地黄饮子对Aβ_(1-42)诱导的SH-SY5Y细胞中RAGE/ROS/凋亡通路的影响

【目的】探讨地黄饮子对Aβ_(1-42)诱导的SH-SY5Y细胞中RAGE/ROS/凋亡通路的影响。【方法】(1)采用四甲基偶氮唑盐(MTT)法检测胎牛血清组、空白组及地黄饮子含药血清低、中、高剂量组的细胞活力,确定地黄饮子含药血清的最佳浓度和作用时间。(2)以0~20μmol/L Aβ_(1-42)寡聚体处理SH-SY5Y细胞24 h和48 h后,采用MTT法检测细胞活力,Annexin V/碘化丙啶(PI)双染法观察细胞凋亡,确定Aβ_(1-42)作用细胞的最佳浓度及时间,以建立阿尔茨海默病(AD)细胞模型。(3)采用MTT法检测空白组、模型组、西药对照组及地黄饮子含药血清低、中、高剂量组细胞活力,采用Annexin V/PI双染法观察各组细胞凋亡,采用二氢乙啶(DHE)染色法检测各组活性氧(ROS)含量,观察地黄饮子对Aβ_(1-42)诱导的SH-SY5Y的细胞损伤的修复作用。(4)采用Western blot法检测空白组、模型组、地黄饮子含药血清中剂量组RAGE蛋白;进一步将Aβ_(1-42)诱导的SH-SY5Y细胞进行RAGE转染后,采用DHE染色法检测各组ROS含量,采用Annexin V/PI双染法检测各组细胞凋亡率,观察地黄饮子对Aβ_(1-42)诱导的SH-SY5Y细胞凋亡及RAGE表达的影响。【结果】作用时间为24 h时,地黄饮子含药血清低、中剂量组细胞活力较空白组均显著增强(P0.05或P0.01)。建立AD体外模型的Aβ_(1-42)浓度为5μmol/L,作用时间为24 h。各给药组Aβ_(1-42)诱导细胞活力较模型组显著增强,细胞凋亡率和ROS含量显著下降(P0.05或P0.01),而地黄饮子中剂量组细胞活力最强,细胞凋亡率最低。地黄饮子中剂量组Aβ1-42诱导细胞RAGE蛋白表达较模型组显著降低(P0.05)。地黄饮子中剂量组能降低RAGE转染的Aβ_(1-42)诱导SH-SY5Y细胞的ROS生成和细胞凋亡率(P0.01)。【结论】地黄饮子可能通过抑制ROS产生和细胞凋亡发挥抗氧化作用,进而抑制RAGE蛋白来防治AD。     

High dietary consumption of trans fatty acids decreases brain docosahexaenoic acid but does not alter amyloid-β and tau pathologies in the 3xTg-AD model of Alzheimer's disease

Dietary consumption of trans fatty acids (TFA) has increased during the 20th century and is a suspected risk factor for cardiovascular diseases. More recently, high TFA intake has been associated with a higher risk of developing Alzheimer's disease (AD). To investigate the impact of TFA on an animal model genetically programmed to express amyloid-β (Aβ) and tau pathological markers of AD, we have fed 3xTg-AD mice with either control (0% TFA/total fatty acid), high TFA (16% TFA) or very high TFA (43% TFA) isocaloric diets from 2 to 16 months of age. Effects of TFA on plasma hepatic enzymes, glucose and lipid profile were minimal but very high TFA intake decreased visceral fat of non-transgenic mice. Importantly, dietary TFA increased brain TFA concentrations in a dose-related manner. Very high TFA consumption substantially modified the brain fatty acid profile by increasing mono-unsaturated fatty acids and decreasing polyunsaturated fatty acids (PUFA). Very high TFA intake induced a shift from docosahexaenoic acid (DHA, 22:6n-3) toward n-6 docosapentaenoic acid (DPA, 22:5n-6) without altering the n-3:n-6 PUFA ratio in the cortex of both control and 3xTg-AD mice. Changes in levels of Aβ₄₀, Aβ₄₂, tau protein, phosphorylated tau protein and synaptic markers were not statistically significant in the three groups of 3xTg-AD mice, despite a trend toward decreased insoluble tau in very high TFA-fed 3xTg-AD animals. In summary, TFA intake modulated brain fatty acid profiles but had no significant effect on major brain neuropathological hallmarks of AD in an animal model.     

