Preparation of Ginsenoside Rb1
Ginsenoside Rb1 (C54H92O23, molecular weight 1109.29, purity >98.0%) was purchased from the MedChemExpress LLC (cas no. 41753-43-9, Shanghai, China). Rb1 was dissolved in dimethyl sulphoxide (DMSO) to make a 1 mM reserve solution, which was deposit in -20˚C.
Animals
APP/PS1 double transgenic mice and wild-type (WT) mice, aged 2 months, were obtained from laboratory animal center of Kunming medical university (Kunming, China). The animals were housed under a room a temperature of 22± 2 °C with light control based on a 12h light-dark cycle. All mice were supplied with unlimited food and water. All experiments were approved and conducted in compliance Guidelines of the Institutional Animal Care Committee of Kunming University, China (license number: kmu-eac-2018021; Kunming, China).
Experimental design
The APP/PS1 mice were randomly divided to the two groups. One group was treated with ginsenoside Rb1 (APP/PS1+Rb1 group), and another group was treated with 10% sterilized DMSO in place of Rb1 (APP/PS1+vehicle group). Each mouse in the APP/PS1+Rb1 group was administered with 5 mg/kg Rb1 once a day for 6 months by gavage. After treatment, the Morris water maze (MWM), the novel object recognition (NOR) task and electroencephalogram (EEG) recordings were performed to detect the potential alterations induced by Rb1 treatment in APP/PS1 mice. One week post the EEG recording, all mice were sacrificed to obtain the cortex and hippocampus for subsequent analyze.
NOR task
The NOR task is last for three days and all mice were placed in a transparent plastic box with size of 40×40×40 cm3. The first day was used as a habituation phase, while the second day was used as exploratory phase and last day was used for testing. During the habituation phase, each mouse were administration in the apparatus without objects in 5 minutes. 24 h later, two identical objects were placed into the box and mice had the opportunity to explore for 5min. On the last day, one of the old objects was substitute for a novel object (NT) in the apparatus. Each mouse has the right to explore novel object (FT) and familiar object (FT) freely, subsequently the time spend was recorded. The cognitive function of mice was evaluated by the discriminant index (NT-FT)/(NT+FT).
MWM tests
The MWM test was carried out as previously described [23]. Mice were trained individually in a circular pool (diameter, 100 cm; height, 50 cm) filled with water (24±1°C, 20 cm depth). The day before the regular testing, all mice were habituated to the water and allowed them step on a white platform (hidden below the water surface) to rescued themselves. Briefly, a mouse was subjected to the swim around for 10 seconds(s) and afterwards allowed to stay the white platform submerged under water for only 1 to 2 s. During the routine testing, the escape platform was placed in different position. The time that elapsed until the mouse reached the escape platform was noted as escape latency, which lasts 5 days. On the 6th day, the escape platform was moved from tank and the probe testing was performed. The mouse swam for 60s to find the platform location.
The time spent by each mouse in the target quadrant, and the numbers that they entered the quadrant were recorded. A computer tracking program was used to track the animal path, and an animal behavior video analysis system (Xinruan Information Technology Co., Ltd., Shanghai, China) was applied to analyzed the data.
EEG recordings
For EEG experiments, recording was conducted in each mouse using the polyimide-based microelectrode array (PBM-array). The protocols in this research used a method published in some literature previously[24].
The mouse was anesthetized with isofluorane (2–3% for induction) mixed with O2 (0.5-1 L/min) for electrode fixation before EEG recordings. The mice were then implanted into the hippocampus (AP-2.1mm, ML-1.5, DV-1.5mm) with three bare electrodes with exposed tips of polyimide coating. Two miniscrews were fixed on the occipital bone above the cerebellum, which were used as ground and reference respectively. Connect the connector to the multi-channel EEG amplification system to collect neuroelectric signals. The CED Micro1401 data acquisition system digitizes the analog signal with a sampling frequency of 1khz. Signal processing ensured that in the epoch every 30 seconds, the integer value showed the dominant frequency (DF) in Hz. The high frequency time is calculated by adding the 30 s epochs numbers for DF> 6 Hz and dividing by the total number of 30 s epochs per mouse (total recording time)[25, 26].
