Soybean meal was purchased from the local market. Anti‐synaptophysin (#36406S), anti‐synapsin I (#5297S), anti-β-actin (#3700S), anti-extracellular signal-regulated kinases 1 and 2 (ERK1/2, #9102S), anti-phospho-protein kinase C (PKC, #9371S), and anti-phospho-ERK1/2 (#4370S) were purchased from Cell Signaling (MA, USA). Anti‐synaptotagmin (#ab13259), anti‐synaptobrevin (#ab18013), anti-syntaxin (#ab188583), anti‐synaptosomal‐associated protein 25 kDa (SNAP 25, #ab41455), anti-PKC (#ab23511), anti-Ca2+/calmodulin-dependent protein kinase II (CaMKII, #ab92332), and anti-phospho-CaMKII (#ab171095) were purchased from Abcam (Cambridge, UK). Anti-phospho-synapsin I site-4,5 (Ser 62, 67) (#GTX82591), anti-phospho-synapsin I site-3 (Ser 603) (#GTX82589), and anti-horseradish peroxidase‐conjugated secondary antibodies (#GTX213110‐01, #GTX213111‐01) were purchased from Gentex (MI, USA). Other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Preparation of SME
The extract was prepared as described in previous studies [26, 27]. Briefly, 50 g of soybean meal was dissolved in 150 mL of ethanol/water (1:1 v/v) by continuously stirring for 2 h at 4°C. Then, the supernatants were obtained by centrifugation (6000 rpm for 20 min at 25 °C) and lyophilized to obtain a powder. The extract powder was then stored at -20 °C for further use in the biological assays.
Isoflavone and phenolic acid of SME were separated on an Agilent Eclipse plus C18 column (100 mm × 4.6 mm, 3.5 μm) at a flow rate of 0.35 mLmin−1 by using an Agilent 1200 series binary pump. Gradient elution was performed with mobile phase A (5% methanol with 0.1% formic acid) and mobile phase B (methanol with 0.1% formic acid). The initial condition was 50:50 mobile phase A/mobile phase B (v/v) for 2.5 min, after which is was changed to 80% mobile phase B in 0.1 min that was maintained for 2 min. Finally, the solvent composition was quickly reverted to the initial conditions and equilibrated for 11 min. Mass spectrometry was operated in multiple ion-monitoring mode (MRM) and negative polarity at −4200 V by using API 3000 (MDS SCIEX, Applied Biosystems, Ontario, Canada).
The International Guidelines for Care and Use of Laboratory Animals were followed for all experiments, and the experimental protocol was approved by the Animal Care Committee of Fu Jen Catholic University (approval number: A11018). Thirty male Sprague-Dawley rats (Taiwan BioLASCO) weighing 160–200 g were used. They were housed in plastic cages and were fed on pellets with free access to tap water. Room temperature was controlled at 22 ± 2°C with a 12-h light:12-h dark cycle. After 3 days of training with MWM, rats were divided into SME and tap water (control) groups; they were orally administered SME solution or an equal volume of 0.9% normal saline, respectively, daily for 2 consecutive weeks. After 2 weeks, the behavioral test was conducted 30 min by using the MWM video analysis system. Next, the rat's body weight was measured, and stool samples were collected and immediately stored at -80°C for gut microbiota analysis. Finally, the rats were deeply anesthetized and killed, and the hippocampus was collected to prepare synaptosomes for glutamate release assay, transmission electron microscopy (TEM), and Western blotting. In addition, the liver and kidney were collected from the rats after sacrifice for hematoxylin-eosin (H&E) staining.
The MWM test was conducted to evaluate the performance of spatial learning and memory, as described by previous study . A circular pool with a diameter of 55 cm and height of 25 cm was filled with opacified water (20 cm depth) at 25 ± 1°C. The pool was divided into four quadrants, and the platform was placed at the center of one fixed quadrant for all trials. Training (days 1‒4) was conducted four times a day, and the escape latency time for each rat to go to the platform was measured for 120 s. Rats reaching the platform were allowed to be remain there for 15 s. Rats that failed to locate the platform were guided to the platform and allowed to stay for 30 s. The latency period of the failed rats was recorded within 120 s. The swimming path from the entry to the hidden platform, escape latency, and movement distance in the coverage zone were recorded using a video-tracking system (Version 1.17, SINGA Technology Corporation, Taipei, Taiwan).