Isolated Amyloid-β(1-42) Protofibrils, But Not Isolated Fibrils, Are Robust Stimulators of Microglia

Senile plaques composed of amyloid-β protein (Aβ) are an unshakable feature of the Alzheimer's disease (AD) brain. Although there is significant debate on the role of the plaques in AD progression, there is little disagreement on their role in stimulating a robust inflammatory response within the context of the disease. Significant inflammatory markers such as activated microglia and cytokines are observed almost exclusively surrounding the plaques. However, recent evidence suggests that the plaque exterior may contain a measurable level of soluble Aβ aggregates. The observations that microglia activation in vivo is selectively stimulated by distinct Aβ deposits led us to examine what specific form of Aβ is the most effective proinflammatory mediator in vitro. We report here that soluble prefibrillar species of Aβ(1-42) were better than fibrils at inducing microglial tumor necrosis factor α (TNFα) production in either BV-2 and primary murine microglia. Reconstitution of Aβ(1-42) in NaOH followed by dilution into F-12 media and isolation with size exclusion chromatography (SEC) revealed classic curvilinear β-sheet protofibrils 100 nm in length. The protofibrils, but not monomers, markedly activated BV-2 microglia. Comparisons were also made between freshly isolated protofibrils and Aβ(1-42) fibrils prepared from SEC-purified monomer. Surprisingly, while isolated fibrils had a much higher level of thioflavin T fluorescence per mole, they were not effective at stimulating either primary or BV-2 murine microglia compared to protofibrils. Furthermore, SEC-isolated Aβ(1-40) protofibrils exhibited significantly less activity than concentration-matched Aβ(1-42). This report is the first to demonstrate microglial activation by SEC-purified protofibrils, and the overall findings indicate that small, soluble Aβ(1-42) protofibrils induce much greater microglial activation than mature insoluble fibrils.     

Salidroside attenuates hypoxia-induced abnormal processing of amyloid precursor protein by decreasing BACE1 expression in SH-SY5Y cells

Hypoxia which is mainly mediated by hypoxia-inducible factor 1 (HIF-1), can greatly contribute to the occurrence of Alzheimer's disease (AD) by increasing β-site APP cleaving enzyme (BACE1) gene expression, protein level and β-secretase activity, resulting in a significant generation of amyloid-beta (Aβ). Salidroside has been reported to have great neuroprotective effects. The aim of this study was to investigate the effects of salidroside on hypoxia-induced abnormal processing of the amyloid precursor protein (APP) in SH-SY5Y cells and its possible mechanism. Western blot analysis showed that 200 μM of salidroside pretreatment significantly decreased BACE1 protein level and promoted the secretion of sAPPα in hypoxic condition. Salidroside had no effect on the level of APP, ADAM10 and ADAM17. ELISA analysis revealed that salidroside was able to inhibit the increase of β-secretase activity and Aβ generation induced by hypoxia, with no effect on γ-secretase activity. Notably, under hypoxia condition, mRNA of BACE1 and protein level of HIF-1α were decreased by salidroside pretreatment. These results demonstrated for the first time that salidroside was able to attenuate abnormal processing of amyloid precursor protein induced by hypoxia in SH-SY5Y cells, providing a new insight into prevention and treatment of Alzheimer's disease.     

