A Study On Immunological Clearance Of The Natural Small Molecule Curcumin Targeting Toxic β-amyloid Oligomers In 5×FAD Mice

Objective:Design and establishment of a curcumin-targeted immunological clearance method for toxic Aβ oligomers using curcumin and targeting peptides as raw materials.further validation of curcumin-mediated immunological clearance in5×FAD mouse model to assess its potential in ameliorating pathological changes and cognitive impairment,ultimately verifying the significance of Aβ oligomer formation in AD pathogenesis.Methods :1.Establishment of curcumin-targeted immunoclearance for Aβ oligomers: A hemoglobin-labeled Aβ16-20 and Aβ22-28 peptide conjugated with curcumin was constructed using chemical synthesis as the active immunoclearance antigen.Female Balb/c mice aged 4-6 weeks were selected,and two immunization procedures,15-day and 55-day immunizations,were used to immunize the mice.The titers and specificity of the immunized mouse serum antibodies were detected using enzyme-linked immunosorbent assay(ELISA),and the binding of purified immunoglobulin G(Ig G)and curcumin injected into the peritoneal cavity of 5×FAD mouse brain tissue slices was detected using immunohistochemical methods.2.Verification of the toxic effects of Aβ oligomers in 5×FAD mice by immunoclearance: Indirect ELISA was used to detect the titer or concentration of synthetic polypeptide serum antibodies produced in 5×FAD mice.Immunohistochemical methods were used to detect Aβ plaques and Aβ oligomers in the brains of 5×FAD mice after treatment,and immunofluorescence was used to co-locate Aβ plaques and Aβ oligomers to investigate whether the formation of Aβplaques was related to the formation of Aβ oligomers.Nissl staining was used to detect neuronal damage in the brains of 5×FAD mice.In addition,changes in the number of astrocytes and microglia in the brains of 5×FAD mice were detected by immunohistochemistry.The cognitive impairments of 5×FAD mice after treatment were assessed by a water maze experiment.Finally,the changes in the body weight of5×FAD mice were monitored over time,and Prussian blue staining was used to preliminarily evaluate the biosafety of the synthetic polypeptide vaccine by detecting microbleeding in the brains of mice.Results :1.Curcumin-targeted Aβ oligomer immune clearance was established by using curcumin-conjugated Aβ-modified short peptides to actively immunize mice,and the ELISA detection of the antibody titer in mouse serum showed that the synthesized curcumin-modified peptides had good immunogenicity and could produce high titers of antibodies.The binding of curcumin and Aβ in vitro was detected by ELISA,and the immunohistochemical detection of the curcumin and Aβ incubation in vivo experiment showed that the produced antibodies had good specificity and could selectively bind to the curcumin and Aβ complex.2.The toxicity of Aβ oligomers was verified in 5×FAD mice through immune clearance.After treatment with curcumin-mediated immune clearance,AD-related pathological indicators were detected in 5×FAD mice.The experimental results showed that the immune clearance induced by curcumin-peptide complex could significantly reduce the content of Aβ oligomers in the brains of mice(p <0.05),and the level of Aβ plaques in the brain also showed a decreasing trend but without significant statistical significance.The water maze experiment on the treated mice showed an improvement in the cognitive function of 5×FAD mice.In addition,after treatment,the expression of glial fibrillary acidic protein(GFAP)and ionized calcium-binding adapter molecule 1(IBA1)in the brains of 5×FAD mice decreased(p<0.05),indicating a decrease in the number of astrocytes and microglia.At the same time,the experimental results showed that compared with the Control group,the expression of Nissl bodies or neuronal damage in the treated group of 5×FAD mice improved.Finally,a preliminary evaluation of the safety of immune clearance in5×FAD mice found that the body weight of the treated mice remained normal,and no obvious micro-bleeding points were observed in the brain tissue.Conclusions :Compared with the 15-day immune program,the 55-day immune program can induce the synthesis of high titer and specificity multi-peptide vaccine antibodies,which can bind to the complex of curcumin and Aβ.The curcumin-mediated Aβoligomer immunoclearance significantly reduced the content of Aβ oligomers in the brains of 5×FAD mice,reduced the number of astrocytes and microglia,alleviated neuronal damage in the brains of 5×FAD mice,and improved cognitive dysfunction in the 5×FAD model mice.