2.1 Animals and Grouping
Animal procedures were approved by the Laboratory Animal Ethics Committee of The Third Affiliated Hospital of Guangzhou Medical University (SL2021-022), which were performed in accordance with guidelines for animal use. Forty adult male C57/B6 mice (8 weeks old, 22g-25g) were housed in 12-hour light–dark cycle (lights on at 7:00 a.m. and off at 7:00 p.m.) at 23 ℃, 60± 10% humidity with ad libitum access to food and water.
The LPS was dissolved in PBS, and similarly, the Tak-242/PDTC was dissolved in saline. All mice were divided into the following four groups randomly: Control group, LPS group, LPS + Tak-242 and LPS + PDTC. In the first three days, LPS or PBS was given by intraperitoneal injection (i.p.) to establish sickness behaviour model at the dose of 250 μg/kg once a day for 3 days. At the same time, mice in the treatment group were treated with Tak-242 or PDTC by gavage. The schematic presentation of the experimental designing, treatment and sacrificed of the animal is presented in Fig. 1.Behavioral tests
Behavioral tests were carried out by experimenters blinded to the grouping.Animals were placed in the laboratory two hours before the experiment to adapt to the environment. Mice underwent test sessions at approximately the same time each day.
The open field(OF) Open-field tests were carried out as described[18].Briefly, The open field (60 × 60 × 40 cm) was located at a laboratory with sound‐attenuating and soft light with a camera installed above field. The 75% alcohol was used to clean the walls and bottom of the filed in order to eliminate odors between animals was tested. The exploratory and motor behaviors of mouse were recorded and analyzed usingEnthoVision XT 7.0 software (Noldus, Netherlands).
Elevated plus-maze (EPM) testThe EPM consisted of two open arms (6 × 30 cm) and two closed arms (6 × 30 cm, surrounded by 40 cm plastic walls) that originated from a common central platform (6 cm × 6 cm) elevated 50 cm from the floor. The mice were placed individually on the central platform with the head facing towards an open arm and allowed to explore for 6 min. The number of entries and the time spent in each arm were recorded by the tracking system and The proportion of time spent in the open arms (the time spent in the open arms / 5 min) were calculated.
Morris Water Maze Test (MWM)
In order to know the changes of spatial learning and memory in mice in the early stage of LPS injection, we performed morris water maze test[19]. MWM testing included a spatial probe test and a place navigation test. The MWM consisted of a circular pool (120 cm diameter and 50 cm deep) flled with water 22±2℃and an escape platform (10 cm diameter) submerged 1 cm below the surface of the water. A video camera mounted overhead the pool was used to record the swimming activity automatically. Every mice underwent three training trials per day for 4 days. Each trial was terminated when the mice found the platform or afer 60s. On the 5th day, the platform was removed and each trial last 60 s in duration. The swimming path length, swimming speed, and time spent in the target quadrant were measured.
2.3 Whole-genome gene expression analysis and Reverse Transcription-qPCR (RT-qPCR)
In order to clarify the change of gene expression in the brain at the early stage of SEA, we performed whole genome sequencing of mice, which receive intraperitoneal injection with 50μg/kgLPS per day for three days. Then the total RNAs of hippocampus were collected using a TRIzol Plus RNA Purification Kit (Cat. No. 12183018A, Invitrogen Life Technologies) for whole-genome gene expression analysis according to the previous reports[20]. Briefly, the quality and quantity of the purified RNA were assessed using Agilent 2100 Bioanalyzer.The method based on fragments per kilobase of exon per million fragments mapped (FPKM) was used to calculate gene expression. The P value of less than or equal to 0.05 was considered significant, and a cutoff of a fold change (FC) of 1.2 was applied to identify differentially expressed genes ( DEGs ) between control and LPS group mice. GO enrichment analysis of functional significance applied a hypergeometric test to map all differentially expressed genes to terms in the GO database, searching for significantly enriched GO terms in DEGs compared with the genomic background[21]. The pathway analysis based on the KEGG database was used to identify significantly enriched metabolic pathways or signal transduction pathways in DEGs relative to the whole genome background. The procedures of real-time PCR were performed as previously described[22]and the primer sequences used in the current study were listed in Supplementary 1(Table 1).
2.4 Immunostaining
After all of the behavioral tests, three mice of every group were deeply anesthetized with 4% tribromoethanol and transcardially perfused with PBS followed by intracardially with 4% paraformaldehyde in 0.01M phosphate-buffered saline (PBS, pH 7.4). The brain was collected into post-fixed in 4% PFA for 24h and dehydrated with 15% sucrose at 4℃overnight, followed by 30% sucrose at 4℃for 24h. For immunofluorescence, a standard protocol was used with minor modifications[20]. Briefly, 20µm frozen coronal sections were washed with PBS three times, permeabilized (PBS with 1.5% Triton X-100), blocked (PBS with 10% goat serum plus 3% bovine serum albumin) and incubated with primary antibodies overnight at 4℃. Then the sections were washed in PBS and incubated with the secondary fluorescent antibodies for 2 hours. The sections were then mounted and kept in the dark. The primary and secondary antibodies were listed in Supplementary 1(Table 2).
2.4 Bio-Plex Pro™ cytokine and ELISA assays
After all of the behavioral tests, blood was collected by eyeball picked. Thereafter, the mice were killed by decapitation, and brains were quickly collected. Lyzates from hippocampus were prepared using RIPA solution including Phenylmethanesulfonyl fluoride (PMSF, 1:100, P8340-1,Solarbio Bioscience & Technology), Protease Inhibitor Cocktail (1:100, 539137-10vlcn, Millipore) and Phosphatase Inhibitor Cocktail (1:100, 539131, Calbiochem). Protein concentrations were determined by the BCA Protein Assay kit (Beyotime Biotechnology, China) and adjust the concentration of total protein from sample to sample by applying additional tissue lysate. Bio-Plex Pro™ cytokine assays (BIO-RAD, Hercules, CA, USA) were carried out for measuring the cytokines in blood and hippocampus using a 23-plex test kit, which included IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17, Eotaxin, G-CSF, GM-CSF, IFN-g, KC, MCP-1, MIP-1a, MIP-1b, RANTES and TNF-a.
2.5 Western blot assay
The aforementioned proteins were also used for western blotting experiments. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes (Merck Millipore, Billerica, MA) followed by immunoblotting and the immunoreactive bands were detected by chemiluminescent HRP detection reagent (Millipore WBLUF0100). Densitometry of the western blot protein bands was analyzed using ImageJ. The primary and secondary antibodies were listed in Supplementary 1(Table 3).
2.6 Statistical analysis
Results were presented as mean ± SD, and statistical analysis was performed using two-independent samples Student’s t-test. Statistical significance (P< 0.05 or P < 0.01) was expressed as * or ** compared with the control group, and # or ## compared with the LPS group, respectively.