Cellular response toβ-amyloid neurotoxicity in Alzheimer's disease and implications in new therapeutics

β-Amyloid(Aβ)is a specific pathological hallmark of Alzheimer's disease(AD).Because of its neurotoxicity,AD patients exhibit multiple brain dysfunctions.Disease-modifying therapy(DMT)is the central concept in the development of AD thera-peutics today,and most DMT drugs that are currently in clinical trials are anti-Aβdrugs,such as aducanumab and lecanemab.Therefore,understanding Aβ's neurotoxic mechanism is crucial for Aβ-targeted drug development.Despite its total length of only a few dozen amino acids,Aβis incredibly diverse.In addition to the well-known Aβ_(1-42),N-terminally truncated,glutaminyl cyclase(QC)catalyzed,and pyroglutamate-modified Aβ(pEAβ)is also highly amyloidogenic and far more cytotoxic.The extracel-lular monomeric Aβ_(x-42)(x=1-11)initiates the aggregation to form fibrils and plaques and causes many abnormal cellular responses through cell membrane receptors and receptor-coupled signal pathways.These signal cascades further influence many cel-lular metabolism-related processes,such as gene expression,cell cycle,and cell fate,and ultimately cause severe neural cell damage.However,endogenous cellular anti-Aβdefense processes always accompany the Aβ-induced microenvironment alterations.Aβ-cleaving endopeptidases,Aβ-degrading ubiquitin-proteasome system(UPS),and Aβ-engulfing glial cell immune responses are all essential self-defense mechanisms that we can leverage to develop new drugs.This review discusses some of the most recent advances in understanding Aβ-centric AD mechanisms and suggests prospects for promising anti-Aβstrategies.

NLRP3炎症小体在阿尔兹海默症中的作用及潜在治疗靶点

阿尔兹海默症(Alzheimer’s disease,AD)是常见的神经退行性疾病,严重影响患者的生存质量。目前尚无有效的针对性治疗措施。AD发病机制复杂,是环境、遗传和年龄影响因素共同作用的结果。大脑中β-淀粉样蛋白(β-amyloid,Aβ)沉积、微管相关蛋白tau过度磷酸化形成的神经纤维缠结及神经元丢失是AD典型病理特征,大量研究证明,Aβ、tau蛋白聚集诱导核苷酸结合寡聚化结构域样受体含pyrin结构域蛋白3(nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3,NLRP3)炎症小体活化是AD炎性机制的核心环节,且以其和上下游分子为靶点的抑制剂和化合物治疗在细胞和动物模型中均发挥神经保护作用,改善空间记忆功能障碍,但临床疗效和安全性仍待研究。因此,抑制NLRP3炎症小体的活化可能是AD的潜在治疗靶点。本文以NLRP3炎症小体的活化机制和影响因素及与AD关系进行综述,并总结以NLRP3炎症小体为靶点的AD治疗药物,以期为AD等NLRP3炎症小体相关疾病提供新的治疗方向。

β-Amyloid对PC12细胞毒性损害的机制研究

目的利用体外PC1 2细胞培养来研究Aβ对神经元细胞毒性作用 ,以探索早老性痴呆 (AD)的发生机制。 方法通过DNA末端标记及电子显微镜超微结构观察研究了Aβ诱导培养的PC1 2细胞的形态学和分子生化改变。 结果Aβ可诱导PC1 2细胞核DNA发生降解 ,出现染色质浓缩成块状 ,胞浆浓缩 ,胞膜内陷 ,凋亡小体形成等。 结论Aβ在AD发生中可能是通过诱导神经元凋亡而引起神经元丢失的。

BACE1 inhibitors:A promising therapeutic approach for the management of Alzheimer’s disease

Alzheimer’s disease is a neurological disorder marked by the accumulation of amyloid beta(Aβ)aggregates,resulting from mutations in the amyloid precursor protein.The enzymeβ-secretase,also known asβ-site amyloid precursor protein cleaving enzyme 1(BACE1),plays a crucial role in generating Aβpeptides.With no targeted therapy available for Alzheimer’s disease,inhibiting BACE1 aspartic protease has emerged as a primary treatment target.Since 1999,compounds demonstrating potential binding to the BACE1 receptor have advanced to human trials.Structural optimization of synthetically derived compounds,coupled with computational approaches,has offered valuable insights for developing highly selective leads with drug-like properties.This review highlights pivotal studies on the design and development of BACE1 inhibitors as anti-Alzheimer’s disease agents.It summarizes computational methods employed in facilitating drug discovery for potential BACE1 inhibitors and provides an update on their clinical status,indicating future directions for novel BACE1 inhibitors.The promising clinical results of Elenbecestat(E-2609)catalyze the development of effective,selective BACE1 inhibitors in the future.

