Aβ1 42

Aβ1–42 (Sigma, St. Louis, MO, USA) was diluted with distilled water and incubated in 37°C water bath for 3 d to prepare the aggregates of Aβ1–42 to 125 mmol/L as the stock solution. Then the stock solution was added to the cultured hippocampal neurons to a final concentration of 2.5 mmol/L. The viability of hippocampal neurons subjected to Aβ1–42 treatment was evaluated by MTT assay and Aβ1–42-induced p-Tau level in hippocampal neurons was also determined.

The hexafluoroisopropanal (HFIP)-pretreated Rodamin B-labeled synthetic β-Amyloid peptide (Aβ_1–42) was purchased from GL Biochem (Shanghai). The lyophilized Aβ_1–42 was dissolved in dimethyl sulfoxide (D2650, Sigma) to a concentration of 2 mM and then was further diluted with Ham’s Nutrient Mixture F12 medium (51651C, Sigma) to a final concentration of 200 μM. The diluted Aβ_1–42 was incubated at 4°C for 24 hours, then aliquoted and stored at −80°C for use.

HT-22 cells (1.0 × 10⁴ cells/well) were seeded into 96-well plates. The second day, the cells were treated with 5 μM Aβ1–42 [oligomer, dissolved in dimethyl sulfoxide (DMSO) and incubated at 37°C for 72 h to induce aggregation, Sigma, Louis, MO, USA] [19 (link)], 5 μM Aβ1–42 + 5 μM CUR (dissolved in DMSO, Sigma), 5 μM Aβ1–42 + 10 μM CUR, and 5 μM Aβ1–42 + 15 μM CUR [20 (link)], respectively. HT-22 cells without any treatment served as control group. After incubation for 24 h and 48 h, respectively, cells in each well were incubated with 10 μL cell counting kit 8 (CCK8, Dojindo Co., Ltd, Tokyo, Japan) for 2 h. Ultimately, absorbance was read at 450 nm using Synergy H4 microplate reader (BioTek, Winooski, VT, USA).

The specific process of AD model preparation was reported in our previously published article, and the process was mainly completed through the administration of Aβ_1–42 (Sigma, St. Louis, MO, USA) via intracerebroventricular injection.
Briefly, Aβ_1–42 was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma, St. Louis, MO, USA), flash-frozen in liquid nitrogen, and then lyophilized to completely remove the solvent. The lyophilized Aβ_1–42 peptides were then dissolved in 100 mM NaOH at 6 mg/mL, aliquoted, flash-frozen in liquid nitrogen, and stored at −80 °C until use [53 (link)].
For the intracerebroventricular injection (i.c.v.) of soluble Aβ_1–42, mice were intraperitoneally anesthetized with isoflurane and then placed into a stereotactic apparatus (Motorized Stereotaxic Stereo Drive; KOPF 900HD, Neurostar, Berlin, Germany). Freshly prepared Aβ_1–42 (0.3 nmol/2 μL in 0.1 M phosphate-buffered saline [PBS]) was injected into bilateral cerebral ventricles (0.3 mm posterior, 1.0 mm lateral, and 2.5 mm ventral to the bregma) using a stepper-motorized microsyringe at 0.2 μL/min. The aggregated Aβ_1–42 in the hippocampus was confirmed via immunostaining with an Aβ-specific antibody [54 (link)]. Control mice were treated in the same way and injected with the same volume of PBS.

Aβ_1-42 (order number H-1368; Bachem, Bubendorf, Switzerland) and AβpE₃ (Sigma Aldrich, Burlington, MA, USA) were suspended in 100% hexafluoroisopropanol (HFIP, Sigma Aldrich) to approximately 1 mg/400 µL HFIP and shaken at 37 °C for 1.5 h. The HFIP was removed by evaporation using a Speedvac for approximately 30 min, and when completely dry, aliquots of 100 µg Aβ_1-42 and 100 µg AβpE3 were stored at −20 °C. The Aβ_1-42 was dissolved in DMSO (Sigma Aldrich) to a concentration of 100 µM with the aid of an ultrasonic water bath. This solution was further diluted using Ringer solution to a concentration of 50 nM Aβ_1-42. AβpE3 was dissolved in H₂O.