2.1 Materials
Paeoniflorin (PF), fluoxetine hydrochloride (FLU), and corticosterone (CORT) (Yuanye Shanghai, China); 3-methyladenine (3-MA) (MCE, China); DMEM medium and fetal bovine serum (FBS) (Gibco, Australia); Mitochondrial Membrane Potential Assay Kit with JC-1 (JC-1) (Solarbio, China); 2, 7-dichlorofluorescein diacetate (DCFH-DA) and BCA kit (Beyotime, China); ELISA kits of IL-1β, BDNF (Enzyme Immunoassay, Jiangsu, China); ELISA kits of 5-HT, CORT (Fantejin, China); anti-LC3 antibody, anti-NLRP3 antibody, anti-Caspase-1 antibody, anti-ASC antibody (Proteintech, Wuhan, China); anti-P62 antibody, anti-Beclin1 antibody, anti-Parkin antibody, anti-GAPDH antibody, HRP Goat Anti-Rabbit IgG (Wanlei, China); DAPI, Alexa Fluor 488-conjugated goat anti-rabbit (Servicebio Wuhan, China); ECL kit (Biosharp, China).
2.2 Cell culture
HT22 cells were cultured in DMEM medium with 10% heat-inactivated FBS, 1% penicillin and streptomycin at 37°C in 5% CO2. HT22 cells in the exponential phase of growth were used for all subsequent experiments.
2.3 Cell viability assay
HT22 cells in were inoculated in 96-well culture plate at a density of 5×104 cells/well and grown at 37°C, 100 µL PF solution with different concentrations (0, 1, 10, 50, 100, 200, 300, 400, and 500 µM) were treated for 24 h to screen the safe concentration of PF. 100 µL CORT solution with different concentrations (0, 1, 5, 10, 20, 50, 100, 200, and 300 µM) were treated for 24 h. Subsequently, cell viability was tested according to the CCK-8 kit installed. In addition, different concentrations of 100 µl PF solution (0, 1, 10, 20, 50, 100 and 200 µM) were added for 24 h after treatment with CORT (100 µL, 100 µM). The cell viability was tested using CCK-8. 10 µL CCK-8 solution was added to each well and measured at a wavelength of 450 nm with a microplate reader (Epoch2; American Burton Instruments Co., Ltd.) after 2 h at 37°C. The cell viability (%) was calculated as follows: Cell viability (100%) =(Adrug-Ablank)/(Acontrol-Ablank) ×100%.
2.4 ROS Assay
Intracellular ROS levels were measured using the fluorescent probe DCFH-DA. HT22 cells were inoculated in 12-well plates at a density of 1×105 cells/well and pretreated with CORT solution (100 µM), followed by incubation with PF solution (50, 100 and 200 µM) for 24 hours. Afterwards, the cells were washed twice with PBS and then incubated with 10 µM DCFH-DA for 30 min at 37°C. Subsequently, cells were washed three times with PBS. The fluorescence intensity of ROS was observed using a fluorescence microscope (Thermo Fisher Scientific, USA).
2.5 Measurement of mitochondrial membrane potential
JC-1 is a cationic dye that crosses the cell membrane and localizes towards the mitochondrial aggregates in response to the mitochondrial membrane potential (MMP), making it an ideal fluorescent probe widely used to detect the mitochondrial membrane potential of HT22 cells is measured with the fluorescent indicator JC-1. In short, HT22 cells were inoculated in 6-well plates at a density of 1×105 cells/well, and the treated cells were washed with PBS once. Cell culture medium and JC-1 staining medium were added and incubated with 37◦C for 20 min. Subsequently, the cells were cleaned twice with JC-1 staining buffer and after the lotion was discarded. Appropriate amount of cell culture medium was added and observed under fluorescence microscope (Thermo Fisher Scientific, USA).
2.6 Animals
Adult male C57/BL6 mice weighing 18-23g, were provided by Changchun Yisi Laboratory Animal Technology Co., Ltd. The mice were placed under standardized laboratory conditions, with a temperature of 23 ± 2℃ and a relative humidity of 55% ± 5%, and maintained a light dark cycle of 12:12h. During the experiment, mice were given free drinking water and standard diet. After one week of adaptive feeding, all mice were randomly separated into 7 groups: control group, CUMS-induced group, CUMS + FLU (10 mg/kg) group [28], CUMS + PF (20, 40 and 80 mg/kg) group, and CUMS + PF (80 mg/kg) + 3-MA (10 mg/kg) group [25]. All experiments were conducted according to the animal experiment guidelines of Jilin Agricultural University.