未知病变类型脑血管中β-amyloid、α-actin、collagen Ⅳ的含量

目的研究未知病变类型脑血管病变的结构特征。方法通过刚果红染色、免疫组织化学染色、计算机图像分析技术对未知病变类型脑血管病变的β-amyloid、α-actin、collagenⅣ的含量进行研究。结果未知病变类型脑血管壁α-actin、collagenⅣ呈少量阳性染色,与正常脑血管存在显著差异(P<0.05);β-amyloid染色呈阴性,与正常脑血管无差异(P>0.05)。病变血管壁中上述三种蛋白的表达特点与脑血管淀粉样变(cerebralamyloidangiopathy,CAA)及小动脉硬化玻璃样变不同。结论未知病变类型脑血管病变具有不同于CAA的病变特征。

Relationship between β-amyloid protein 1-42, thyroid hormone levels and the risk of cognitive impairment after ischemic stroke

BACKGROUND Post-stroke cognitive impairment(PSCI)is not only a common consequence of stroke but also an important factor for adverse prognosis of patients.Biochemical indicators such as blood lipids and blood pressure are affected by many factors,and the ability of evaluating the progress of patients with PSCI is insufficient.Therefore,it is necessary to find sensitive markers for predicting the progress of patients and avoiding PSCI.Recent studies have shown thatβ-amyloid protein 1-42(Aβ1-42)and thyroid hormone levels are closely related to PSCI,which may be the influencing factors of PSCI,but there are few related studies.AIM To investigate the relationship between serum levels of Aβand thyroid hormones in acute stage and PSCI and its predicted value.METHODS A total of 195 patients with acute cerebral infarction confirmed from June 2016 to January 2018 were enrolled in this study.Baseline data and serological indicators were recorded to assess cognitive function of patients.All patients were followed up for 1 year.Their cognitive functions were evaluated within 1 wk,3 mo,6 mo and 1 yr after stroke.At the end of follow-up,the patients were divided into PSCI and non-PSCI according to Montreal cognitive assessment score,and the relationship between biochemical indexes and the progression of PSCI was explored.RESULTS Compared with patients with non-PSCI,the levels of Aβ1-42,triiodothyronine(T3)and free thyroxin were lower in the patients with PSCI.Repeated measures analysis of variance showed that the overall content of Aβ1-42 and T3 in PSCI was also lower than that of the non-PSCI patients.Further analysis revealed that Aβ1-42(r=0.348),T3(r=0.273)and free thyroxin(r=0.214)were positively correlated with disease progression(P<0.05),suggesting that these indicators have the potential to predict disease progression and outcome.Cox regression analysis showed that Aβ1-42 and T3 were important factors of PSCI.Then stratified analysis showed that the lower the Aβ1-42 and T3,the higher risk of PSCI in patients who were aged over 70,female and illiterate.CONCLUSION Aβ1-42 and T3 have the ability to predict the progression of PSCI,which is expected to be applied clinically to reduce the incidence of PSCI and improve the quality of life of patients.

Mutations of beta-amyloid precursor protein alter the consequence of Alzheimer's disease pathogenesis

