical fitness, promote SOD, GR, GSH-Px activities and anti-inflammatory factor GSH level in the brain, reduce the levels of MDA, IL-1β and TNF-α in the brain, decrease the level of Aβ1-42 and increase the level of APP protein in the brains of AD rats compared with the AD model group (P<0.05, P<0.01), besides, within the dose range of 1-9 g·kg-1·d-1, HW has a more obvious effect with the increase of dose. Conclusion::HW has the effects in preventing and treating AD, which is related to the HW' s mechanisms in enhancing the function of antioxidant system in brain, reducing neuroinflammatory reaction and deposition of Aβ1-42 induced by oxidative stress, and maintaining the expression level of APP protein.

Effect of Electro-acupuncture on Behavior Alterations in Mice with Isoflurane-induced Alzheimer's Disease and Its Mechanism / 广州中医药大学学报

Objective To investigate the effect of electro-acupuncture on behavior alterations,the expression of amyloid precursor protein(APP),amyloid β-protein(Aβ) proteins and neuroapoptosis in the cerebral cortex in the APPswe/PS1dE9 double transgenic mice with Alzheimer's disease (AD) induced by isoflurane.Methods Sixmonth-old AD mice and wild-type mice at the same age were randomly divided into wile-type control group,AD group,isoflurane group,electro-acupuncture group (N =8).The mice were given pretreatment with electro-acupuncture at Baihui(GV20) acupoint and Yongquan(KI 1) acupoint once a day for 3 successive days,15 min each time.And then the mice in electro-acupuncture group and isoflurane group were exposed to a box full of 1.2％ isoflurane for 4 hours.Morris water maze was used to test the learning and memory abilities of the mice,Western blotting method was used to detect the expression of APP-C83,APP-C99 and Aβ,and terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL) was used to detect neuroapoptosis in the cerebral cortex.Results The escape latency of AD group was longer than that of wild-type mice group(P＜0.05),and the latency of isoflurane group was longer than that of AD group (P ＜ 0.05),while the latency of electro-acupuncture group was shorter than that of isoflurane group(P ＜ 0.05).The percentage of retention time in the target quadrant and the times for crossing the target quadrant in isoflurane group were lower than those of AD group (P ＜ 0.05),but were higher in electro-acupuncture group than those in isoflurane group (P ＜ 0.05).APPC83 expression level in isoflurane group was significantly lower than that in AD group (P ＜ 0.05),while APP-C83 expression level in electro-acupuncture group was higher than that in isoflurane group (P ＜ 0.05).APP-C99 expression level in isoflurane group was significantly higher than that in AD group (P ＜ 0.05),and APP-C99 expression level in electro-acupuncture group was lower than that in isoflurane group (P ＜ 0.05).The cortical apoptosis index in isoflurane group was significantly higher than that in AD group (P ＜ 0.05),and the cortical apoptosis index in electro-acupuncture group was lower than that in isoflurane group (P ＜ 0.05).The expression level of Aβ in AD group,isoflurane group and electro-acupuncture group was significantly higher than wild-type control group (P ＜ 0.05).Conclusion Electro-acupuncture can relieve the AD-like neurotoxicity induced by isoflurane and inhibit the decline of learning and memory abilities of AD mice,and the mechanism is probably related with suppressing the overexpression of APP-C99 and reducing the production and accumulation of Aβ,thereby alleviating the neuroapoptosis.

Over-expressed human amyloid precursor protein depressed M cholinergic receptor binding in SH-SY5Y cells / 基础医学与临床

Objective To observe the effect of over-expressed amyloid procursor protein(APP) gene on the cholinergic receptor binding and the ChAT activity in human neuroblastoma cell line SH-SY5Y.Methods SH-SY5Y cells were transfected with pCMV695 plasmid containing wild type human APP695 gene by Lipofectamine 2000 method.The expression of APP was detected by Western blot.A? contents were test by ELISA assay in over-expression SH-SY5Y cell clones(SH-SY5Y-APP).The special binding of muscarinic and nicotinic cholinergic receptors in those A?-overproducing cell clones was determined by radio-ligand binding method.The cholinergic acetyl transferase(ChAT) activity was assayed by radiao-immunoassay.Results No evident morphologic changes of cytotoxicity were detected after transfection.When A? production was 2～2.6 times as much as that of normal cells,muscarinic receptor binding was decreased from 18.5 % to 21.9 %(P

Therapeutic Potential of ?-Secretase in Alzheimer's Disease / 生物化学与生物物理进展

Alzheimer!s disease (AD) is one of the commonest neurodegenerative diseases affected mainly theelderly. AD is characterized by the formation of neuritic plaque in brain, which is composed mainly of extracellular?amyloid deposion, the A?. A?is deprived from serial hydrolysis of amyloid precursor protein (APP) by twosecretases, the ?and ?-secretase respectively. Alternatively, APP can also be sequential processed by ?-secretaseand ?-secretase, which not only preclude the formation of A?, but also generate a large ectodomain (sAPP?) whohas several neuroprotective properties. Thus the secondary processing pathway has become the focus of ADresearch. Many results have indicated that members of the adamalysin family of proteins, mainly the ADAM 10,ADAM 17 and ADAM 9, fulfill some of the criteria required of ?-secretase. Here the biological characteristics of?-secretase, its activity regulation and its potential function as targets for the treatment of AD were summerized.