Liver or kidney tissues were fixed in 4% PFA, dehydrated with graded alcohol, and embedded in paraffin wax. A series of paraffin sections (5 μm) were cut using a Leica rotation microtome and stained with H&E, and images were captured under a microscope with 400x magnification. Histological changes in the liver and kidney sections were determined in terms of cytoplasmic color fading, vacuolization, nuclear condensation, nuclear fragmentation, nuclear fading, and erythrocyte stasis .
Rat hippocampal synaptosomes from each group were placed in an electron microscope fixative solution for 1 day. Samples were then postfixed in 1% osmium tetraoxide for 2 h, followed by gradient ethanol dehydration, soaking, and embedding in pure epoxy resin. Samples were cut into 70-nm-thick sections and stained with uranium and lead. Finally, sections were observed under a TEM (JEM-1400, JEOL, Japan).
Western blotting analysis
Western blotting was performed as described by previous reported . Briefly, hippocampal synaptosomes were homogenized and the concentrations of proteins were determined using Bradford's method, with bovine serum albumin (BSA) as a standard. Equal protein amounts (20 mg) were subjected to sodium dodecylsulfate polyacrylamide (SDS-PAGE) gel electrophoresis and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 3% BSA in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBST) for 1h at room temperature and incubated overnight at 4°C with primary antibodies. The antibodies used were anti-synaptophysin (1:200000), anti-synaptotagmin (1:1000), anti-synaptobrevin (1:800), anti-syntaxin (1:10000), anti-synapsin I (1:50000), anti-SNAP 25 (1:50000), anti-PKC (1:700), anti-phospho-PKC (1: 2000), anti-CaMKII (1:10000), anti-phospho-CaMKII (1:2000), anti-ERK1/2 (1:5000), anti-phospho-ERK1/2 (1:2000), anti-phospho-synapsin I site-4, 5 (1:2000), anti-phospho-synapsin I site-3 (1:2000), and anti-b-actin (1:1000). Next, the membrane was washed with TBST three times and incubated with a secondary horseradish peroxidase-conjugated antibody (1:5000) at room temperature for 1 h. Protein bands were visualized using a chemiluminescence reagent (Amersham, Buckinghamshire, UK). The intensity of the protein bands was analyzed using ImageJ software (Synoptics, Cambridge, UK).
Gut microbiota analysis was conducted by the Biotools Microbiome Research Center (Taipei, Taiwan). Briefly, DNA was extracted from fecal samples using the QIAamp PowerFecal DNA kit (Qiagen, CA, USA). The 16s rDNA amplicon sequencing of the V4 hypervariable region was performed with an Illumina HiSeq (paired-end 250 bp). Primers was designed to target the V4 region of the 16S rDNA (position 319 of the bacterial 16s rRNA gene to position 806). Each reaction was denatured at 95°C for 3 min followed by 25 cycles of (95°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec), followed by a final extension at 72°C for 5 min . Reactions each contained a unique sequence index to enable pooling. Pools were purified with the AMPure XP beads and sequenced on an Illumina HiSeq platform. The 16S rDNA data were analyzed with the open-source bioinformatics pipeline Quantitative Insights into Microbial Ecology (QIIME). The sequences were grouped into operational taxonomic units (OTUs) by UCLUST at a minimum of 97% sequence similarity. Representative sequences from each OTU were aligned using the PyNAST software (v.1.2). Taxonomy was assigned using the Silva database (v.132).
Statistical analysis was done using the SPSS.16.0 software. The data were expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was run followed by Tukey post hoc comparisons test. The criterion for the statistical significance was p < 0.05.