Alzheimer's disease is pathologically defined by accumulation of extracellular amyloid-β(Aβ). Approximately 25 mutations in β-amyloid precursor protein(APP) are pathogenic and cause autosomal dominant Alzheimer's disease. To date, the mechanism underlying the effect of APP mutation on Aβ generation is unclear. Therefore, investigating the mechanism of APP mutation on Alzheimer's disease may help understanding of disease pathogenesis. Thus, APP mutations(A673T, A673 V, E682 K, E693 G, and E693Q) were transiently co-transfected into human embryonic kidney cells. Western blot assay was used to detect expression levels of APP, beta-secretase 1, and presenilin 1 in cells. Enzyme-linked immunosorbent assay was performed to determine Aβ_(1–40) and Aβ_(1–42) levels. Liquid chromatography-tandem mass chromatography was used to examine VVIAT, FLF, ITL, VIV, IAT, VIT, TVI, and VVIA peptide levels. Immunofluorescence staining was performed to measure APP and early endosome antigen 1 immunoreactivity. Our results show that the protective A673 T mutation decreases Aβ_(42)/Aβ_(40) rate by downregulating IAT and upregulating VVIA levels. Pathogenic A673 V, E682 K, and E693 Q mutations promote Aβ_(42)/Aβ_(40) rate by increasing levels of CTF99, Aβ_(42), Aβ_(40), and IAT, and decreasing VVIA levels. Pathogenic E693 G mutation shows no significant change in Aβ_(42)/Aβ_(40) ratio because of inhibition of γ-secretase activity. APP mutations can change location from the cell surface to early endosomes. Our findings confirm that certain APP mutations accelerate Aβ generation by affecting the long Aβ cleavage pathway and increasing Aβ_(42/40) rate, thereby resulting in Alzheimer's disease.

Key gene and protein changes in the beta-amyloid pathway following Longyanshen polysaccharides treatment in a mouse model of Alzheimer's disease

BACKGROUND： During onset and development of Alzheimer＇s disease, β-amyloid （Aβ） precursor protein （APP）, β-site amyloid precursor protein cleaving enzyme （BACE）, and β-amyloid are key genes and proteins in the Aβ pathway, and over-expression of these genes can lead to Aβ deposit/on in the brain. OBJECTIVE： To observe the influence of Longyanshen polysaccharides on expression of BACE, APP, and Aβ in the senescence-accelerated mouse prone/8 （SAMP8） brain, and to compare these effects with huperzine A treatment. DESIGN, TIME AND SETTING： A randomized, controlled, neurobiochemical experiment was performed at the Department of Pharmacology and Scientific Experimental Center of Guangxi Medical University from September 2005 to January 2008. MATERIALS： Longyanshen polysaccharfdes powder was extracted from the dried slices of the medicinal plant Longyanshen. The active component, Longyanshen polysaccharides, was provided by the Department of Pharmacology, Guangxi Medical University; huperzine A was purchased from Yuzhong Drug Manufactory, China. METHODS： Healthy SAMP8 mice were used to establish a model of Alzheimer＇s disease. A total of 50 SAMP8 mice were randomly assigned to 5 groups （n = 10）： SAMP8, huperzine A, low-, middle-, and high-dose polysaccharides. In addition, 10 senescence-accelerated mouse resistant 1 （SAMR1） mice were selected as normal controls. SAMP8 and SAMR1 mice were administered 30 mL/kg normal saline; the huperzine A group was administered 0.02 mg/kg huperzine A; the low-, middle-, and high-dose polysaccharides groups were respectively administered 45, 90, and 180 mg/kg Longyanshen polysaccharides. Each group was treated by intragastric administration, once per day, for 50 consecutive days. MAIN OUTCOME MEASURES： One hour after the final administration, immunohistochemical analysis was used to determine Aβ expression in the cortex and hippocampus of SAMP8 mice. Reverse-transcription polymerase chain reaction was used to determine mRNA levels of BACE and APP in SAMP8 brain tissue. RESULTS： Compared with the SAMR1 group, Aβ expression in the cerebral cortex and hippocampus, as well as expression of BACE, APP mRNA in the brain was significantly increased in the SAMP8 group （P 〈 0.05-0.01）. Compared with the SAMP8 group, Aβ expression, as well as BACE and APP mRNA expression, were significantly decreased in the cerebral cortex and hippocampus of huperzine A and low-, middle-, and high-dose polysaccharides groups （P 〈 0.05-0.01）. In particular, the effect of high-dose polysaccharides was the most significant （P 〈 0.05-0.01 ）. CONCLUSION： Longyanshen polysaccharides reduced or inhibited over-expression of BACE, APP, and Aβ in SAMP8 mice in a dose-dependent manner, and the effect was not worse than huperzine A.

