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Inhibition of Heparin Oligosaccharides on the Cytotoxic Effect of β-amyloid Peptide

Author: SunXiaoZuo
Tutor: JinZuo; LingPeiXue
School: Shandong University
Course: Microbial and Biochemical Pharmacy
Keywords: heparin oligosaccharides β-amyloid peptide cytotoxicity MTT CD
CLC: R96
Type: Master's thesis
Year: 2013
Downloads: 17
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Abstract


The purpose of this project is to study the inhibition effects of heparin oligosaccharides on the cytotoxicity of β-amyloid peptide, including the preparation and purification of heparin oligosaccharides, the pre-protection and repariation against Aβ-induced injury on neurons, and the study of the effects of the heparin oligosaccharides on structural changes of Apby CD.Heparin, a mucopolysaccharide sulfuric acid ester, is consisted of alternating glucosamine, L-iduronic acid, N-acetyl glucose amine and D-glucuronic acid. The relative molecular weight (Mr) distribution is about1200-40000. It is an acidic mucopolysaccharide, mainly produced by mast cells and basophils. Heparin, member of glycosaminoglycans, has many functions. In addition to the classical antithrombotic and anticoagulant activities, it has some other functions, such as immunomodulatory, inhibiting proliferation of smooth muscle, anti-inflammation activity, and antineoplastic activity. Heparin oligosaccharides can be produced by different degradation methods. The average molecular weight is about1000-4000. Heparin oligosaccharides have different biological functions due to various monosaccharide combinations.Alzheimer’s disease (AD), the main cause of dementia in the elderly, is becoming an ever-increasing problem as the population ages. Its basic pathological mechanism is represented by conformational changes in Aβ and tau proteins. These two proteins are normally expressed but can be self-assemble into toxic P-pleated sheet aggregates. The main histopatholgical features are neuritic plaques formed by the extracellular deposition of Aβ and neurofibrillary tangles, which consist of intracellular aggregates of hyperphosphorylated tau proteins in the cytoplasm of neurons.Given the central role of AP in the pathogenesis of AD, research in the last decade was aimed at developing therapies that target amyloid production, aggregation, clearance, or toxicity. The neuropathology of AD is very complex and has not been completely understood. AD pathogenesis is also accompanied by a series of inflammatory reactions. The complexity of AD pathology provides numerous potential targets for therapeutic interventions. Componds that interact directly with Aβ or interfere with its production or aggregation can reduce the inflammatory and neurotoxic effects of Aβ. The present study was designed to observe the pre-protection and repariation against Aβ-induced injury in neurons. Effects of heparin oligosaccharides on structural changes of AP were studied by CD.1Preparation and purification of the heparin oligosaccharidesThe hydrolysis conditions were optimized by HPLC. The optimum conditions of enzymatic digestion were as follows:Heparin:100mgThe heparanase I solution:100ul (1IU)Sodium acetate solution (0.1M, pH=7.0):10mlCalcium acetate solution (0.01M):1500ulReaction temperature:35℃Bio-gel P10and G-10chromatography were used to purify the digestion mixture. Samples were confirmed by mass spectrometry and PAGE to be tetrasaccharides (dp4), hexasaccharide (dp6), octasaccharide (dp8) and decasaccharide (dp10). 2The pre-protection and repariation against Aβ-induced injury in neuronsMTT assay was used to observe the pre-protection and repariation against Aβ-induced injury in both SH-SY5Y and PC12cell lines. We observed that both Ap1-40and Ap10-35can cause toxic effect on neural cells. When cells were pre-treated with different concentrations of four oligosaccharides for24h, the cell survival fraction was obviously increased. The greater the concentration of drugs was used, the more obvious the inhibition was detected, indicating that heparin oligosaccharides might pre-protect the neural cells. Furthermore, we observed that heparin oligosaccharides have better effect in repairing damage caused by Aβ.3The effects of the heparin oligosaccharides on structural changes of Aβ by CDTo probe the secondary structure of AP at this stage and to determine whether heparin oligosaccharides affect later conformational changes of the peptide monomer or its oligomers, CD was used to monitor peptide assembly. Aβ, incubated alone in37℃for1day, produced initial spectra characteristic of random coils and a-helix. A significant conformational transition occurred during the subsequent6days, producing a β-sheet conformer. No such transitions were observed in the presence of heparin oligosaccharides. All spectra of oligosaccharides-treated Aβ revealed populations of conformer that were largely random coil and a-helix. The dp6showed the best results.

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