Protective Effects Of Astragalosides Against Synergistic Hippocampal Neurotoxicity Of Amyloid β-Protein And Glucocorticoids In Rats

Background:Alzheimer's disease(AD) is the major neurodegenerative disorder of the elderly, and is characterized by progressive cognitive deficits such as impairment of memory. One of the pathological hallmarks of Alzheimer's disease is extracellular accumulation of senile plaques composed primarily of aggregated amyloid ?-protein(Aβ). A report from WHO showed that the incidence rate of AD is 5-20% among the people older than sixty five year old. Although mutations in three different genes are known to underlie some cases of the rare, inheritable forms of the disease, the etiology of the more common sporadic cases remains unknown, and up to now, there is no reliable methods to prevent and treat the diesase.Therefore, many researchers focus on studying the machnism and reliable methods to prevent and treat the diesase.Histopathologically, AD is characterized by intraneuronal neurofibrillary tangles, synaptic loss , neuronal death and senile plaques composed primarily of aggregated amyloid ?-protein(Aβ). Aβis a heterogeneous 39-43-amino acid peptide generated by sequential cleavage of amyloid precursor protein by ?-secretase andγ-secretase. It is generally considered that Aβplays a pivotal role in the pathogenesis of AD.Glucocorticoids(GCs) are important adrenal steroids that affect numerous physiological processes in the brain and body, but long-term elevations of GCs are associated with decreased cognitive performance, attenuated synaptic efficacy and neuronal atrophy. Many studies in the world are focusing on the relationship between the GCs, Aβand pathogenesis of AD, but whether GCs could enhance Aβ-inducing hippocampal neuronal injury is not well determined.Astragaloside (AST) is an active compound extracted from the root of astragalus membranaceus, a valuable Chinese Herbs. Our previous studies showed that the AST not only have significant immunomodulatory, anti-inflammatory and anti-stress effect , but also improves the learning and memory impairment induced by cytoxan and dexamethasone in two-month old mice and senescent mice respectively as well as protect against cerebral ischemia- reperfusion injury. However, whether AST could protect hippocampal neuron against the injury induced by Aβor DEX plus Aβwas not elucidated.Previous studies showed that Aβand GCs all play a important role during the progress of AD. For example, Aβcould induce apoptosis of hippocampal neuron in vitro, injection of A?25–35 into CA1 field of rat hippocampus could damage the brain tissue of rats in vivo, and prolonged exposure to high concentration of DEX induced obvious memory impairment as well as severe histological damage in CA1 field of hippocampus in senescent mice. But synergetic effects of Aβand GCs on hippocampal neuronal injury was not well known. On the other hand, there are no effective methods for prevention and treatment of AD. Therefore, in this study, we first examine whether DEX could potentiate Aβ-induced learning and memory impairment in SD rats in vivo, and, if so, what is the underlying mechanism. Then we want to study the protective effects of AST on hippocampal neuronal injury induced by A? or DEX plus A?, and its relative mechanism.Methods PartⅠSynergetic Effect of DEX and A ? on Hippocampal Neurotoxicity and Its Related MechanismsMorris water maze test was used to investigate whether DEX could potentiate Aβ-induced learning and memory impairment in SD rats in vivo, and the histopathologic changes in CA1 field of hippocampus was examined under a light microscope. Primary hippocampal neurons derived from 18 day embryonic rat were cultured. Cultured cells were treated with 0.01-10μM DEX for 48 h or 1-40μM A?25–35 for 24 h and the suboptimal concentrations of DEX and A?25–35 which producing suboptimal effects on neuronal activity were defined using MTT(3-(4,5-Dimethylthiazol -2-yl)-2,5-Diphenyl Tetrazolium Bromide) assay. Then cultured cells were treated DEX alone at 10μM for 48 h or A?25–35 alone for 24 h in serum-free DMEM, or cultured cells were pretreated with DEX at 10μM for 24 h followed by A?25–35 at 1μM or 5μM for various time in serum-free DMEM. Colorimetric MTT assay and TUNEL(Terminal- Deoxynucleotidyl Transferase Mediated Nick End Labeling) staining were used to investigate the influence of DEX on hippocampal neuronal cell death with Aβ. It was determined the effect of DEX on intracellular calcium ([Ca2+]i) with Aβ25-35 by fluorescence imaging with a confocal laser microscope using fluo-3 acetoxymethylester (AM) as a fluorescent dye. The effects of DEX on Aβ25-35-induced p53 protein, phospho-tau and nuclear factorκB (NF-κB) were analyzed by western blot.PartⅡNeuroprotective Effects of AST Against Synergistic Hippocampal Neurotoxicity of Aβand DEX in RatThe AD (Alzheimer,s Disease)model rats were established by injecting Aβ25-35 into the CA1 field of hippocampus. The effect of AST on learning and memory impairment in the model rats were studied by Morris water maze,and the brain protection of AST in model rats were observed through pathomorphologic changes. It were used the methods of MTT assay to investigate the influence of AST on hippocampal neuronal cell death with amyloid ?