Beta-amyloid precursor protein cleavage enzyme-1 expression in adult rat retinal neurons in the early period after lead exposure

Previous studies have reported that non-human primates and rodents exposed to lead during brain development may become dependent on the deposition of pre-determined β-amyloid protein （Aβ）,and exhibit upregulation of β-site amyloid precursor protein expression in old age.However,further evidence is required to elucidate the precise relationship and molecular mechanisms underlying the effects of early lead exposure on excessive Aβ production in adult mammals.The present study investigated the effects of lead exposure on expression of β-amyloid precursor protein cleavage enzyme-1 （BACE-1） in the rat retina and the production of Aβ in early development,using the retina as a window for studying Alzheimer＇s disease.Adult rats were intraocularly injected with different doses of lead acetate （10μmol/L,100μmol/L,1 mmol/L,10 mmol/L and 100 mmol/L）.The results revealed that retinal lead concentration,BACE-1 and its cleavage products β-C-terminal fragment and retina Aβ1-40 were all significantly increased in almost all of the lead exposure groups 48 hours later in a dose-dependent manner.The only exception was the 10μmol/L group.The distribution of BACE-1 in the retina did not exhibit obvious changes,and no distinctive increase in the activation of retinal microglia was apparent.Similarly,retinal synaptophysin expression did not exhibit any clear changes.These data suggest that lead exposure can result in the upregulation of retinal neuron BACE-1 expression in the early period of development and further increase the overproduction of Aβ1-40 in the retina.Our results provided novel insight into the molecular mechanisms underlying environmentally-induced Alzheimer＇s disease.

<i>Ratanasampil</i>(Tibetan Medicine, RNSP) Reduces <i>β</i>-Amyloid Protein (Aβ) and Pro-Inflammatory Factor Levels and Improves Cognitive Functions in Mild-to-Moderate Alzheimer’s Disease (AD) Patients Living at High Altitude

Ratanasampil (RNSP) is a traditional Tibetan medicine used for the treatment of stroke and cerebrovascular diseases. Previous discoveries that RNSP can reduce β-amyloid protein levels and increase learning and memory in Alzheimer’s mouse models (Tg2576) led us to investigate whether RNSP can improve cognitive functions in Alzheimer’s patients. In this study, 146 AD patients living in Qinghai province received either one gram or 0.33 gram daily of RNSP for 16 weeks. Placebo patients received Piracetam. Serum Aβ40 and Aβ42 levels were measured at the beginning of the study and after 4 and 16 weeks of treatment. Compared to the same group before treatment, MMSE scores, ADAS-cog scores and ADL scores were significantly improved (p 0.05, p > 0.05). After 16-week treatment, serum TNF-α, IL-1β, IL-6 and Aβ42 levels were significantly decreased (p < 0. 01) in the high-dose RNSP group, whereas no significant differences were found in the low-dose and placebo groups. The Aβ42/Aβ40 ratio was significantly decreased after 4-week and 16-week treatment in the high-dose RNSP group (p < 0. 05, p < 0.01). Furthermore, serum Aβ42 concentrations had a strong positive correlation with TNF-α, IL-1β and IL-6 levels. There were no observable adverse effects in either treatment or control groups. We conclude that further clinical trials of RNSP in Alzheimer disease are warranted.

Brain-derived neurotrophic factor prevents beta-amyloid-induced apoptosis of pheochromocytoma cells by regulating Bax/Bcl-2 expression

Brain-derived neurotrophic factor was utilized in the present study to treat cell injury models induced by aggregated β-amyloid（25 35）. Methylthiazolyldiphenyl-tetrazolium bromide assay and western blot analysis showed that brain-derived neurotrophic factor provided neuroprotection against cellular apoptosis by suppressing the decline in β-amyloid（25 35）-induced cell activity and the increasing ratio of Bax/Bcl-2. After treating pheochromocytoma cells with tyrosine kinase receptor B receptor inhibitor K252a, brain-derived neurotrophic factor reverses the above- mentioned changes. The experimental findings suggested that brain-derived neurotrophic factor prevented β-amyloid peptide-induced cellular apoptosis by modulating Bax/Bcl-2 expression, and this effect was associated with binding to the specific tyrosine kinase receptor B receptor.

