| Backgrounds:The prevention and treatment of Alzheimer’s disease is becoming more and more important as the growing of aging population worldwide. The pathogenesis of Alzheimer’s disease is complex and the causes are still unclear. However, there are a few major theories in this matter including:amyloid hypothesis, hyperphosphorylated tau hypothesis, cholinergic neuronal apoptosis hypothesis, free radical damage hypothesis, glutamate excitotoxicity and so on. Calcium dysregulation was first proposed in 1984 by Khachaturian, increasing lines of evidence have recently claimed that calcium dysregulation plays a central role in AD pathogenesis. IP3 receptor-mediated Ca2+ signals are subverted to cause cell death by activating mitochondrial pathway or Ca2+ sensitive enzymes. Hence, factors that modulate or disrupt IP3-mediated Ca2+ signaling are expected to exert powerful effect on AD.It has been known for about 20 years, that heparin binds to IP3 receptors. However, heparin is considered to be a cell-impermeant agent. When it is used as a conventional IP3 receptor antagonist to study the signal transduction pathways, it is usually applied in direct microinjection into the cytoplasm. Ultra-low-molecular-weight heparin (ULMWH) is currently under development which is synthesized from unfractionated heparin with an average MW of 2.2 kDa. It exhibits more advantages compared to heparin, including low average molecular weight, a much narrower distribution, better bioavailability and lower risk of bleeding. It may provide a novel and effective target for the prevention and treatment of AD. Objective:The present study was designed to investigate the effects of ULMWH on Aβ25-35-induced apoptosis and intracellular Ca2+ elevation induced by Ca2+ channel agonist, and to clarify the underlying mechanisms. It will provide the theoretical and experimental basis for clinical application.Methods1. Aβ25-35 induced cell damage and the identification of fiber formation(1) To observe the neuron morphology by optical microscope and to determine the effects of different concentrations of Aβ25-35 (10,25,35,50μM) for 24h on cell viability by MTT assay.(2) Aβ25-35 was incubated at 37℃for 7 days, then investigated the formation of fibril structures during the process of "aging" of the peptide using transmission electron microscopy or fluorescent indicator thioflavin T.2. Effects of ULMWH on Aβ25-35 induced apoptosisCells were pretreated with LMWH (10μg/ml) or ULMWH (2,10,50μg/ml) for 2h, followed by co-treatment with Aβ25-35 for 24h.(1) The effects of ULMWH on cell viability were evaluated by MTT assay. LDH release was detected with an assay kit according to the manufacturer’s protocol.(2) The apoptosis ratio of cells was quantitative analyzed by flow cytometry. The effects of ULMWH on morphology of apoptotic cells were observed by Hoechst 33258 staining. TUNEL assay was used to detect apoptotic morphology and the apoptotic ratio was calculated.(3) The expression of Bcl-2 and Capase-3 was detected by western blotting. The intracellular calcium concentration was measured by a fluorescent dye, Fura-2/AM.3. Effects of ULMWH on intracellular calcium concentration (1) Effects of ULMWH at different concentrations (1,10, 100μg/ml) on resting neuronal [Ca2+]i in normal culture medium or in Ca2+-free culture medium (a modified DMEM containing 1 mmol/l EGTA) were observed.(2) Effects of ULMWH on KC1 or glutamate-induced increase in [Ca2+]i were observed. The cells were pretreated with ULMWH (1,10, 100μg/ml) in normal culture medium for 5 min, and then fluorescence intensity was measured after administration of KC1 (20mM) or glutamate (500μM).(3) Effects of ULMWH on IP3 evoked increase in [Ca2+]i were examined. 10μM saponin was added to the cells to increase the permeability of IP3. Then, the cells were preincubated with ULMWH (1,10,100μg/ml) in Ca2+-free culture medium (a modified DMEM containing 1 mmol/1 EGTA) for 5min, and fluorescence intensity was measured after administration of IP3 (10μM).(4) The mitochondria and ER were firstly isolated, then different concentrations of ULMWH (1,10,100μg/ml) were added to the preparations. After 5 min of incubation, fluorescence intensity was measured after administration of 10μM IP3 to the culture medium.Results1. Aβ25-35 induced cell damage and the identification of fiber formation(1) The primary cultured neurons were maintained at 37℃in a humidified atmosphere containing 5% CO2 for 7 days prior to experiment. The neurons enlarged and neurite increased to form a dense network. The neurons migrated closer to each other and some of them gatherd into groups.The neurons were in good condition and can be used for experiments.