The emergence of designed multiple ligands for neurodegenerative disorders

The incidence of neurodegenerative diseases has seen a constant increase in the global population, and is likely to be the result of extended life expectancy brought about by better health care. Despite this increase in the incidence of neurodegenerative diseases, there has been a dearth in the introduction of new disease-modifying therapies that are approved to prevent or delay the onset of these diseases, or reverse the degenerative processes in brain. Mounting evidence in the peer-reviewed literature shows that the etiopathology of these diseases is extremely complex and heterogeneous, resulting in significant comorbidity and therefore unlikely to be mitigated by any drug acting on a single pathway or target. A recent trend in drug design and discovery is the rational design or serendipitous discovery of novel drug entities with the ability to address multiple drug targets that form part of the complex pathophysiology of a particular disease state. In this review we discuss the rationale for developing such multifunctional drugs (also called designed multiple ligands or DMLs), and why these drug candidates seem to offer better outcomes in many cases compared to single-targeted drugs in pre-clinical studies for neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Examples are drawn from the literature of drug candidates that have already reached the market, some unsuccessful attempts, and others that are still in the drug development pipeline.     

Mitochondrial dysfunction and Alzheimer's disease

To date, one of the most discussed hypotheses for Alzheimer's disease (AD) etiology implicates mitochondrial dysfunction and oxidative stress as one of the primary events in the course of AD. In this review we focus on the role of mitochondria and mitochondrial DNA (mtDNA) variation in AD and discuss the rationale for the involvement of mitochondrial abnormalities in AD pathology. We summarize the current data regarding the proteins involved in mitochondrial function and pathology observed in AD, and discuss the role of somatic mutations and mitochondrial haplogroups in AD development.     

川芎嗪与葛根素合用对海马神经元损伤后的影响

目的:探讨β-淀粉样蛋白(amyloid-beta protein,Aβ)对原代培养海马神经元丙二醛(MDA)含量、超氧化物歧化酶(SOD)活力及乳酸脱氢酶(LDH)漏出量的影响及葛根素与川芎嗪合用对Aβ25~35诱导神经元损伤的保护作用。方法:通过Aβ25~35诱导体外培养的海马神经元,建立阿尔茨海默(AD)细胞模型,检测空白组(正常海马神经元)、模型组、脑复康组(终浓度为50μmol.L-1)、川芎嗪组(终浓度为50μmol.L^-1)、葛根素组(终浓度为60μmol.L^-1),川芎嗪加葛根素组(葛根素注射液终浓度为30μmol.L^-1,川芎嗪注射液终浓度为25μmol.L^-1)对海马神经元SOD活性、MDA含量及LDH漏出量的影响。结果:成功建立海马神经元AD细胞模型,经检测模型组MDA含量及LDH漏出量显著高于空白对照组且SOD活力明显低于空白对照组,差异具有显著意义(P<0.01);脑复康组、川芎嗪加葛根素组MDA含量及LDH漏出量均低于模型组,且SOD活力明显高于模型组,差异均具有显著意义(P<0.01);川芎嗪与葛根素合用组在降低MDA含量及LDH漏出量及提高SOD活性方面的效果显著强于川芎嗪与葛根素单用,差异具有显著意义(P<0.01)。结论:Aβ25~35诱导海马神经元存在自由基损伤,葛根素、川芎嗪能抗自由基损伤,对神经元具有保护作用,且二者合用在抗自由基损伤上具有协同作用。     

Nitric oxide inhibits insulin-degrading enzyme activity and function through S-nitrosylation

Insulin-degrading enzyme (IDE) is responsible for the degradation of a number of hormones and peptides, including insulin and amyloid β (Aβ). Genetic studies have linked IDE to both type 2 diabetes and Alzheimer's disease. Despite its potential importance in these diseases, relatively little is known about the factors that regulate the activity and function of IDE. Protein S-nitrosylation is now recognized as a redox-dependent, cGMP-independent signaling component that mediates a variety of actions of nitric oxide (NO). Here we describe a mechanism of inactivation of IDE by NO. NO donors decreased both insulin and Aβ degrading activities of IDE. Insulin-degrading activity appeared more sensitive to NO inhibition than Aβ degrading activity. IDE-mediated regulation of proteasome activity was affected similarly to insulin-degrading activity. We found IDE to be nitrosylated in the presence of NO donors compared to that of untreated enzyme and the control compound. S-nitrosylation of IDE enzyme did not affect the insulin degradation products produced by the enzyme, nor did NO affect insulin binding to IDE as determined by cross-linking studies. Kinetic analysis of NO inhibition of IDE confirmed that the inhibition was noncompetitive. These data suggest a possible reversible mechanism by which inhibition of IDE under conditions of nitrosative stress could contribute to pathological disease conditions such as Alzheimer's disease and type 2 diabetes.     

