STS was purchased from Shanghai NO.1 Biochemical & Pharmaceutical CO.. Kits used for detection of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Primary antibodies including anti-postsynaptic density 95 (PSD95), anti-postsynaptic density 93 (PSD93), anti-synaptophysin (SYP), anti-nerve growth factor (NGF) and anti-brain-derived neurotrophic factor (BDNF) were purchased from Cell Signaling Technology, Inc. Anti-glucose transporter isoform 1 (GLUT1), anti-low-density lipoprotein receptor-related protein 1(LRP1), anti-receptor for advanced glycation end products (RAGE) and anti-β-actin were purchased from Sigma-Aldrich. All secondary antibodies (horseradish peroxidase conjugated anti-rabbit IgG and anti-mouse IgG) were purchased from Cell Signaling Technology, Inc. Liberase Blendzyme 2 was purchased from Roche, Dulbecco’s PBS (D-PBS) was purchased from EuroClone. Penicillin-streptomycin was purchased from Gibco.
Animal and Treatment
APP/PS1 (APPswe/PSEN1dE9) double transgenic mice and wild-type mice (non-transgenic mice) were purchased from the Model Research Centre of Nanjing University with the same background and age. Animals were housed at a standard temperature (22 - 25 °C) with automatic light cycles (12-h light/dark) and a relative humidity of 50 - 60 %. All the animal experiments were approved by the Guiding Principles for the Care and Use of Laboratory Animals adopted and promulgated by United States National Institutes of Health. Twelve-month-old mice were randomly divided into 5 groups (n = 10 each group): WT group (wild-type, 0.9 % saline), WT+STS-H group (wild-type, STS 20 mg/kg/day), APP/PS1 group (APP/PS1, 0.9 % saline), STS-L group (APP/PS1, STS 10 mg/kg/day), and STS-H group (APP/PS1, STS 20 mg/kg/day). Mice were treated with saline or STS by intraperitoneal injection once a day for 8 weeks.
Morris Water Maze Test
Using Morris water maze test to evaluate the spatial learning and memory ability was mentioned in previous study (Xu et al. 2019). The Morris Water Maze Animal Behavioral Analysis System (Guangzhou Feidi Biology Technology Co., Ltd., Guangzhou, China) consisted of a black circular tank filled with water at 22- 26°C, a hidden platform, and a recording system. The pool was spatially divided into 4 imaginary quadrants (NE, SE, SW, NW) by a computerized tracking/image analyzer system. A circular transparent escape platform (10 cm diameter) was positioned 1- 2 cm below the opaque water surface in the middle of the target quadrant of the pool. The learning and memory abilities of mice were assessed by the Morris water maze test in a dark room. Mice were given orientation navigation tests for 6 consecutive days. Before the measurement, mice were trained once to find the platform. For each daily trial, there were 4 sequential training trials beginning with placement of the animal in the water facing the wall of the pool with the drop location changing for each trial randomly; the recording system then started to record the time. The escape latency and the swim path tracking until the mice landed on the platform were recorded on video tape. If the mouse failed to locate the platform within 60 s, it was guided to the platform and kept there for 10 s. For the probe trials, the mice were allowed to swim freely in the pool for 60 s with platform removal. The time required to cross to the original platform position, the time spent in the target quadrant, and the escape latency were measured.
Y-maze tests were used to assess cognitive changes, short-term spatial working memory (by spontaneous alternation), and exploratory activity (by total number of arm choices) of mice. The method was mentioned in previous study (Wang et al. 2018). The Y-maze is a three-arm horizontal maze (30 cm long and 8 cm wide with 15 cm high walls) in which the arms are symmetrically disposed at 120° angles from each other. The maze of floors and walls are made of opaque polyethylene plastic in a black environment. The test consisted of two phases with 1-hour intervals. During the training, the new arm was separated with a baffle and the mice were placed into the starting arm for 10 minutes, during which they had free access to the starting arm and the other arm. The test was performed 1 hour after the training. For this, the baffle in the new arm was removed and the mice were placed into the starting arm. The number of mice access into the three arms within 5 minutes was recorded. An alternation was recorded when mice made consecutive visits to the three different arms. The Y-maze spontaneous alternation was calculated as follows: Alternation behavior (%) = number of alternations / (total arm entries − 2) × 100%. At the end of the experiment, arms were cleaned with alcohol so as to remove the scent of previous mice. All mice were anesthetized and decapitated after behavioral experiments immediately. The hippocampus and cortex were carefully dissected from brains for examination. All the processes were performed on ice-cold plate. Tissues were rapidly stored at - 80°C.
Measurement of ChAT and AChE Activity
The hippocampus and cortex tissues were homogenized with ice-cold saline. The homogenate was centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was collected for the assay of the ChAT and AChE activities according to the manufacturer’s instructions.
ROS, MDA, and SOD Assays
The hippocampus and cortex tissues were homogenized with cold saline. The homogenate was centrifuged at 12,000 × g for 15 minutes at 4 °C. Supernatant was collected to detect the levels of ROS, which was measured by DCFH-DA as a redox sensitive fluorescent dye. DCFH was oxidized to strong green, fluorescent substance DCF in the presence of ROS, which has a maximum peak at excitation wavelength of 502 nm and emission wavelength of 530 nm and intensity is proportional to intracellular reactive oxygen species. Supernatant was collected to detect the levels of MDA and the activity of SOD using Universal Microplate Spectrophotometer (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions.
Isolation of Brain Microvascular Endothelial Cells (BMECs)
The BMECs were isolated from brain as described before (Navone et al. 2013). Transfer the brain into a sterile glass dish with D-PBS with 0.1% (vol/vol) penicillin-streptomycin. By using sterile scissors and a scalpel, fragment the tissue sample into pieces. Centrifuge at 276g for 10 min. Aspirate the supernatant and resuspend the homogenate pellet with Liberase Blendzyme 2 at a concentration of 0.625 mg/ml. Incubate the suspension at 37 °C on a rotator for 1 h. Resuspend the homogenate with D-PBS. Centrifuge the suspension at 276g for 10 min. Aspirate the supernatant. Coat a 25 cm2 flask with collagen type I and incubate it for 20 min at 37 °C. Aspirate the collagen solution from the flask, then wash with D-PBS. Resuspend the pellet in culture medium and transfer the suspension to the flask. Maintain the cellular suspension at 37 °C in an atmosphere of 5% CO2. The BMECs were collected were rapidly stored at - 20°C.
Western Blot Analysis
The BMECs, hippocampus and cortex tissues were homogenized and lysed. The lysate was centrifuged at 12,000 × g for 10 min at 4 °C and then denatured by boiling at 100 °C with 1 : 4 loading buffer. The protein was fractionated and subsequently transferred onto PVDF membranes. The membranes were blocked in 5 % skim milk for 1 h at room temperature. The membranes containing the protein were incubated with anti-SYP, anti-PSD93, anti-PSD95, anti-BDNF, anti-NGF, anti-GLUT1, anti-LRP1, anti-RAGE and mouse anti-β-actin overnight at 4 °C. Then the membrane was incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse for 1 h at room temperature. Routinely, protein load was monitored by using a super enhanced chemiluminescence reagent (Applygen Technologies Inc., Beijing, China).
Experimental values were given as means ± SEM. SPSS 19.0 statistical software (IBM, Endicott, NY) was evaluated to perform all statistical analysis. Two-way analysis of variance (ANOVA) was applied among the different groups to analyze differences in data for the biochemical parameters, followed by Dunnett’s significant post hoc test for pairwise multiple comparisons. Differences were considered as statistically significant at p < 0.05.