(2) The cell survival decresed followed by the increase of the concentrations of Aβ25-35. However, the survival rate was only 43.17% and the neurons were seriously damaged in the Aβ25-35 (50μM) group. The survival rate was 60.66% in the Aβ325-35 (35μM) group, combined with the cell morphology,35μM Aβ25-35 was chosen to induce cell injury in the following experiments.(3) The Aβwere viewed under a transmission electron microscope using 35μM peptide assembly buffer (pH7.4). After incubation for 30min, the control samples exhibited the flocculus and appeared as amorphous structure, no fiber structure formation. On the other hand, after incubation for days, the samples had clear-cut fibrils with a flat and ribbon-like morphology.(4) Aβ25-35 at final concentrations of 35μM incubated for days in the assembly buffer had significantly higher thioflavin T fluorescence intensity (82.99), while the value of the control group was only10.88.2. Effects of ULMWH on Aβ25-35 induced apoptosis(1) Cell viability was decreased while LDH release significantly increased after exposure to 35μM Aβ25-35 for 24h (P＜0.01). However, when cells were preincubated with ULMWH at different concentrations (2,10,50μg/ml) for 2 h, and then exposed to Aβ25-35, cell toxicity was attenuated and LDH release was inhibited significantly compared with the Aβ25-35 group (P<0.01). The results suggested that ULMWH has the protective effect on Aβ25-35-induced cell damage.(2) The apoptotic rate was significantly increased in Aβ25-35 treated group by flow cytometry analysis (P＜0.01), and decreased when treated with ULMWH in a dose-dependent manner (P＜0.01). Many cells with smaller nuclei and condensed chromatin were seen in Aβ25-35-treated group by Hoechst 33258 staining, and ULMWH improved morphological characterization and decreased the number of apoptotic cells (P＜0.01). In control group, most of the cellular nucleus exhibited the uniform blue staining by TUNEL assay, and there were no obvious apoptotic cells. Meanwhile, many cells in ULMWH treatment group showed the brown nuclear staining and nuclear shrinkage. The number of apoptosis reduced in ULMWH treatment groups (P<0.01). The results suggested that ULMWH inhibited Aβ25-35-induced apoptosis.(3) In Aβ25-35-treated group, Caspase-3 expression markedly increased while Bcl-2 decreased compared with control group (P＜0.01). However, ULMWH significantly decreased the expression of Caspase-3 and enhanced the expression of bcl-2 (P＜0.01).(4) Aβ25-35 can remarkably increase intracellular Ca2+ concentration (P<0.01), and co-treatment with different concentrations of ULMWH reduced [Ca2+]i significantly compared with AP25-35 group (P<0.01).3. Effects of ULMWH on intracellular calcium concentration(1) The neuronal resting [Ca2+]i is 174.2±4.7nM in control group. ULMWH decreased the [Ca2+]i in both normal culture medium and in Ca2+-free culture medium in a dose-dependent manner (P＜0.01).(2) KC1 or glutamate induced [Ca2+]i increase (P＜0.01) is through Ca2+ influx. However, pretreatment with ULMWH (1,10, 100μg/ml) had no evident effect on KCl-induced [Ca2+]i elevation while partially inhibited glutamate induced [Ca2+]i elevation.(3) IP3 significantly increased [Ca2+]i in primary cultured neurons (P＜0.01). IP3_induced [Ca2+]i elevation was markedly inhibited by ULMWH in a dose-dependent manner (P＜0.01).(4) The mitochondria and ER from neurons were isolated. It showed that IP3 induced the Ca2+ release from mitochondria or ER (P＜0.01). When preincubated with ULMWH for 5min, then added IP3 to evoke calcium release, the [Ca2+]i elevation was remarkably inhibited (P＜0.01).Conclusions (1) ULMWH can improve cell suvival, inhibit LDH release and has protective effect on Aβ25-35-induced cell injury.(2) ULMWH can improve cell morphology and decrease the number of apoptotic cells through enhancing the expression of Bcl-2, decreasing the expression of Caspase-3 and inhibiting [Ca2+]i elevation. ULMWH can inhibit A(325-35-induced apoptosis.(3) ULMWH has no effect on KCl-induced calcium influx, however, it can partially reduce glutamate-induced calcium release.(4) ULMWH can significantly inhibit IP3-induced [Ca2+]i elevation at cellular and subcellular level. |
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