Effect Of Danggui Shaoyao Powder On Aβ Aggregation And MT3 Expression Mediated By Copper Ion In AD Model

Alzheimer’ s disease(AD),a progressive neurodegenerative disease,The main clinical symptoms are cognitive impairment progressively,accompanied by behavioral and mental disorders.AD,which is characterized by neurofibrillary tangle(NFT),formation of the senile plaques(SP)and neuronal loss in cortex and hippocampus.And SP is mainly composed of β-amyloid protein(Aβ);abnormal hyperphosphorylation of microtubule-binding protein(Tau protein)is the main component of NFT.The pathogenesis of AD is inconclusive and numerous,there are genetic theory,infectious theory,inflammatory theory,cholinergic theory,aluminum poisoning theory,free radical theory,calcium overload theory,Tau protein theory,β-amyloid Protein(β-Amyloid,Aβ)cascade hypothesis and so on.And the Aβ cascade hypothesis is one of the most recognized pathogenesis of AD,which hold a view that the abnormal aggregation of Aβ is a key link in the Aβ cascade pathway.In addition,For the research and development of therapeutic drugs about AD,Aβ metabolic is the most important target.In the recent studies of AD,the effect of metal ions on Aβ aggregation has been attracted more and more attention.Imbalance of internal steady state about metal ion and the low expression of metallothionein-3(MT3),which are known to been a close relationship between the development and progression of AD[3].ObjectiveTo revealed the mechanism of Danggui Shaoyao Powder on the metabolic balance of copper ion and the expression mechanism of MT3 expression preliminary,to explore the new target of Danggui Shaoyao Powder on AD treatment,provide new ideas and experimental basis for the prevention and treatment of AD by traditional Chinese medicine compound.MethodsExperiments in vitro1.Inhibitory and depolymerization of β-amyloid protein by Danggui Shaoyao San and Cu2+.The Aβ aggregation and depolymerization experiments of Danggui Shaoyao Powder and Cu2+were carried out by Thioflavin T(ThT)method.2.Antagonizes the neurotoxicity of Aβ in Danggui Shaoyao Powder.To detect the effect of Aβ on the viability of SH-SY5Y cells,the effects of Cu2+ on the neurotoxicity of Aβ,and the protective effect of Danggui Shaoyao Powder on SH-SY5Y cells,MTT assay was used.3.Effects of Danggui Shaoyao Powder on the expression of MT3 and the aggregation and depolymerization of Aβ.Western blot was used to detect the effect of Danggui Shaoyao Powder on the expression of MT3 and the metabolism of extracellular Aβ in SH-SY5Y cells which are injured by Aβ.Experiments in vivo1.Effect of Danggui Shaoyao Powder on learning and memory ability of SAMP8 mice.7-month-old male mice(SAMP8)were randomly divided into model group,DSS low-dose group and DSS high-dose group;the male mice with the same age anti-fast aging(SAMR1)as the normal control group.The low dose group and the high-dose group was 3.2 g/kg d,6.4 g/kg·d,the normal and model groups were given the same amount of dd·H2O once a day.After 4 weeks,Morris water maze was used to test the learning and memory abilities of each group.2.Effects of Danggui Shaoyao Powder on Oxidative Stress in SAMP8 Mice.Methane dicarboxylic aldehyde(MDA)content and superoxide dismutase(SOD)activity in the cerebral cortex of mice were detected by the kits.3.Effects of Danggui Shaoyao Powder on the expression of MT3 and Aβ in brain of SAMP8 mice.Fluorescence quantitative PCR and Western blot were used to detect the expression of MT3 in mouse cortex respectively from gene and protein levels,and the content of Aβ in cortex was detected by Western blot.4.Effect of Danggui Shaoyao Powder on copper and iron in brain of SAMP8 mice.The contents of copper and iron in serum and brain were detected by atomic absorption spectroscopy(AAS).Results1.inhibition and depolymerization of Danggui Shaoyao San and Cu2+ on β-amyloid proteinDanggui Shaoyao San could not inhibit the aggregation of Aβ,but could promote the depolymerization of fiber Aβ;Cu2+ has the ability to inhibit Aβ aggregation,but could not promote the defolymerization of Aβ in the fibrous state.2.Study on antagonizion of the neurotoxicity of A 0 about Danggui Shaoyao PowderWhen the concentration of Danggui Shaoyao was 1.25‰,2.5‰ and 5‰,it has a protective effect on Aβ induced SH-SY5Y cells injury.Cu2+ has the ability to increase the toxicity of Aβ,and when the concentration of Danggui Shaoyao Powder was 2.5‰,5‰ and 10‰,there is a protective effect to improve the viability of(Cu2+Aβ)injury of SH-SY5Y cells.3.Effect of Danggui Shaoyao Powder on MT3 Expression and MetabolismCompared with the normal group,the expression of MT3 protein in the model group was not significantly decreased,which indicated that Aβ had no effect on the expression of MT3 protein in SH-SY5Y cells.The Aβ in the cell was mainly in the form of monomer,dimer,oligomer and multimer compared with the model group,the band of intracellular Aβ protein showed thicker stripping and darker color,and the larger molecular weight of Aβ was higher than that of the model group.The extracellular Aβ was mainly in the form of smaller molecular weight such as monomer,polymer and other molecular weight,the treatment groups of extracellular Aβ protein were decreased compared with the model group.4.Effect of Danggui Shaoyao Powder on learning and memory ability of SAMP8 miceDanggui Shaoyao San could significantly shorten the escape latency of SAMP8 mice(P<0.05).It was suggested that Danggui Shaoyao San can improve the spatial learning and memory ability of SAMP8 mice to a certain extent.5.Effect of Danggui Shaoyao Powder on oxidative stress in SAMP8 miceDanggui Shaoyao San significantly increased the SOD activity in SAMP8 mice and decreased the content of MDA in brain(P<0.01;P<0.01).Which indicated that Danggui Shaoyao San could reduce the oxidative stress induced SAMP8 mice injury.6.Effect of Danggui Shaoyao Powder on MT3 and Aβ in brain of SAMP8 miceThe expression of MT3 protein in the model group was significantly lower than that of normal group(P<0.05),and Danggui Shaoyao Powder increased the expression of MT3 in brain of SAMP8 mice(P<0.01).while the level of not significantly different between the model group and the normal group.7.Effect of Danggui Shaoyao Powder on Copper in Brain of SAMP8 Mice There is no significant difference in the content of copper in cortex of each group.The content of copper in the hippocampus of each group is different,and the group of SAMP8 mice was higher than that of the normal group(P<0.05),compared with the model group,the content of copper is decreased in the dose of Danggui Shaoyao Powder(P<0.05).Conclusion1.Danggui Shaoyao San significantly reduced Aβ-induced SH-SY5Y cell damage;2.Cu2+ increased the toxicity of Aβ;3.Danggui Shaoyao powder improved the SAMP8 mice spatial learning and memory ability to a certain extent;4.Danggui Shaoyao Powder relieved the injury on SAMP8 Mice induced by oxidative stress;5,Danggui Shaoyao San can reduce the content of copper in hippocampus of SAMP8 mice.