Neuroprotective effect of the Chinese medicine Tiantai No. 1 and its molecular mechanism in the senescence-accelerated mouse prone 8

Tiantai No. 1, a Chinese medicine predominantly composed of powderedRhizoma Gastrodiae,Radix Ginseng, and Ginkgo leaf at a ratio of 2:1:2 and dissolved in pure water, is neuroprotective in animal models of various cognitive disorders, but its molecular mechanism remains unclear. We administered Tiantai No. 1 intragastrically to senescence-accelerated mouse prone 8 (SAMP8) mice (a model of Alzheimer’s disease) at doses of 50, 100 or 150 mg/kg per day for 8 weeks and evaluated their behavior in the Morris water maze and expression of Alz-heimer’s disease-related proteins in the brain. Tiantai No. 1 shortened the escape latency in the water maze training trials, and increased swimming time in the target quadrant during the spatial probe test, indicating that Tiantai No. 1 improved learning and memory in SAMP8 mice. Immunohistochemistry revealed that Tiantai No. 1 restored the proliferation potential of Ki67-positive cells in the hippo-campus. In addition, mice that had received Tiantai No. 1 had fewer astrocytes, and less accumulation of amyloid-beta and phosphorylated tau. hTese results suggest that Tiantai No. 1 is neuroprotective in the SAMP8 mouse model of Alzheimer’s disease and acts by restoring neuronal number and proliferation potential in the hippocampus, decreasing astrocyte inifltration, and reducing the accumulation of amy-loid-beta and phosphorylated tau.     

Amyloid-beta peptide decreases glutamate uptake in cultured astrocytes: involvement of oxidative stress and mitogen-activated protein kinase cascades

Alzheimer's disease (AD) is a progressive neurodegenerative disorder primarily characterized by excessive deposition of amyloid-beta (Abeta) peptides in the brain. One of the earliest neuropathological changes in AD is the presence of a high number of reactive astrocytes at sites of Abeta deposition. Disturbance of glutamatergic neurotransmission and consequent excitotoxicity is also believed as implicated in the progression of this dementia. Therefore, the study of astrocyte responses to Abeta, the main cellular type involved in the maintenance of synaptic glutamate concentrations, is crucial for understanding the pathogenesis of AD. This study aims to investigate the effect of Abeta on the astrocytic glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST), and their relative participation to glutamate clearance. In addition we have also investigated the involvement of mitogen-activated protein (MAP) kinases in the modulation of GLT-1 and GLAST levels and activity and the putative contribution of oxidative stress induced by Abeta to the astrocytic glutamate transport function. Therefore, we used primary cultures of rat brain astrocytes exposed to Abeta synthetic peptides. The data obtained show that Abeta(1-40) peptide decreased astroglial glutamate uptake capacity in a non-competitive mode of inhibition, assessed in terms of tritium radiolabeled d-aspartate (d-[(3)H]aspartate) transport. The activity of GLT-1 seemed to be more affected than that of GLAST, and the levels of both transporters were decreased in Abeta(1-40)-treated astrocytes. We demonstrated that MAP kinases, extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase, were activated in an early phase of Abeta(1-40) treatment and the whole pathways differentially modulated the glutamate transporters activity/levels. Moreover it was shown that oxidative stress induced by Abeta(1-40) may lead to the glutamate uptake impairment observed. Taken together, our results suggest that Abeta peptide downregulates the astrocytic glutamate uptake capacity and this effect may be in part mediated by oxidative stress and the differential activity and complex balance between the MAP kinase signaling pathways.