2.1 Animals and grouping
Sixty-six male C57BL/6J mice aged 3 months were purchased from Shanghai Jihui Laboratory Animal Care Company (Shanghai, China) and housed in a standard specific pathogen-free animal laboratory (temperature 20-26 °C; relative humidity 40-60%; 12 h light−dark cycle) with access to food and liquid ad libitum. All procedures were performed in accordance with the Guidelines in the Care and Use of Animals and with the approval of the Soochow University Animal Welfare Committee (No. 202009A661). After 1 week of acclimation, the mice were randomly divided into four groups: the CON (n=18), WD (n=18), Lf (n=18), and f+AB (n=12). The mice in the CON group were fed with a standard diet (AIN93G, kcal%: protein 20.3%, carbohydrate 63.9%, and fat 15.8%) and the other three groups were fed with WD (D18061501, kcal%: protein 17%, carbohydrate 43%, and fat 40%). Both diets were purchased from Dyets Inc., (Wuxi, Jiangsu, China) (Table A.1). The mice in the Lf group were intragastrically administered with Lf at 500 mg/kg body weight for five times a week. Native bovine Lf (92.5% purity) was obtained from Hilmar Cheese Company (CA, USA). Lf was prepared as a 65 mg/mL solution in distilled water, and orally administered to mice (i.e., 25–30 g) as an approximately 0.2 ml solution. The mice in the Lf+AB group were administered with Lf and drank solution with antibiotics (ampicillin 1 g/L, vancomycin 0.25 g/L, neomycin 1 g/L,and metronidazole 1 g/L). Drinking water was changed every 2 days. Body weight, diet, and water amounts were recorded weekly. A short-term experiment was performed in the CON, WD, and Lf groups (each group n=6) at 2 weeks. After the cognitive behavior tests, the mice were euthanized and their cecal contents were collected for gut microbiota analyses. The remaining mice in the four groups (each group n=12) were maintained under the same conditions, and the experiments were ended at 16 weeks. The experimental design and animal groups are shown in Fig. A.1.
2.2 Behavioral tests
Nesting behavior test, novel object recognition (NOR) test, and Morris water maze (MWM) test were used to examine recognition memory and spontaneous rodent behaviors. The results were recorded by a video tracking system (SuperMaze software, Shanghai Xinruan Information Technology Co., Ltd., China). In reference to previous studies [17-19], the tests were briefly described as follows.
Nesting behavior test: Approximately 1 h before the dark phase, the mice were transferred to individual testing cages with an unscented paper towel of uniform size. The next morning, the nests were assessed on a rating scale from 1 to 5: 1) paper towels intact; 2) paper towels remain largely intact (>90% complete); 3) paper towels are mostly chopped but no recognizable nest position; 4) a flat nest can be seen and the paper towels are arranged in a certain direction; and 5) the nest is constructed higher than the mouse, and the circumference can completely surround the mouse.
NOR test: The mice were habituated to the testing arena with two identical objects for 10 min for 2 consecutive days. At day 3, the mice were permitted to explore the arena for 10 min for the familiarization phase and then returned to their home cages. After 30 min, one of the objects was replaced with a novel object of similar size and the mice were placed in the testing arena. Short-term object memory was assessed by object exploration defined as an animal's nose approaching the object inside a 2 cm radius around the objects.
MWM test: MWM test was routinely performed using a water maze device with 150 cm diameter and 35 cm high circular pool (XR-XM101, Shanghai Xinruan Information Technology Co. Ltd., Shanghai, China). The mice were individually housed for 1 day and then acquisitively trained over 5 consecutive days with four trials per day. At day 6, the platform was removed and the mice were returned to the device. Latency to the platform, amount of time spent in the target quadrant, and number of platform crossings were recorded to indicate the degree of memory consolidation.
2.3 Biochemical analyses
At the end of experiment, the mice were deprived of food for 12 hours and sacrificed. Serum was separated by centrifugation (3000 g, at 4 °C for 15 minutes) and stored at −80 °C. Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were detected using commercial kits (Applygen Technologies Inc., Beijing, China). Serum TNF-α and IL-6 were analyzed by ELISA kits (MultiSciences (Lianke) Biotech Co., Ltd., Hangzhou, China).
2.4 Hematoxylin-eosin (HE) staining and immunofluorescence double labeling
The mice from the CON, WD, and Lf groups were perfused via the tips of the heart with phosphate-buffered saline (PBS, pH 7.2) and 4% paraformaldehyde. The mouse brains were then maintained in 4% paraformaldehyde for 48 hours and embedded into paraffin block. The paraffin-embedded brains were sagittally cut into 4 μm-thick slice. For histopathological examination, the sections including cortex and hippocampus (cornuammonis (CA) and and dentate gyrus (DG)) were assessed by routine HE staining. For immunofluorescence double labeling, the sections were blocked by bovine serum albumin and then incubated with primary antibody mixture including mouse anti-BrdU (1:500, Wuhan Servicebio Technology Co., Ltd., Wuhan, China) and rabbit anti-Ibα1 (1:500, Servicebio) overnight at 4 °C. After incubation with the secondary antibodies for 1 h at room temperature in the darkness, DAPI (Invitrogen, CA, USA) was applied for counterstaining and images were taken using Pannoramic Midi (3D Histech Ltd., Budapest, Hungary).