2.7 Animal experimental design
CUMS treatment was performed on the basis of previous studies with slight modifications [29]. In order to ensure the unpredictability of the stimulation protocol, the CUMS stimulation modalities were randomly arranged and the same stimulation modality was not used consecutively for 3 days. Stressors included (1) cage tilt (45˚) for 12 h, (2) fasting for 12 h, (3) water fasting for 12 h, (4) ice water swimming (4°C) for 5 min, (5) warm water swimming (28°C) for 20 min (6) shaking the cage for 10 min, (7) reversing the light-dark cycle (overnight lighting, 12 h), (8) moist bedding for 12 h, (9) foreign body exposure for 12 h, (10) white noise for 6 h, (11) tail suspension for 10 min, and (12) heat stress for 5 min.
Mice that had not been induced by CUMS were placed in a separate room and had no contact with CUMS-induced mice throughout the experiment. Animals were induced with CUMS for 7 weeks, followed by daily gavage with PF or FLU starting from the fourth week. Subsequently, 3-MA was injected intraperitoneally for 7 consecutive days until the end of the experiment. Finally, behavioral tests were performed on all mice, and the blood and brain tissue were collected at the end of the experiment. The experimental design is shown in Fig. 3a.
2.8 Behavioral testing
2.8.1 Sugar water preference test (SPT)
As stated before, SPT is a method to evaluate lack of pleasure [30]. Two bottles of sterile water containing 1% sucrose were placed to acclimate the mice to the taste of sucrose. All animals were fasted and deprived of water 24 h before the SPT. We prepared two pre weighed bottles, one containing 1% sucrose solution and the other containing sterile water. During the test, the position of the bottle was changed every 2 hours to avoid potential preferences affecting their selection. After 4 hours of experiment, the consumption of sucrose solution and sterile water was recorded. Sugar water preference (100%) = sugar water consumption (sugar water consumption + pure water consumption) × 100%.
2.8.2 Tail suspension test (TST)
The degree of depression in mice was judged by the immobile time of the TST. Mice were suspended at a distance of 50 cm above the floor. Each mouse was suspended for 6 min and acclimated for 2 min. Simultaneously, the sum of immobility time during the last 4 min was recorded. When mice were passively suspended, we consider it to be stationary. All tests were recorded directly by camera.
2.8.3 Forced swimming test (FST)
The mice were forced to swim in a glass cylinder (height: 40 cm; diameter: 18 cm) containing 30 cm-high water and maintaining the water temperature at 23 ± 2°C. The test time is 6 min, the first 2 min were the adaptation time and the immobility time during the last 4 min was recorded. Whenever mice passively float in the water and only made slight movements or hold its head above the water line, it was considered immobile. All tests were recorded directly by camera.
2.8.4 Field mine experiment (OFT)
The OFT is routinely used to study anxiety-like behaviors in mice [31]. The device consists of two parts: an open field reaction box and analysis system of camera recording. The length, width and height of the reaction box are 80 cm, without cover, and the inner wall is black. The camera above it covers the entire reaction box. The bottom plate was set to 25 cells, and set the active area. The immobility time, the distance of movement and the time of central residence within 5 min were measured. The experiment was conducted in a quiet environment, and alcohol was used to remove feces and odors so as not to affect the experimental results.
2.8.5 Elevated plus maze (EPM)
Since this rodent prefers the dark environment, they tend to move in the closed arms. But curious, they will move in the open arm, the conflicting behaviors of inquiry and avoidance generate anxiety. Therefore, EPM is often used to evaluate the effects of depression models and antidepressants. The specified active area was set before the experiment, and the behavior of mice was analyzed by the camera recording. After that, the mice were placed in the central area of maze and allowed to move freely for 5 min in the maze. The duration and trajectory of mice in the open and closed arms were recorded. The experiment requires a quiet environment, continuous experiments in different mice, cleaning the feces in the device and using alcohol to eliminate odor, so as not to affect the experimental results.
