Chemicals and reagents
A10E was synthesized on the basis of our previous study [24]. Tacrine, huperzine A, dithiobisnitrobenzoic acid (DTNB), ethopropazine hydrochloride, and acetylthiocholine iodide (ATCI) were obtained from the company of Sigma-Aldrich (St. Louis, MO, USA). Aβ peptide was obtained from GL Biochem (Shanghai, China).
Preparation of Aβ oligomers
Aβ oligomer preparation was described in previous studies reported by us [27-29]. Briefly, Aβ peptide was dissolved in hexafluoroisopropanol (HFIP, Aladdin, Shanghai, China) to form Aβ monomers. Afterwards, 100 μL Aβ monomers were added to a tube and diluted with 900 μL Milli-Q water. HFIP in the solution was evaporated under nitrogen till a final concentration of Aβ monomers at 60 μM. After being persistent agitated for two days at room temperature, the Aβ solution was centrifuged at 14,000 g for 15 min at 4 °C. And the supernatant was collected and mainly contained Aβ oligomers.
Animals
Use and care of laboratory animals followed the National Institutes of Health (NIH) Guide (NIH Publications No. 8023, revised 1978) and were authorized by the Animal Care and Use Committee of Ningbo University. Male Institute of Cancer Research (ICR) mice (around 30g, 3 months old) were purchased from Zhejiang Academy of medical Sciences (Hangzhou, Zhejiang, China), wild-type (WT) C57BL/6 mice and APP/PS1 mice (around 25g, 1 month old) were purchased by Southern Model Biological Research Center (Nanjing, China). Mice were raised with a 12-h light/dark cycle (humidity: 50 ± 10 %, temperature: 22 ± 2 °C).
Intrahippocampal injection (i.h.p.) of Aβ oligomers
In advance of i.h.p., mice were given sodium pentobarbital [50 mg/kg, intraperitoneal injection (i.p.)] to be anesthetized, and then using a stereotaxic instrument (RWD Life Science, Shenzhen, China) to fix their heads tightly. After the skull was exposed, two holes were drilled using the following coordinate: AP - 0.4 mm from bregma; ML ± 1.0 mm from the midline; and DV − 2.0 mm from pia mater [30]. Aβ oligomers (1 μL/side, 0.6 μg) were individually injected into both holes at a constant speed of 0.2 μL/min using an UltraMicroPump (RWD Life Science). Control mice were given an equal volume of 0.9% normal saline as vehicle (i.p.).
Animal group and drug treatment
A10E were dissolved in 0.9% normal saline. 4-week-old APP/PS1 mice were randomly assigned into 3 groups (n = 7). Two A10E-treated groups were treated with A10E at the dose of 0.36 and 0.72 μmol/kg (i.p.), respectively, into APP/PS1 mice every third day for 28 weeks. APP/PS1 and WT mice were injected with equal volumes of 0.9% normal saline daily (i.p.).
For Aβ oligomers-injection experiments, mice were randomly assigned into 7 groups (n = 8): vehicle (0.9% normal saline, i.h.p.) plus vehicle (0.9% normal saline, i.p.), Aβ oligomers (i.h.p.) plus vehicle (0.9% normal saline, i.p.), Aβ oligomers (i.h.p.) plus A10E (0.18 μmol/kg, i.p.), Aβ oligomers (i.h.p.) plus A10E (0.54 μmol/kg, i.p.), Aβ oligomers (i.h.p.) plus A10E (1.1 μmol/kg, i.p.), Aβ oligomers (i.h.p.) plus tacrine (7.9 μmol/kg, i.p.) and Aβ oligomers (i.h.p.) plus huperzine A (4.1 μmol/kg, i.p.). Drugs was treated daily until the day of sacrifice.
Open field (OF) test
OF test is used to analyze the exploratory and locomotor activities of animals [31]. The test was conducted in an open plastic box (50×50×39 cm) in which the floor was divided into four equal quadrants (25×25 cm) by crossed black lines as a previous study described [32]. Mice were placed in the center of the floor and permitted to explore it for 5 min. The number of rearing and line crossing of mice was recorded. The floor was cleaned between two individual tests using 10 % ethanol in order to avoid influence of the urine and odor on behavior.