Western blot
One week later, after the EEG recording, the mice were killed by cervical dislocation and decapitation. Thereafter, the brain was taken out and stored at -80 ºC for western blot and Cell-surface biotinylation assay. The levels of Nav1.1α, Nav1.2 and Nav1.6 were analyzed using Western blot. Briefly, obtained protein of cortex and hippocampus from mouse were extracted with RIPA buffer (Beyotime, Jiangsu, China). The homogenate was centrifuged at 12,000g at 4°C for 10 minutes. Then the BCA protein assay quantifies proteins. The precipitated proteins were separated on 4-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. They were then transferred to PVDF membranes and combined with Nav1.1a (1:800; cat. no. ASC-001; Alomone), Nav1.2 (1:200; cat. no. ASC-002; Alomone), Nav1.6 (1:500; cat. no. ab65166; Abcam) and GAPDH (1:800; Catalog no. Sc-365062; Santa Cruz, California, Delaware, USA). The membrane was then incubated with an appropriate secondary antibody (goat anti-rabbit IgG and goat anti-mouse IgG; ZSGB-BIO, Beijing, China, 1:5000) at room temperature for 2 hours. Finally, protein quantification was performed using enhanced chemiluminescence Luminol reagent (Beyotime, Shanghai, China). The proteins were developed by Bio-Rad Gel Imaging System (ChemiDoc™ XRS+; Bio-Rad Laboratories, Inc., Hercules, CA, USA) with Quantity One software v4.6.6 (Bio-Rad Laboratories, Inc.)
Cell-surface biotinylation assay
Cell surface biotinylation and NeutrAvidin pull-down was performed as previously published work[25, 26]. Briefly, the brain tissues were incubated in ice-cold Krebs solution and the preparation of solution was carried out with modification (in mM) of described previously[25]: 120 NaCl, 4.5 KCl, 1.5 KH2PO4, 10 glucose, 1.5 MgSO4, 26 NaHCO3 and 1.5 CaCl2. Biotinylated proteins were pulled down by Neutravidin-agar-agar beads (Pierce, USA). Later, the surface and intracellular levels of Nav1.1α were determined using SDS-PAGE/Western blot (as described above).
Cell culture
The primary cultures of cortical or hippocampus tissues from C57BL/6J mice (one-day-old) were prepared as described [27]. The prepared cortex and hippocampus tissues was incubated in 0.25% trypsin (Gibco) for 10 minutes at the 37˚C and then the pancreatin was inactivated by fetal bovine serum (Gibco). A 70-μm cell strainer used to purify the tissues and leave the neuron cells. The cells were maintained in Neurobasal medium, with 2% B27 supplement (Gibco), 0.5% penicillin-streptomycin solution and 0.25% GlutaMax (Gibco), and then seeded onto 6-well culture plates. Finally, half of culture medium was substituted with fresh portions every three days.
Preparation of oligomer form of Aβ1-42
The Aβ1-42 peptide was dissolved in 1, 1, 1, 3, 3, 3- Hexafluoro-2-propanol (HFIP) at 1mM concentration. Afterwards, the solution was evaporated in the fume hood at room temperature for 24 h and the final clear peptide film stored at -20˚C. For oligomer preparation, films were resuspended in DMSO to 5mM, diluted at 5 mM in neural basal medium for 5h.
Model preparation and drug deliver
To explore the effects of Rb1 on excitability in Aβ1-42-treated neuronal cells, the Aβ1-42-treatment cultured cells were treated with 5µM Rb1 (name as Rb1 group), and the vehicle for Rb1 was DMSO (name as DMSO group). Besides, the Aβ1-42- treatment cells treated only with medium were treated as blank groups (blank group), and the baseline group is the primary neural cells untreated by Aβ1-42 and Rb1. 24h after the Rb1 treatment, the treated cells were then processed for the next detection.
Cell viability assay
The viability of primary neuronal cells was assessed using by MTT assay. Briefly, the primary neuron cells (2×104 cells/well) were plated in 96-well culture dishes. After that, 10mL MTT regent were added to each well at 37˚C and incubated for 4 hours. Then, the 100 mL DMSO displace the culture medium to dissolve the formazan crystals. Finally, Absorbance was read by microplate reader (Microplate reader, Bio-Rad, 3550) at 570 nm.
Electrophysiological patch clamp recording
Electrophysiological patch clamp recording was using to investigated potential effect of Rb1 on neuronal excitability. Electrophysiological measurements are used to measure the action potential or sodium current of cultured neurons. The electrophysiological patch clamp recording details was performed as previously described[24]. In short, Cell capacitance was measured from a transient current evoked by a -80mV depolarizing step from a holding potential of − 70 mV. Then a series of depolarizing current pulses are used and the inherent excitation is checked by constructing an input-output (I-O) function.
Statistics
Data were analyzed using the SPSS 19.0 (IBM, Armonk, New York) for Windows covariance software package. Data are presented as mean ± s.e.m. Comparisons between the two groups were performed using the student T test. Statistical differences between groups were assessed using analysis of variance (ANOVA) and Bonferroni postmortem tests. The MWM test was analyzed using bidirectional repeated measurement (RM) analysis of variance (ANOVA) and Tukey test. Use bidirectional variance analysis to analyze NOR data. The NOR data were analyzed by one-way ANOVA, and then the paired groups (two tails) were tested by student T test. Statistical significance was set as P<0.05.