血清β-Amyloid水平在膝骨关节炎发生评估中的应用价值

目的:探讨膝骨关节炎(KOA)患者血清中β-Amyloid水平与KOA发生及预后的相关性。方法:从温州医科大学附属第二医院育英儿童医院住院患者中选取56例经全膝关节置换或膝关节镜手术治疗的KOA患者为试验组和25例非KOA患者为对照组。试验组根据X线Kellgren-Lawrence(K-L)分级方法分级,同时收集手术患者术前术后血清标本,采用双抗体夹心酶联免疫吸附法(ELISA)测定血清β-Amyloid水平后,绘制受试者工作特征(ROC)曲线评价血清β-Amyloid对KOA的预测价值,采用Spearman秩相关分析患者血清β-Amyloid水平与KOA的相关性,应用Pearson相关性分析患者手术前后血清β-Amyloid变化值与患者住院时间的相关性。结果:试验组患者血清β-Amyloid水平高于对照组(P<0.001);Logistic回归分析显示β-Amyloid是KOA患病的危险因素(OR=15.122,P<0.05);ROC曲线分析显示,β-Amyloid cut off值等于0.770,曲线下面积(AUC)为0.752,95%CI=0.636~0.867,敏感度85.5%,特异度60.0%;K-L2组患者血清β-Amyloid低于K-L3/4组患者(P=0.041);β-Amyloid的表达与KOA严重程度分级呈显著正相关(r=0.332,P=0.013);术前KOA患者血清β-Amyloid水平高于术后(P=0.002),KOA患者手术前后血清β-Amyloid变化值与患者住院时间呈显著负相关(r=-0.949,P<0.001)。结论:β-Amyloid是KOA患病的危险因素,血清β-Amyloid水平可能成为一种潜在的评估KOA发生及预后的生物标志物。

Protective effects of proanthocyanidins on beta-amyloid peptide (25-35)-induced PC12 cell apoptosis by blocking S-phase and increasing p53 gene expression

BACKGROUND： Current studies related to the effects of proanthocyanidins on Alzheimer＇s disease have focused primarily on the signal transduction pathway of cellular apoptosis. However, the influence of p53 gene expression on cell cycle regulation, with regard to the protective mechanisms of proanthocyanidins, has not been reported. OBJECTIVE： To observe the effect of proanthocyanidins on cell cycle distribution, cellular apoptosis and p53 gene expression in β-amyloid peptide （25-35） （Aβ25-35）-induced PC12 cells cultured in serum-free media, and to investigate the molecular neuroprotective mechanisms of proanthocyanidins with regard to cell cycle regulation. DESIGN, TIME AND SETTING： A parallel, controlled, at the Institute of Biochemistry and Molecular Biology cellular, and molecular study was performed Guangdong Medical College from July 2006 to July 2008. MATERIALS： Proanthocyanidins were provided by Nanjing Xuezi Medical and Chemical Research Center, China; Aβ25-35 was provided by Sigma, USA; PC12 cells were provided by the Institute of Basic Medical Science, Academy of Military Medical Sciences; and rabbit anti-p53 polyclonal antibody was provided by Santa Cruz Biotechnology, USA. METHODS： PC12 cells were cultured in serum-free media for 24 hours. Cells from the model group were treated with 25 μmol/L Aβ25-35 for 24 hours. Cells in the drug protection group were pre-treated with 30 mg/L proanthocyanidins for 1 hour and then treated with 25 μmol/LAβ2^-35 for 24 hours. The control group was not treated. MAIN OUTCOME MEASURES： Flow cytometry was used to detect cell cycle distribution and rate of apoptosis; reverse-transcriptase polymerase chain reaction was used to detect p53 mRNA expression; and Western blot was used to detect p53 protein expression. RESULTS： After treating with 25 μmol/LAβ25-35 for 24 hours, the rate of apoptosis and the percentage of cells in S phase were significantly increased （P 〈 0.01 ）, and p53 mRNA and protein expressions were decreased. Pretreatment with proanthocyanidins for 1 hour blocked the increase in apoptosis and the percentage of cells in S phase in Aβ25-35-induced PC12 cells （P 〈 0.01 ） and increased p53 mRNA and protein expressions. CONCLUSION： Proanthocyanidins blocked apoptosis and S-phase arrest in Aβ25-35-induced PC12 cells cultured in serum-free media. The protective mechanism could be related to increased p53 mRNA and protein expressions.