-protein (A?). The effect of AST on intracellular calcium ([Ca2+]i) with A?25–35 was detected by fluorescence imaging with a confocal laser microscope using fluo-3/acetoxymethylester (AM) as a fluorescent dye, and the effect of AST on hippocampal neuron apoptosis induced by A?25–35 were determined with TUNEL staining. The effects of AST on DEX and Aβ25-35-induced phospho-tau protein were analyzed by western blot. The level of p53 mRNA was obsersed by RT-PCR(Reverse Transcriptase Polymerase Chain Reaction ). The effects of AST on reactive oxygen species(ROS) in vitro were detected by NBT reduction and hydroxylation of benzoic acid.ResultsPartⅠSynergetic Effect of DEX and A ? on Hippocampal Neurotoxicity and Its Related Mechanisms1. Microinjection of A?25–35 (5μg,each CA1) bilateralis into the CA1 region of adult rats and DEX(5 mg/kg/d, sc×7d)could slightly increase the escape latency and the swim distances in SD rats during training session in the Morris water maze test, but there was no statistic significant compared with vehicle-treated control. DEX(1 mg/kg/d, sc, 7d) did not decreased the escape latency and the swim distances in SD rats during training session in the Morris water maze test, but DEX(5 mg/kg/d, sc×7d) could potentiate Aβ(5μg,each CA1)-induced learning and memory impairment in SD rats. Severe histological damage was observed in the CA1 cell fields of the hippocampus in DEX plus Aβ-treated group. These neuropathological changes were characterized by decreased cell number, soma shrinkage and condensation, or nuclear pyknosis. Our results suggest that DEX could potentiate Aβ-induced learning and memory impairment and neuropathological abnormalities.2. Treatment for 48 h with DEX(0.01-10μM) alone did not cause a significant reduction in MTT compared with vehicle-treated control cultures. Treatment with aggregated A?25–3(51-40μM)alone decreased cell viability in a concentration-dependent manner. A 24-h preincubation with DEX(1 or 10μM)further decreased the viability of hippocampal neuron induced by A?25–35(1 or 5μM), suggesting synergetic hippocampal neurotoxicity of DEX and A?.3. DEX( 10μM) alone failed to increase the number of cells stained positively for TUNEL in the hippocampal neurons,but pretreatment with DEX(10μM) significantly prompted the A?(5μM) -induced apoptosis in hippocampal neurons. This result suggests that DEX could enhances apoptosis in hippocampal neurons induced by A?.4. A?25–35(5μM) could significantly increased the length of the whole comet in alkaline single-cell gel electrophoresis. DEX( 10μM) did not influence the the length of the whole comet, but the length of the whole comet in DEX ( 10μM) plus A?25–35(5μM) treated group was markedly longer than that in A?25–35 alone treated group. This result futher suggest that DEX increase the vulnerability of the hippocampal neuron to A?.5. Treatment with A? 25-35(5μM) alone induced a slight increase in [Ca2+]i in neurons 10 min later. [Ca2+]i then declined but remained higher than basal level until analyzed over 60 min. DEX(10μM ) alone did not affect [Ca2+]i in hippocampal neurons 24 h after DEX was added to the culture, but pre-incubation of neurons with DEX (10μM) markedly increased the A? 25-35(5μM) -triggered elevation in [Ca2+]i. These results imply that DEX could potentiate the neurotoxic action of A? mediated by increasing the level of intracellular Ca2+.6. A?25–35(5μM) causes a time-dependent increase in the level of nuclear NF-κB p65 proteins. A 24 h preincubation with DEX (10μM)could down-regulate the elevated level of nuclear NF-κB p65 proteins induced by A?25–35, suggesting that DEX increase the vulnerability of the hippocampal neurons to A? mediated by down-regulating the level of nuclear NF-κB proteins. A?25–35(5μM) alone could decrease the cytoplasmic level of IκBαprotein 4 h after A?25–35 was added to the culture. Increased levels of cytoplasmic IκBαprotein were observed in hippocampal neurons 28 h after incubation with DEX(10μM), and pretreatment of hippocampal neurons with DEX(10μM) for 24 h could slow the disappearance of cytoplasmic IκBαprotein mediated by A?25–35, suggesting that DEX down-regulation of the elevated level of nuclear NF-κB p65 proteins induced by A? might be associated with increased rate of IκBαprotein synthesis.7. A? 25–35 (5μM) treatment resulted in small but significant increases in the level of total protein and nuclear protein of p53 24 h after A? was added to the culture. Treatment with DEX (10μM) alone for 48 h did not increase the levels of total protein and nuclear protein of p53. Pretreatment with DEX for 24 h didn't promote the increased total protein