2.10 Immunofluorescent staining
Immunofluorescent staining was used to label hippocampal neurons in our study. Seven days after surgery, the rats were euthanized with pentobarbital sodium solution (100 mg/kg) and their brains were fixed in 4% paraformaldehyde solution (PH 7.4). Their brains were then dehydrated and cryosectioned into 20µm slices using a Leica ryostat. Rat hippocampal sections were immersed in PBS containing 0.2% Triton X-100 (Solarbio, Beijing, China) and 5% Bovine Serum Albumin (BSA, Beyotime, Shanghai, China) for 30 min. Anti-NeuN antibody (1: 200; Abcam, Cambridge, USA) was incubated overnight at 4°C. Goat anti-rabbit IgG H&L (Cy3®) (1:500; Abcam, Cambridge, USA) was then incubated with the secondary antibody for two hours at room temperature. The stained sections were observed and photographed using a fluorescence microscope.
2.11 TUNEL staining
TUNEL staining is a highly sensitive method for detecting apoptosis[26]. The One-Step TUNEL Apoptosis Assay Kit (Beyotime, Shanghai, China) was used in this study. NeuN-stained hippocampal sections were washed three times with PBS. PBS containing 0.5% Triton X-100 was added, and the mixture was incubated for five min at room temperature. TUNEL detection solution was prepared with TDT enzyme and fluorescent labeling solution, dropped onto the slices, and incubated at 37°C for 60 min. After the sections were washed with PBS, they were photographed under a fluorescence microscope.
2.12 Cell culture
The mouse hippocampal neuronal cell line HT-22 used in this study was obtained from ATCC (Manassas, VA, USA). The medium was formulated with high-glucose DMEM (Thermo Fisher Scientific, MD, USA) and 10% fetal bovine serum (FBS, Invitrogen, CA, USA) with 1% penicillin-streptomycin mixture (Sigma-Aldrich, MO, USA) added to prevent bacterial contamination. The cells were maintained at 37 ℃ in humidified atmosphere containing at 5% CO2. The medium was changed every three days.
2.13 Cell model of oxygen-glucose deprivation/reoxygenation (OGD/R)
OGD/R is a classic cellular model for ischemia-reperfusion injury. The cell culture medium was replaced with glucose-free deoxygenated DMEM (Thermo Fisher Scientific, MD, USA) with the other components unchanged, and the culture conditions were changed to 95% N2 and 5% CO2 for 4 h after which the cell culture conditions were returned to normal.
2.14 Cell Viability Assay
The Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China) exhibits different absorbances by reacting with dehydrogenases in the mitochondria of cells to assess cell number. After adding different concentrations of CPCGI to the OGD/R HT-22 cells for 72 h, the cells were transferred into a 96-well plate, and 10µl CCK-8 detection solution was added to each well. After two hours of incubation at 37°C, the liquid absorbance was measured at a wavelength of 450 nm to assess cell viability.
2.15 Western blotting analysis
Rat hippocampal tissue after seven days of treatment and HT-22 cells after 72 h of treatment were collected, and the total protein was extracted by complete lysis with a sonicator, and protein concentrations of the supernatant were determined by using the BCA Protein Assay Kit (Pioneer Biotechnology, Xi’an, China). After adding SDS-PAGE sample loading buffer (Beyotime, Shanghai, China), the protein samples were separated on 8% or 10% SDS polyacrylamide gels (NCM Biotech, Shanghai, China) for electrophoresis. The proteins were then transferred to a PVDF membrane (Millipore, MO, USA) with a pore size of 0.22µm. The PVDF membrane was cut into multiple bands according to protein molecular weight, and the following protein antibodies were used: GAPDH (Proteintech, Wuhan, China), IL-1β (Proteintech, Wuhan, China), TNF-α (Proteintech, Wuhan, China), IKKβ (Cell Signaling Technology, MA, USA), p-IKKβ (Affinity, Jiangsu, China), p65 (Cell Signaling Technology, MA, USA), and p-p65 (Affinity, Jiangsu, China). After overnight incubation at 4°C, the corresponding secondary antibodies, HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, Wuhan, China) and HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, Wuhan, China), were added according to the primary antibody species. Immunoreactivity was detected using an enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific, MD, USA).
2.16 Statistical analysis
The data were evaluated using GraphPad Prism 5 (San Diego, USA) and are presented as the mean ± standard deviation (SD). Statistical differences between groups were analyzed using the t-test or one-way analysis of variance (ANOVA). Statistical significance was set at P < 0.05.