THE PROTECTIVE EFFECTS OF THE TOTAL SAPONIN OF DIPSACUS ASPEROIDES ON THE APOPTOSIS OF HIPPOCAMPAL NEURONS INDUCED BY β-AMYLOID PROTEIN

Objective To investigate the effects of the total saponin of Dipsacus asperoides (tSDA) and ginsenoside Rb1 (GRb1) on the apoptosis of primary cultured hippocampal neurons induced by β-amyloid protein (Aβ). Methods Primary cultured hippocampal neurons, the cultures were pretreated with tSDA and GRb1 on 10d for 24 hours respectively. Then the cultures were treated with 35 μmol·L -1 Aβ25-35 for 24 hours, observed the changing of survival rate of neurons and the apoptosis of neurons with biochemical analysis combining immunofluorescent cytochemical double-staining technique. Results Hippocampal neurons were treated with 35 μmol·L -1 Aβ for 24 hours, and survival rate of neurons downed to 52.6%. When neurons were pretreated by tSDA and GRb1, survival rate of neurons increased 11% to 15%. The findings of immunofluorescent cytochemical double-staining indicated that apoptotic neurons were obviously more than that of the blank group, reaching 43.9%.When neurons were pretreated by tSDA and GRb1, apoptotic neurons were downed to 16.6%, 10.8% respectively. Conclusion tSDA had the same effects as GRb1, protecting the neurons, antagonizing neurotoxicity of Aβ, increasing survival rate of neurons, and reducing apoptotic neurons induced by Aβ.

Multifaceted neuroprotective effects of(-)-epigallocatechin-3-gallate(EGCG)in Alzheimer's disease:an overview of pre-clinical studies focused onβ-amyloid peptide

Alzheimer’s disease(AD)is the most common neurodegenerative disease characterized by cognitive decline and memory impairment.Many lines of evidence indicate that excessiveβ-amyloid peptide(Aβ)generation and aggregation play pivotal roles in the initiation of AD,leading to various biochemical alteration including oxidative damage,mitochondrial dysfunction,neuroinflammation,signaling pathway and finally resulting in neuronal death.AD has a complex pathogenic mechanism,and a single-target approach for anti-AD strategy is thus full of challenges.To overcome these limitations,the present study focused to review on one of multiple target-compounds,(-)-epigallocatechin-3-gallate(EGCG)for the prevention and treatment of AD.EGCG is a main bioactive polyphenol in green tea and has been reported to exert potent neuroprotective properties in a wide array of both cellular and animal models in AD.This review demonstrated multiple neuroprotective efficacies of EGCG by focusing on the involvement of Aβ-evoked damage and its Aβregulation.Furthermore,to understand its mechanism of action on the brain,the permeability of the blood-brain barrier was also discussed.

Comparison ofβ-Amyloid Plaque Labeling Methods:Antibody Staining,Gallyas Silver Staining,and Thioflavin-S Staining

Objective To evaluate senile plaque formation and compare the sensitivity of three differentβ-amyloid(Aβ)labeling methods(antibody staining,Gallyas silver staining,and thioflavin-S staining)to detect Aβdeposition.Methods APPswe/PSEN1dE9 transgenic mice(APP/PS1)of different ages were used to examine spatiotemporal changes in Aβplaque deposition.Antibody staining,Gallyas silver staining,and thioflavin-S staining were used to detect Aβplaque deposition in the same brain region of adjacent slices from model mice,and the results were compared.Results With aging,Aβplaques first appeared in the cortex and then the deposition increased throughout the whole brain.Significantly greater plaque deposition was detected by 6E10 antibody than that analyzed with Gallyas silver staining or thioflavin-S staining(P<0.05).Plaque deposition did not show significant difference between the APP/PS1 mice brains assayed with Gallyas silver staining and ones with thioflavin-S staining(P=0.0033).Conclusions The APP/PS1 mouse model of Alzheimer’s disease could mimick the progress of Aβplaques occurred in patients with Alzheimer’s disease.Antibody detection of Aβdeposition may be more sensitive than chemical staining methods.