2.9 Enzyme-linked immunosorbent assay (ELISA)
The expressions of 5-HT, BDNF and IL-1β in the suspension of HT22 cells induced by CORT were detected. Blood samples were collected after behavioral tests, and serum was obtained by centrifugation at 10,000 rpm for 10 min at 4°C. The supernatant of hippocampal tissue homogenate was collected by centrifugation at 12,000 rpm for 10 min at 4°C. The IL-1β and CORT levels in serum and 5-HT and BDNF levels in hippocampal were measured by using the ELISA kit according to the manufacturer's instructions.
2.10 H&E staining
The pathological morphology of the hippocampus was evaluated by H&E staining. Mice hippocampus was taken and fixed with 4% paraformaldehyde at room temperature. Dehydrated with alcohol, embedded in paraffin and cut into 5 mm slices. H&E staining was then performed, and the sections were stained in hematoxylin solution, washed, dehydrated in 70% and 90% alcohol, and stained in alcohol-eosin solution. The stained sections were dehydrated in pure alcohol and then made transparent with xylene. An optical microscope (Olympus, BX51) was used to observe and take pictures.
2.11 Transmission electron microscopy (TEM) analysis
Hippocampus tissues of mice in each group were sliced (1–3 mm) and incubated in 2.5% glutaraldehyde and 2% paraformaldehyde at 4°C for 2 h. Microglia were precipitated and fixed in 1% glutaraldehyde. After post-fixation with 1% osmium tetroxide, the sections were treated with aqueous uranyl acetate, then dehydrated and embedded with epoxy resin and treated with lead citrate. Images were observed under TEM (HITACHI HT7800, Japan).
2.12 Immunofluorescence (IF)
HT22 cells were inoculated at 1×105 cells/well in 12-well glass slides and allowed to stand overnight for proper attachment. Then, the cells were treated as designed. After treatment, cells were washed twice with PBS and fixed with 4% paraformaldehyde, permeabilized with 0.01% Triton X-100 and closed in 5% BSA. Cells were then incubated with primary antibody overnight. Secondary fluorescent antibody was added for 1 h. DAPI was used for nuclear re-staining. Samples were then imaged by fluorescence microscopy (NIKON Eclipse ci, China).
Hippocampus sections were permeated by 0.5% Triton X-100 at room temperature for 20 min and blocked by normal goat serum at room temperature for 30 min. The sections were incubated by glial fibrillary acidic protein (LC3 or P62) antibody at 4◦C overnight and incubated by Alexa Fluor 488-conjugated goat anti-rabbit for 1 h. DAPI was used for nuclear re-staining. Images were captured under a BX53 fluorescence microscope. The results were quantified with ImageJ software.
2.13 Immunohistochemistry (IHC)
Brain tissue specimen was fixed via 4% paraformaldehyde and embedded in paraffin. The section was cut into 4 µm. After xylene deparaffinization, hydration and antigen retrieval, the section was sealed through normal goat serum lasting 20min at room temperature. Then, the section was treated with rabbit anti-NLRP3 at 37◦C for 90 min and incubated with secondary antibody for 30 min at room temperature. The color was developed by DAB reagent for 5 min. The nucleus was counterstained through hematoxylin for 3 min, and the sections were washed by running water. After dehydration, the sections were transparent and sealed with neutral gum. Images were acquired through fluorescence microscopy (NIKON Eclipse ci, China). The results were quantified with ImageJ software.
2.14 Western blotting analysis
Brain tissue and microglia are lysed by RIPA on ice for 30 minutes. Samples were centrifuged at 12,000 g for 10 min at 4°C. Supernatants were collected and protein concentrations were quantified using the BCA protein assay kit. Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. Then, the membranes were sealed in skimmed milk powder containing 5% TBST for 1 h at room temperature and incubated with primary antibody at 4°C overnight. After that, membranes were washed and incubated with secondary antibodies for 1 h. The signals were detected by ECL kit and protein was visualized with enhanced chemiluminescence with Chemiluminescence Imager (Analytik Jena). The results were quantitative analysis using ImageJ software.
2.15 Graphing and statistical analysis
All data were expressed as mean ± standard deviation, and one-way analysis of variance (ANOVA) was used to statistically analyze the data, with Tukey post hoc test for comparison between multiple groups. Values of p < 0.05 were considered statistically significant for the analysis. Statistical analyses were performed using SPSS software version 20.0 and graphs were generated using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA).