Preparation and identification of amyloid-β protein 1-42 oligomers and their effect on astrocytes

Objective To explore the preparation and identification of amyloid-β protein 1-42 (Aβ1-42) oligomers and their effect on in vitro cultured astrocytes in mice. Methods (1) Aβ1-42 peptides were dissolved and 100 μmol/L Aβ1-42 polypeptide was chosen as mother liquor; and then, they were incubated under different conditions (4 °C for 24 h, 4 °C for 72 h, 37 °C for 24 h and 37 °C for 72 h); the form of Aβ1-42 was observed under electron microscope and the degrees of Aβ1-42 polymerization were detected by Western blotting. (2) Aβ1-42 oligomers of different concentrations (Aβ1-42 polypeptide as mother liquor being incubated under condition of 4 °C for 24 h) were added into the in vitro cultured astrocytes; CCK-8 assay was used to detect the effects of Aβ1-42 oligomers (0, 0.1, 0.5, 1, 5, 10, 50, and100 μmol/L) on astrocytic viability; after 0, 1, 10, and 50 μmol/L Aβ1-42 oligomers treatment, immunofluorescence was employed to detect the morphological changes of astrocytes and Western blotting was used to detect the glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQP4) expressions. Results (1) Different forms and degrees of polymerization of Aβ1-42 could be observed by electron microscope and Western blotting: 100 μmol/L Aβ1-42 polypeptides could induce 10 nm granulated mixture of Aβ1-42 oligomers at 4 °C incubation for 24 h; proteins with relative molecular mass of 10 000 had decreased expression, and those of 15 000-25 000 had increased expression. (2) Twenty-four h after Aβ1-42 oligomers treatment, the viability of astrocytes was increased gradually: as compared with the 0 μmol/L Aβ1-42 oligomer treatment group, the 10, 50 and 100 μmol/L Aβ1-42 oligomer treatment groups had significantly increased viability of astrocytes (P<0.05); immunofluorescent staining indicated that as compared with the 0 μmol/L Aβ1-42 oligomer treatment group, the one, 10, and 50 μmol/L Aβ1-42 oligomer treatment groups had activated astrocytes: enlarged soma, increased cell processes and increased GFAP fluorescence intensity were noted; Western blotting indicated that following the increased oligomer concentrations, the protein expressions of GFAP and AQP4 increased: as compared with the 0 μmol/L Aβ1-42 oligomer treatment group, the 10 and 50 μmol/L Aβ1-42 oligomer treatment groups had significantly increased GFAP protein expression (P<0.05); and as compared with the 0 μmol/L Aβ1-42 oligomer treatment group, the one, 10, and 50 μmol/L Aβ1-42 oligomer treatment groups had significantly increased AQP4 protein expression (P<0.05) Conclusions The Aβ1-42 oligomers could be prepared with 100 μmol/L peptide under 4 °C for 24 h. Aβ1-42 oligomers could activate astrocytes and up-regulate the AQP4 expression, which might be a self protective mechanism. Key words: Alzheimer's disease; Amyloid-βprotein; Astrocyte; Aquaporin-4