Protective effect of paeonol on beta-amyloid 25-35- induced toxicity in PC12 cells

BACKGROUND：Paeonol is a primary phenolic component of the Chinese medicinal herb Cortex moutan. Recent studies have shown that paeonol has anti-inflammatory, analgesic, and antioxidative effects as well as a significant cardioprotective effect against myocardial ischemia. OBJECTIVE： To investigate the protective effect of paeonol on β-amyloid 25-35-induced toxicity in PC12 cells and analyze its mechanism of action. DESIGN, TIME AND SETTING： A controlled repeated-measures cell-based study was performed in the Department of Pharmacology of Guangdong Medical College between September 2006 and December 2007. MATERIALS： Paeonol was supplied by Xuancheng Baicao Plant Industry and Trade Company, China. PC12 cells were a kind gift from Dr. Haitao Zhang at Guangdong Medical College. β-amyloid 25-35 was purchased from Sigma Company, USA. Lactate dehydrogenase （LDH） and malondialdehyde （MDA） kits were purchased from Nanjing Jiancheng Bioengineering Research Institute, China. METHODS： PC12 cells were maintained in Dulbecco＇s modified eagle＇s medium （DMEM） supplemented with 100 mL/L heat-inactivated horse serum and 50 mL/L fetal bovine serum at 37 ℃ and cultured in an incubator with 5% CO2. The medium was renewed every other day. Batches of cells were assigned into three groups. （1） Paeonol group： cells were preincubated with different concentrations of paeonol （12, 25 or 50 μmol/L） for one hour and β-amyloid 25-35 was added to the medium; （2） control group： cells were cultured in DMEM supplemented with 100 mL/L heat-inactivated horse serum and 50 mL/L fetal bovine serum; and （3） β-amyloid 25-35 group： β-amyloid 25-35 was added to the medium. MAIN OUTCOME MEASURES： When PC12 cells in each group were cultured for 24 hours, the cell viability was determined using the MTT reduction assay, LDH release into the culture media was measured by 2,4-dinitrophenylhydrazine chromatometry and MDA content was measured using a thiobarbituric acid assay. RESULTS： When PC12 cells were treated withβ-amyloid 25-35 （50 μmol/L） for 24 hours, their viability was significantly lower compared with the control group （P 〈 0.01）. When the cells were treated with paeonol for one hour prior to incubation withβ-amyloid 25-35, their viability was significantly increased compared with theβ-amyloid 25-35 group （P 〈 0.05–0.01）. LDH activity and MDA level in the β-amyloid 25-35 group were significantly increased compared with the control group （P 〈 0.01）. When the cells were treated with different concentrations of paeonol, LDH activity and MDA level in PC12 cells were significantly decreased compared with theβ-amyloid 25-35 group （P 〈 0.01）. CONCLUSION： Paeonol protects PC12 cells againstβ-amyloid 25-35-induced toxicity and the protective effect of paeonol is probably achieved through its antioxidative effects.

Mesenchymal stem cells:As a multi-target cell therapy for clearingβ-amyloid deposition in Alzheimer’s disease

Extracellularβ-amyloid(Aβ)plaques and neurofibrillary tangles(NFTs)are the pathological hallmarks of Alzheimer’s disease(AD).Studies have shown that aggregates of extracellular Aβcan induce neuroinflammation mediated neurotoxic signaling through microglial activation and release of pro-inflammatory factors.Thus,modulation of Aβmight be a potential therapeutic strategy for modifying disease progression.Recently,a large number of reports have confirmed the beneficial effects of mesenchymal stem cells(MSCs)on AD.It is believed to reduce neuroinflammation,reduce Aβamyloid deposits and NFTs,increase acetylcholine levels,promote neurogenesis,reduce neuronal damage,and improve working memory and cognition.In this review,we focus on the role of MSCs in clearing Aβdeposition.MSCs have the potential to modulate Aβ-related microenvironments via enhancement of autophagy,proteolysis of Aβaggregates,phagocytic clearance of Aβby microglial M2 polarization,decrease oxidative stress(OS),and correction of abnormal sphingolipid(SL)metabolism.With advantages in clinical applications,these data suggest that the use of MSCs as a multi-target modulator of Aβwould be an effective therapeutic approach in AD.

