Preparation Of Chondroitin Sulfate Nanoselenium And Its Multi-targeted Antialzheimer’s Disease Activity

Objective:Alzheimer’s disease（AD）is a complex progressive neurodegenerative disorder of the brain characterized by neural degeneration and impairment of cognitive function.To further explore the pathogenesis of AD,numerous studies have indicated that a number of factors,including amyloid-β（Aβ）deposits,tau hyperphosphorylation,low levels of acetylcholine,inflammation,oxidative damage,mitochondrial dysfunction and metal dy-shomeostasis,may play important roles in the pathogenesis of AD.Chondroitin sulfate is a sulfated glycosaminoglycan（GAG）that is present in the extracellular matrix.CS exhibits various biological activities,such as anti-inflammation,antioxidation,neuroprotection and anti-neuroinflammation.Selenium（Se）is an essential micronutrient for cellular function in many organisms and has the biological functions of antioxidant and free radical scavenging.Se can effectively reduce the oxidative stress damage of free radicals on cells and play an important role in the treatment of neurodegenerative diseases including AD.It has been found that nano-Se has low toxicity,high immune activity,free radical scavenging and antioxidant activities,but it is unstable in aqueous solution and easy to aggregate.In this study,chondroitin sulfate nano-selenium（CS@Se）was prepared by L-cysteine reduction with CS as a stable dispersant.The drug not only improves the disper-sibility and stability of nano-selenium,but also combines the anti-AD effects of both nano-selenium and CS.It confers anti-AD effects on a variety of potential targets of CS@Se,and examines the anti-AD activity of CS@Se obtained from the cell and animal levels.Method:1.CS@Se nanoparticles were successfully obtained by L-cysteine reduction,and then characterized by Fourier-transform infrared spectroscopy（FTIR）,zeta potential,trans-mission electron microscope（TEM）and inductively coupled plasma mass spectrometry（ICP-MS）and cell viability assay.2.The inhibitory effect of CS@Se on the amyloid fibril formation of Aβ_1-42has been demonstrated by ThT binding assay,Congo red binding assay,ANS fluorescence assay,circular dichroism,TEM and fluorescence microscope.3.The inhibitory effect of CS@Se on the cytoskeleton damage of SH-SY5Y cells induced by Okadaic acid（OA）was investigated by confocal laser microscopy.DCFH-DA fluorescent probe was used to detect the level of reactive oxygen species（ROS）in cells.Thiobarbituric acid（TBA）was used to determine the content of lipid oxidation（MDA）.The glutathione peroxidase（GSH-Px）level in SH-SY5Y cells was determined based on the reduction of absorbance at A₃₄₀ nm induced by hydrogen peroxide in the presence of reduced glutathione（GSH）and NADPH.The inhibitory effect of CS@Se on oxidative stress injury induced by Aβ_1-42 in SH-SY5Y cells were investigated by mensurating the levels of ROS,MDA and GSH-Px.The regulatory effects of CS@Se on p-Tau（ser396）,p-Tau（ser404）and p-GSK-3βwere investigated by Western blot.4.The AD mice model was established by subcutaneous injection of D-galactose and intragastric administration of Al Cl₃.The effects of CS@Se on the learning and memory ability of AD mice were investigated by behavioral tests such as open field,water maze,new object recognition and cross elevation.The changes of hippocampal ultrastructure of AD mice were observed by immunohistochemistry and TEM technique.The aim of this study was to investigate the effects of CS@Se on hippocampal neurons and synaptic ultrastructural damage in AD mice.Result:1.When the CS/L-cys/Na₂Se O₃ ratio was maintained at 2:2:1,the monodispersed and homogeneous spherical of the formed CS@Se nanoparticles particle size of 89.1±4.5 nm,uniform particle size,uniform distribution and good physical stability,so we choose CS/L-cys/Na₂Se O₃ ratio was maintained at 2:2:1 as the optimum ratio for the preparation of CS@Se.The zeta potential of CS@Se is-41.7m V,and the absolute value of zeta potential of CS@Se is higher than that of nano-Se（-32.8m V）,which increases the dispersion of CS@Se nanoparticles in aqueous solution.The results of SH-SY5Y cyto-toxicity study showed that when the concentration of CS@Se was up to 100μg/m L,the cell viability of SH-SY5Y cells was more than 80%,and CS@Se was much lower than Na₂Se O₃.2.ThT fluorescence binding experiment,Congo red binding experiment,ANS fluor-escence experiment,CD,TEM and other methods proved that CS@Se could delay the beta-folding of Aβ_1-42,improve the water solubility of Aβ_1-42 and inhibit the aggregation of Aβ_1-42 in vitro from the aspects of growth kinetics of Aβfibers,formation of secondary structure of Aβfibers,hydrophobicity of Aβfibers and ultrastructure of Aβfibers.ThT fluorescence binding assay and cytotoxicity assay demonstrated that CS@Se could reduce the transformation from low toxic free Aβ_1-42 to high toxic Aβ_1-42 aggregates after Aβ_1-42entered the cell interior,thereby inhibiting the SH-SY5Y neurotoxicity induced by Aβ_1-42.3.CS@Se significantly decreased the actin cytoskeleton instability and protected SH-SY5Y cells from Okadaic acid induced alterations to the actin cytoskeleton structures.Moreover,CS@Se could decreased the levels of intracellular reactive oxygen species（ROS）and malondialdehyde（MDA）,and increased the levels of GSH-Px.Western Blot results indicated that CS@Se could attenuate hyperphosphorylation of Tau at Ser396 and Ser404 sites by regulating the expressions of GSK-3β.4.The results of behavioral tests in AD mice showed that CS@Se alleviated the anxiety of AD mice,improved the spatial learning and memory ability and spatial cog-nitive level of AD mice.HE staining and TEM results showed that CS@Se could reduce mitochondrial apoptosis,improve the abnormal synaptic ultrastructure of hippocampal neurons in AD mice,and increase the survival of neurons in AD mice.Conclusion:In this study,CS@Se nanoparticles were successfully prepared by L-cysteine reduction method,and the optimal preparation process was obtained.CS@Se can inhibit the aggregation of Aβ_1-42,maintain the stability of cytoskeleton,antagonize antioxidant stress damage and regulate the over-phosphorylation of Tau protein.CS@Se can reduce mitochondrial apoptosis,improve the abnormal changes of synaptic ultrastructure of hippocampal neurons and increase the survival of neurons in AD mice.Furthermore,CS@Se can improve the spatial learning and memory ability and ameliorate the anxiety of AD mice.CS@Se might be a potent multi-functional agent for the treatment of AD and thus warrants further research and evaluation.