Nf κb

The cells were lysed with RIPA buffer containing 1% protease inhibitor (Sigma‒Aldrich, St. Louis, MO, USA) on ice for 20 minutes, and the total protein in the lysate was separated by approximately 8% to 12% SDS–polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Next, the membranes were blocked with 5% skim milk at room temperature for 2 hours, followed by incubation with primary antibodies at 4°C overnight. The membranes were then incubated with 3% skim milk containing secondary antibodies at room temperature for 1 hour. Finally, the proteins were visualized using an ECL system (KeyGEN, Nanjing, China), and the protein density was analyzed using ImageJ (National Institutes of Health, Bethesda, MD, USA). The primary antibodies used were as follows: Cyclin B1 (Cell Signaling Technology, Danvers, MA, USA), phospho-cdc2 (Cell Signaling Technology, Danvers, MA, USA), cleaved PARP (Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-9 (Cell Signaling Technology, Danvers, MA, USA), PI3K (Cell Signaling Technology, Danvers, MA, USA), phospho-PI3K (Cell Signaling Technology, Danvers, MA, USA), AKT (Abcam, Cambridge, UK), phospho-AKT (Abcam, Cambridge, UK), NF-κB (Servicebio, Wuhan, Hubei), phospho-NF-κB (Servicebio, Wuhan, Hubei), and actin (Proteintech, Beijing, China).

Total protein in the hippocampus was extracted for Western blot detection. The protein samples were mixed with RAF-1, MEK-2, ERK1/2, NF-κB, and β-actin as an internal reference (all obtained from Servicebio, Wuhan, Hubei, China).