Novel object recognition (NOR) test
NOR test is used to test the cognitive function of animals, and carried out in a black open plastic box (50×50×39 cm) described previously [33]. Briefly, the test consisted of training section and exploring session. In the training session, mice were placed in the center of the box and permitted to explore two identical stone cubes (5×5×5 cm) freely for 5 min. The exploring session was conducted after 24 h and one stone cube was replaced by a stone square pyramid (5×5×7 cm). Mice were placed in the center of the box and permitted to explore the two different stone cubes freely for 5 min. The behavior that Mice looked towards and sniffed the object was considered exploratory behavior while walking around the identified object or just moving around the recognized object was not considered exploratory behavior. The field was cleaned between two individual tests using 10% ethanol in order to avoid influence of the urine and odor on behavior. The cognitive function was evaluated by a recognition index, which was the exploration time involving either of the two objects (training session) or the novel object (exploring session) compared with the total exploration time. Because rodents have the nature to explore new things, the increase in cognitive index can be used to reflect an increase in cognitive function.
Morris water maze (MWM) test
MWM test is a classic method to evaluate the spatial learning and memory of mice described in a previous study [34]. The water maze was a circle poor (radius at 75 cm, height at 50 cm), and was filled with water (depth at 30 cm, temperature at 25 °C). Various objects were set outside the pool and could be observed by mice to identify the spatial orientation. The pool is divided into four identical quadrants, and the platform was located in one of the pool quadrants and submerged 1 cm below the water surface. The training session lasted for four consecutive days. Mice were allowed to find the platform up to 90 s, and permitted to remain on it for 10 s to be familiar with the surrounding. The time of mice required to enter the hidden platform called escape latency was recorded, while the mice who could not reach the platform within 90s would be guided to the platform gently and permitted to remain on it for 10s. In the probe session, the platform was removed and mice were allowed to explore freely in the pool for 90 s. Duration in the target quadrant and number of platform area crossing, and motion path of the mice were recorded for analysis.
Brain tissue preparation
The mice were sacrificed to prepare brain tissue the next day after the MWM test. Mice were anesthetized by injection of sodium pentobarbital (50 mg/kg, i.p.) and cardiac-perfused phosphate-buffered saline (PBS). The hippocampus and the whole brain tissue were dissected for biochemical analysis. Furthermore, as for immunohistochemical (IHC) staining, after perfusion with saline, mice were fixed by perfusion with 4% paraformaldehyde. The brain tissues were carefully removed and placed in a centrifuge tube containing 4% paraformaldehyde overnight at 4 °C, then placed into a centrifuge tube containing 30% sucrose solution for dehydration.
Measurement of AChE and ChAT activity
The measurement of AChE activity was conducted according to the protocol described in a previous study [35]. Briefly, the whole brain was added with 10 times volume of lysis buffer [10 mM HEPES (pH 7.5), 1 mM EGTA, 1 mM EDTA, 150 mM Triton X-100 and 1 mM NaCl]. In order to obtain AChE, the mixture was homogenized on ice for 15 min and then centrifuged for 15 min at 3000 rpm at 4 °C, and the supernatant was collected. Afterwards, the brain lysate was incubated with 0.1 mM ethopropazine hydrochloride at 37 °C for 5 min to inhibit butyrocholinesterase (BuChE) activity. Then the test compound was added to the assay medium [0.1 M Na2HPO4 (pH 7.5), 10 mM DTNB and 1 mM ATCI] and pre-incubated at 37 °C with the enzyme for 15 min. Then ATCI was added, and incubated at 37 °C for 30 min. AChE activity was determined by measuring the absorbance at 412 nm.
The measurement of ChAT activity was conducted by using ChAT assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). According to the manual, briefly, brain tissue homogenate was prepared by normal saline according to the ratio of weight to volume, afterwards, supernatant of homogenate was added after the prepared mixture was bathed at 37 °C for 5 min. After bathing at 37°C for 20 min, mixture was centrifuged for 10 min at 4000 rpm, the supernatant was collected and R7 reagent was added. Finally, the active unit of ChAT was measured with the absorbance at 324 nm.