Effects of Yizhi Capsule (益智胶囊) on Learning and Memory Disorder and β-amyloid Peptide Induced Neurotoxicity in Rats

To explore the effects of Yizhi Capsule （益智胶囊, YZC） on learning and memory disorder and β-amyloid peptide induced neurotoxicity in rats. Methods： Various doses of YZC were administered to Sprague-Dawley （SD） rats for 8 consecutive days, twice a day. On the 8th day of the experiment, scopolamine hydrobromide was intraperitoneally injected to every rat and Morris water maze test and shuttle dark avoidance test were carried out respectively to explore the changes of learning and memory capacities in the rats. Resides, after the cerebral cortical neurons of newborn SD rats aged within 3 days were cultured in vitro for 7 days, drug serum containing YZC was added to the cultured neurons before or after β amyloid peptide25-35 （Aβ25-35） intoxication to observe the protective effect of YZC on neurotoxicity by MTT assay and to determine the LDH content in the supernatant. Results： Compared with those untreated with YZC, the rats having received YZC treatment got superiority in shorter time of platform seeking in Morris water maze test, as well as elongated latent period and less times of error in shuttle dark avoidance test. On the cultured neurons, YZC drug serum could effectively increase the survival rate of Aβ25-35 intoxicated neurons and reduce the LDH contents in cultured supernatant. Conclusion： YZC has an action of improving learning and memory disorder, and good protective effect on Aβ25-35 induced neurotoxicity in SD rats. KEY WORDS

Effect of Panax notoginseng saponins on the expression of beta-amyloid protein in the cortex of the parietal lobe and hippocampus, and spatial learning and memory in a mouse model of senile dementia

BACKGROUND： The pharmacological actions of Panax notoginseng saponins （PNS） lie in removing free radicals, anti-inflammation and anti-oxygenation. It can also improve memory and behavior in rat models of Alzheimer＇s disease. OBJECTIVE： Using the Morris water maze, immunohistochemistry, real-time PCR and RT-PCR, this study aimed to measure improvement in spatial learning, memory, expression of amyloid precursor protein （App） and β -amyloid （A β ）, to investigate the mechanism of action of PNS in the treatment of AD in the senescence accelerated mouse-prone 8 （SAMP8） and compare the effects with huperzine A. DESIGN, TIME AND SETTING： A completely randomized grouping design, controlled animal experiment was performed in the Center for Research ＆ Development of New Drugs, Guangxi Traditional Chinese Medical University from July 2005 to April 2007. MATERIALS： Sixty male SAMP8 mice, aged 3 months, purchased from Tianjin Chinese Traditional Medical University of China, were divided into four groups： PNS high-dosage group, PNS low-dosage group, huperzine A group and control group. PNS was provided by Weihe Pharmaceutical Co., Ltd. （batch No.： Z53021485, Yuxi, Yunan Province, China）. Huperzine A was provided by Zhenyuan Pharmaceutical Co., Ltd. （batch No.： 20040801, Zhejiang, China）. METHODS： The high-dosage group and low-dosage group were treated with 93.50 and 23.38 mg/kg PNS respectively per day and the huperzine A group was treated with 0.038 6 mg/kg huperzine A per day, all by intragastric administration, for 8 consecutive weeks. The same volume of double distilled water was given to the control group. MAIN OUTCOME MEASURES： After drug administration, learning and memory abilities were assessed by place navigation and spatial probe tests. The recording indices consisted of escape latency （time-to-platform）, and the percentage of swimming time spent in each quadrant. The number of A β 1-40, A β 1-42 and App immunopositive neurons in the brains of SAMP8 mice was analyzed by immunohistochemistry. The mRNA content ofApp, tau, acetylcholinesterase, and synaptophysin （Syp） was tested by real time PCR and RT-PCR. RESULTS： The PCR results show that PNS can downregulate the expression of the App gene and upregulate the expression of the Syp gene in the parietal cortex and hippocampus of SAMP8 mice. The therapeutic effects of the PNS high-dosage group were greater than those of the PNS low-dosage group and the huperzine A group （P 〈 0.05）. The results of the Morris water maze and immunohistochemistry indicated that PNS can improve the capacity for spatial learning and memory in SAMP8 mice, and reduce the content of A β 1-40, A β 1-42 and expression of App in the brains of SAMP8 mice. The therapeutic effects of the PNS high-dosage group were greater than that of the PNS low-dosage group and the huperzine A group （P 〈 0.05）. CONCLUSION： These results support the hypothesis that PNS plays a therapeutic and protective role on the pathological lesions and learning dysfunction of Alzheimer＇s disease. The therapeutic effects of PNS for Alzheimer＇s disease are possibly achieved through downregulating the expression of the App gene and upregulating the expression of the Syp gene. The therapeutic effects of PNS are dose-dependent and are greater than the effect of huperzine A.