and nuclear protein of p53 induced by A?25–35..8. Aβ25-35(0-20μM) dose-dependently induced phosphorylation of tau at Thr-231 in primary hippocampal neurons cocultured for 1 h with Aβ25-35, whereas maximal phosphorylation was obtained with Aβ25-35(10μM). DEX treatment of neurons in primary culture for 24 h could results in an slight elevation in tau phosphorylation at Thr-231, and pretreated with DEX at 10μM for 24 h could promote the increased level of phospho-tau at Thr-231 induced by Aβ25-35(5μM). These results suggest that DEX increasing the vulnerability of the hippocampal neurons to Aβwas mediated by promoting Aβ-induced tau phosphorylation at Thr-231.PartⅡNeuroprotective Effects of AST Against Synergistic Hippocampal Neurotoxicity of Aβand DEX in Rat1. AD was modeled by microinjection of A?25–35 (10μg/CA1)bilateralis into the CA1 region of adult rats under stereotaxic guidance. AST(20, 40, 80mg/kg, ig×7 d) could decreased the escape latency and the swim distances of AD model rats, suggesting that AST could improve the ability of learning and memory in AD model rats in the Morris water maze test. 2. Injection of A?25–35 (10μg/CA1) into CA1 field of rat hippocampus could damage the brain tissue of rats in vivo and the neuropathological changes were characterized by decreased cell number, soma shrinkage and condensation, or nuclear pyknosis. AST( 40 and 80mg/kg )improved histopathologic change in the CA1 field of rats hippocampus.3. AST(10μg/ml, 20μg/ml and 40μg/ml) could protect hippocampal neurons against A?25-35(10-40μM)-induced hippocampal neuronal injury in vitro. AST(10μg/ml, 20μg/ml and 40μg/ml)could also protect hippocampal neurons against DEX(10μM) plus A?25-35(5μM)- induced hippocampal neuronal injury. These results suggest that AST could protect hippocampal neuron against synergistic neurotoxicity of Aβand DEX.4. It was used the method of TUNEL staining to investigate the influence of AST on hippocampal neuronal cell apoptosis induced by A?25-35 or DEX plus A?25-35 . Results showed that AST (20μg/ml) could significantly inhibited the apoptosis of the hippocampal neurons induced by A?25-35(10μM) or DEX(10μM) plus A?25-35(5μM).5. Treatment with A? 25-35(10μM) alone induced a increase in intracellular calcium( [Ca2+]i) in neurons 10 min after A? was added into the culture. [Ca2+]i then declined but remained higher than basal level until analyzed over 60 min. AST (20μg/ml) could inhibit the increased levels of the [Ca2+]i induced by A?25–35. Pre-incubation of neurons with DEX(10μM) markedly increased the A? 25-35(5μM )-triggered elevation in [Ca2+]i , and AST (20μg/ml) could also inhibit the increased levels of the [Ca2+]i induced by DEX(10μM) plus A?25-35(5μM).6. Aβ25-35(10μM) could increase the level of phospho-tau at Thr-231 in primary hippocampal neurons. Pretreatment with DEX at 10μM for 24 h could further promote the increased level of phospho-tau at Thr-231 induced by 5μM Aβ25-35. AST( 10, 20μg/ml) could significantly inhibit the level of phospho-tau at Thr-231 induced by Aβ25-35(10μM) or DEX(10μM) plus Aβ25-35(5μM).7. The level of p53 mRNA increased 18 h after A?25-35(5μM) was added to the cultured. AST( 20μg/ml) could significantly inhibit the level of p53 mRNA 18 h after A?25-35 was added to the culture.8. AST(5- 80μg/ml) could inhibit NBT reduction induced by both xanthine-xanthine oxidase and non-enzyme generated superoxide anions ( ), but the inhibitory effect of this compound on activity of xanthine oxidase was obtained only at high concentration (more than 80μg/ml). It was also found that AST(0.5-80μg/ml) could dose-dependantly inhibit the hydroxylation of benzoic acid induced by Fenton reaction generated . OH.Conclusions(1) In vivo, DEX could potentiate Aβ-induced learning and memory impairment and pathological damage in CA1 field of hippocampus in SD rats.(2) AST could protect against A?-induced learning and memory impairment. Injection of A? into CA1 field of rat hippocampus could damage the brain tissue of rats in vivo, and AST could improve histopathologic change in the CA1 field of SD rats.(3) in vitro, DEX could potentiate the neurotoxic action of A? and could enhances apoptosis in hippocampal neurons induced by A?, which might be related to its effects in increasing the A?-triggered elevation in [Ca2+]i, down-regulating the elevated level of nuclear NF-κB p65 proteins, slowing the disappearance of cytoplasmic IκBαprotein as well as promoting the increased level of phospho-tau at Thr-231 with Aβ. In contrary, DEX didn't promote the increased levels of total protein and nuclear protein of p53 induced by A?.(4) AST could protect against A? or DEX plus A?-induced hippocampal neuronal injury , which might be related to its down-regulating the increased levels of the [Ca2+]i and phospho-tau at Thr-231, inhibiting the level of p53 mRNA induced by Aβor DEX plus Aβand scavenging and . OH generated by xanthine-xanthine oxidase system, non-enzyme system and Fenton reaction.