2.5 Analysis of the synapse ultrastructure in the hippocampus
After undergoing transcardial perfusion with saline, the brain tissues were taken out and a 1 mm3 tissue block was cut from the hippocampus. After being dehydrated in ascending graded ethanol series and embedded in epoxy resin, the block was fixed in 2% paraformaldehyde-2.5% glutaraldehyde mixture for 24 h and treated post-fixation with 1% osmium tetroxide (OsO4) for 2 h. Sections (70 nm) were cut and stained with 4% uranyl acetate and 0.5% lead citrate. Images were observed under a JEM-1230 transmission electron microscope (TEM) equipped with a side inserted BioScan camera (Veleta, EMSIS GmbH, Germany). Synaptic morphometrics (postsynaptic density, synaptic clefts width, and the curvature of the synaptic interface) were analyzed by the previously described methods (Pan et al., 2021) using Image J software.
2.6 Measurements of colon length and colonic mucus layer thickness
The distal colon was removed quickly, and its length was measured using a high-precision electronic digital caliper (Deli, dl-150, Ningbo, Zhejiang). The colon was cleaned and a part of colon tissues was stored at −80 °C for further use. The remaining descending colons were fixed in Carnoy's solution. After being washed in anhydrous methanol, the tissues were placed in cassettes and stored in anhydrous methanol. For the detection of colonic mucus layer thickness, the tissues were embedded in paraffin and cut into 5 μm-thick slice. After mounting on glass slides with Alcian blue staining, the thickness of colonic mucus layer was observed under an eclipse microscope (Nikon, Tokyo, Japan).
2.7 Quantitative real-time polymerase chain reaction (qRT-PCR) analysis
Total mRNA was extracted from the tissues using the RNA quick purification kit (Yeasen Biotechnology, Shanghai, China), and cDNA was synthesized using Hifair®Ⅱ1st Strand cDNA Synthesis SuperMix (Yeasen Biotechnology, Shanghai, China) and GeneAmp PCR system 9700 (Applied Biosystems) following the manufacturer’s instructions. qRT-PCR was performed using Hieff® qPCR SYBR Green Master Mix (Yeasen Biotechnology, Shanghai, China) and QuantStudio 6 Flex Real-Time PCR System (Thermo, USA). Relative mRNA expression level was determined using the 2-ΔΔCt method with GAPDH as the internal reference control [20]. Table A.2 shows the gene-specific mouse primers.
2.8 Western blot analysis
The tissues were homogenized in ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) and supplemented with complete EDTA-free protease inhibitor cocktail and PhosSTOP Phosphatase Inhibitor. The protein samples were mixed with 5× dual color protein loading buffer (Fudebio, Hangzhou, China) and boiled at 98 °C for 10 min. Equal amounts of protein (30 μg/lane) were loaded on a SDS-PAGE gel at a constant voltage and then transferred to a polyvinylidene difluoride membrane (Millipore, MA, USA). After being blocked and incubated with the primary and secondary antibodies, the bands were visualized using a chemiluminescence image analysis system (Tanon, Shanghai, China) with FDbioFemto ECL (Fudebio, Hangzhou, China). The band intensities were quantified using Gel-Pro Analyzer software (Media Cybernetics, Maryland, USA). The primary antibodies were included the following: synaptosomal associated proteins 25 (SNAP-25 ; Abcam, ab109105), postsynaptic density proteins 95 (PSD-95; CST, 3450), Ibα1 (Abcam, ab178846), TNF-α (Abcam, ab215188), IL-6 (Abcam, ab233706), NLRP3 (Abcam, ab263899), IL-18(Abcam, ab243091), IL-1β (Abcam, ab254360), caspase-1 (Abcam, ab207802), TLR4 (Abcam, ab22048), myeloid differentiation factor 88 (MyD88; Abcam, ab133739), aNF-κB p65 (Abcam, ab32536), GAPDH (ABclonal, AC033), ZO-1 (ABclonal, A11417), occludin (ABclonal, A12621) and β-actin (ABclonal, AC026).
2.9 16S rRNA Gene Sequencing and Analysis
DNA was extracted by a PowerMax extraction kit (MoBio Laboratories, CA, USA) following the product introduction. The V3-V4 region of the bacteria’s 16S rRNA gene was amplified by a thermocycler PCR system. PCR products were purified using AMPure XP Beads (Beckman Coulter, IN, USA) and quantified using PicoGreen dsDNA Assay Kit (Invitrogen); Finally the sequencing was conducted using 2×150 bp pair-end configuration on Illumina HiSeq4000 platform. The 16S rRNA data were analyzed using Quantitative Insights into Microbial Ecology (QIIME) and R package (v3.2.0). Quality filtering was performed to obtain the effective sequence, and the effective sequence clustering with 97% confidence threshold was grouped into operation taxonomic units (OTUs) by Vsearchv2.4.4. Representative sequences of OTUs were selected and annotated based on SILVA128. The community composition of each sample and the abundance of OTUs were counted at the levels of kingdom, phylum, class, order, family, genus, and species. Alpha diversity index (including Chao1, abundance-based coverage estimator (ACE), Shannon and Simpson indexes) was calculated with QIIME software to compare OTUs abundance, evenness, and difference between groups. Linear discriminant analysis effect size (LefSe) was used to select the biomarkers for comparison among the groups.
2.10 Statistical analysis
Data were analyzed using the statistical package SPSS (Version 20, Chicago, USA). Differences among the intervention groups were determined using one-way analysis of variance (ANOVA) followed by least significant difference (LSD, if homogeneity of variance was satisfied) or Tamhane’s T2 (if homogeneity of variance was not satisfied) for multiple comparisons. A p value of < 0.05 was considered to be statistically significant. For 16S rRNA gene sequencing analysis, all reads were deposited and grouped into OTU at a sequence identity of 97%. The taxonomic affiliation of the OTUs was determined with quantitative insights into microbial ecology (QIIME, version 1.8.0) against the Greengenes database version 13.8.