Western blotting analysis
Western blotting assay was carried through on the basis of a protocol described previously [32]. The brain tissues were homogenized in lysis buffer containing RIPA, protease inhibitors and phosphatase inhibitors (Sangon Biotech, Shanghai, China). Afterwards, the mixture was centrifuged at 13200 rpm for 15 min, and the supernatant was taken. The concentration of protein was determined by using a BCA Protein Kit (Beyotime Biotechnology, Shanghai, China). Sample was added with appropriate amount of loading buffer and heated to denature the protein, and stored at -20 °C. According to the size of the target protein, the corresponding concentration of the separation gel and the concentrated gel were prepared, and the loading quantity of sample was determined on basis of the protein concentration. In electrophoresis process, firstly the concentrated gel was run with a constant pressure of 80 V, and the separator gel was run with a constant pressure of 120 V until target protein band was separated. The separated proteins in gel were then transferred to polyvinylidene fluoride membrane in a transfer conditions of 100 V for 1.5 h. After transfection, 5% BSA was used to block the membrane at room temperature for 2h, followed by incubating the membrane with primary antibodies ChAT (Santa Cruz, USA, 1:1000), BDNF, tau (Abcam, UK, 1:1000), TrkB, p-TrkB, Akt, p-Akt, p-tau (Cell Signaling Technology, USA, 1:1000), and β-actin (Affinity bioscience, USA, 1:1000) respectively overnight at 4 °C.
The next day, the membrane was washed for 3 times (15 min for each time) using TBST buffer (2 mM NaCl, 10 mM Tris-HCl and 0.1 % Tween-20; Shanghai Aladdin Biochemical Technology). Then the secondary antibodies were applied to specifically bind to corresponding primary antibody at room temperature for 1 h. After washing the samples three times with TBST, the development of the protein bands was using ECL luminescent substrate. The amount of protein was evaluated by analyzing the intensity of each band using Image J software (NIH Image, Bethesda, MD, USA).
IHC staining
IHC staining was conducted according to a protocol described in a previous study [36]. Briefly, after blotting the dehydrated brain with absorbent paper, the brain tissue was fixed on the sample plate with an embedding agent at -20 °C. Brain tissue was cut into slices with 25 μm thick using a freezing microtome. Brain slices were incubated with antigen repair fluid at 60 °C for 30 min, followed by incubation with the immunized blocking solution at room temperature for 1 h. Afterwards, the brain slices were incubated with primary antibodies Iba-1 and GFAP (Cell Signaling Technology, 1:400) at 4 °C overnight. The next day, the brain slices were washed for 3 times (15 min for each time) using PBS, and then added with corresponding fluorescent secondary antibody, and incubated at room temperature for 1 h in the dark. Subsequently, the slices were washed for 3 times (15 min for each time) using PBS, and nuclei were stained with 4′,6-dimidyl-2-phenylindole (DAPI). The slices were eventually sealed with anti-quenching seals. Fluorescent marks in brain slices were observed using a confocal fluorescence microscope.
As for staining of Aβ, brain slices were incubated with antigen repair fluid at 60 °C for 30 min, followed by incubation with the immunized blocking solution at room temperature for 1 h. Afterwards, the brain slices were incubated with primary antibodies Aβ (Sigma-Aldrich, 1:400) at 4°C overnight. The next day, the brain slices were washed for 3 times (15 min for each time) using PBS, and then added with secondary antibody working solution including Horseradish Peroxidase and Goat Anti-rabbit IgG in 1:200 solution, and incubated at 37°C for 30 min. Subsequently, the slices were washed for 3 times (15 min for each time) using PBS, and colored by using diaminobenzidine color development kit. The images were captured by the camera of the light microscope.
Enzyme linked immunosorbent assay (ELISA)
The concentration of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in hippocampus was detected by ELISA. The sample including 10 mg of mouse hippocampus tissue and 90 μl of PBS was totally homogenized. After centrifugation at 4 °C for 20 min (2000–3000 rpm), the supernatant was carefully collected for ELISA. According to the ELISA kit (Meibiao Biology, Jiangsu, China) detection method, the OD value of each well was measured by adjusting the blank control hole at a wavelength of 450 nm on the microplate reader. A standard curve was prepared based on the concentration and OD value of the standard, and then the concentrations of IL-6 and TNF-α in the hippocampus of each group of mice were calculated according to the standard curve equation.
