Aβ31 35

The mouse hippocampal nerve cell line (HT22) were purchased from Guangzhou Jennio Biotechnology Co., Ltd. The HT22 cells were cultured in Dulbecco's Modified Eagle Medium complete medium (HyClone) supplemented with 10% foetal bovine serum (FBS; Sciencell) and were kept in a constant‐temperature incubator at 37°C and 5% CO₂. After adhesion, cells with 80% density and adequate growth conditions were selected for the synchronization treatment in each group. The complete medium for culturing cells was replaced with a starvation medium supplemented with 1% FBS. HT22 cells were synchronized by serum deprivation (1% FBS) for 1 h, which was regarded as circadian time 0 (CT0), and then treated with the complete medium again. The synchronized cells were cultured for n hours, denoted as CTn.¹⁷ Next, different treatments were performed on synchronized cells. The control group cells were cultured in the complete medium. The cells of the Aβ31‐35 group were treated with 5 μmol/L Aβ31‐35 (Abcam). The cells in the APN +Aβ31‐35 group were first pretreated with 10 ng/ml APN (Pepro Tech)for 1 h and then treated with 5 μmol/L Aβ31‐35. The cells in the APN group were treated with 10 ng/ml APN alone. In the LiCl (Sigma) + Aβ31‐35 group, the cells were pretreated with 30 μmol/L LiCl (a specific inhibitor of GSK3β¹⁸) for 20 min, followed by the addition of 5 μmol/L Aβ31‐35 (Figure 1B).