SH-SY5Y cells
SH-SY5Y cells (Chinese Academy of Sciences, Shanghai, China) were cultured in a mixed medium containing 90% high glucose modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), and 1% penicillin (100 U/mL)/ streptomycin (100 µg/mL). Culture medium was replaced every two days. The culturing condition was 37°C with 5% CO2. The culture medium of SH-SY5Y was replaced with DMEM with 1% FBS and continue culture for 24 h before experiments.
Measurement of cell viability
3 (4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) assays was applied to evaluate the cell viability as previous study described [27]. In short, 10 μL MTT solution (5 mg/mL) was added to 96-well plates and incubated at 37 °C for 4 h. Then 100 μL solvating solution (10 % SDS solution supplemented with 0.01 N HCl) was added, and the plate continued incubating tranquilly for 16-20 h. A spectrophotometrer (Thermo Fisher Scientific, Waltham, MA, USA) was applied to evaluate the cell viability according to the absorbance at 570 nm with 655 nm as a reference wavelength.
Dot blotting analysis
Dot blotting analysis was also carried out according to the methods we reported previously [37]. Firstly, the mixture of equal volume of different concentrations of the drugs and Aβ solution were prepared. The mixture was agitated to form the sample. Afterwards, 2 μl of the sample were spotted onto the nitrocellulose membrane, and were dried by air. After blocking the membrane 5% BSA diluted in 1% TBST solution overnight, the membrane was incubated with anti-oligomer antibody A11 (Thermo Fisher Scientific, 1:1000) or anti-Aβ1-17 antibody 6E10 (Sigma-Aldrich, 1:1000) with gentle shaking lasting for 1 h. Afterwards, the membrane was washed by 1% TBST for 15 min before it was incubated with second antibody for 1 h. After 3 washes in 1% TBST, the membranes were visualized by using the ECL detection reagents (Tanon 5200 Automatic chemiluminescence imaging analysis system, Shanghai, China).
Preparation of Aβ fibril and thioflavin-T (ThT) assay
Aβ fibrils preparation was also described previously [38]. Briefly, Aβ monomer was dissolved in HFIP, and diluted with Milli-Q water. After fully evaporating HFIP with nitrogen, Aβ monomer was diluted with sodium hydroxide solution to form 1 mM Aβ solution. Next, 2 μL Aβ solution with chemicals or vehicle were added to PBS at 197 μM with 5 μM ThT (Sigma-Aldrich). The system was incubated in the dark for 3 days at 37°C. The quantity of Aβ fibrils was evaluated by fluorescence intensity at the excitation and emission wavelengths of 440 and 485 nm, respectively.
Immunoblotting analysis
The Immunoblotting analysis of Aβ oligomers formation was performed as previously described [34]. To begin, Aβ monomer was dissolved in HFIP to 2.5mg/ml, evaporated under nitrogen until the final concentration of Aβ1-42 becomes 60 µM, and mixed with an equal part of A10E sample. then the sample was shook for 48 h at 27 °C. Subsequently, samples were electrophoresed and transferred. Membranes were boiled for 10 min for 3 times, and blocked with 5% BSA for 1 hour at room temperature, incubated with rabbit anti-Aβ1-42 antibody 6E10 (1:1000, Invitrogen, California, USA) followed by corresponding secondary antibody, and developed in an automatic chemiluminescence imaging system.
Data analysis and statistics
The results were exhibited as mean ± SEM. The data was analyzed by GraphPad Prism (version 6.0, GraphPad Software, Inc., San Diego, CA, USA). The difference between groups was evaluated by one-way analysis of variation (ANOVA) and statistical comparison was evaluated by Tukey's test. However, the group variance of the body weight of mice and recognition index were analyzed by two-way repeated measures ANOVA. Values of p < 0.05 were considered statistically significant. In figures, % or folds of control was calculated by separating each control value on the average of total control group values for a particular protein, and then the values of treated group were normalized to control (100% or 1.0).