Preparation of drugs
GEPT (called GETO in our previous publications), consists of 35% ginsenoside extracted from ginseng, 35% flavonoid glycoside extracted from epimedium, 15% tenuifolin extracted from polygala, and 15% curcumin from tuber curcumae. GEPT was provided by Henan Wanxi Pharmaceutical Company Limited (batch no. 20010923, China). Hydrochloric acid donepezil tablets were provided by Eisai Pharmaceutical Company Limited (batch no. 090508A, China). GEPT was dissolved in 0.5% carboxymethyl cellulose (CMC) (Sigma, USA) at a concentration of 30 mg/mL, and donepezil tablets were crushed and dissolved in 0.5% CMC at a concentration of 0.092 mg/mL.
Animals and medicine administration
Three-month-old APPV717I mice and C57BL/6 J mice (non-transgenic inbred mice, used as vehicle controls) were purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences & Peking Union Medical College (Beijing, China). The APP/V717I transgenic mice had a C57BL/6 J genetic background and carried mutated human APP-CT100 containing the London mutation, V717I, which results in increased generation of Aβ42 and AD-like pathological changes. All animals were housed in the Pharmacological Experiment Center of Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China. They were maintained in a temperature-controlled (24°C) pathogen-free vivarium, on a 12:12-h light:dark cycle (12-h light [06:00 to 18:00], 12-h dark [18:00 to 06:00]) with free access to food and water. All experimental procedures were performed in compliance with the Provision and General Recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Research Ethics Board of Beijing University of Chinese Medicine.
APPV717I transgenic mice were arbitrarily divided into 10 groups (n = 12 per group) and received intragastrically administrated vehicle or medicines. APP groups were given 0.5% CMC and donepezil groups were given donepezil (APP + D; 0.92 mg/kg/d) for 4 or 8 months. GEPT was administered at three dosage levels, with a separate group for each: low dose (APP + Gl; 0.075 g/kg/d), medium dose (APP + Gm; 0.15 g/kg/d), and high dose (APP + Gh; 0.30 g/kg/d), also for 4 or 8 months. Male C57BL/6 J mice served as vehicle controls (n = 12) and were given 0.5% CMC for 4 or 8 months.
Spatial learning and memory were assessed in orientation navigation tests using the Morris water maze (MWM) as described previously. After 4 or 8 months of treatment, all mice underwent testing in the MWM, which consisted of two days of learning and memory training, followed by three days of probe trials. The animals’ swim paths and the numbers of annulus crossings were recorded on videotape; the percentage of time spent in each quadrant and the average swim speed were determined from these videotapes.
All behaviorally tested mice were deeply anesthetized with 10% chloral hydrate (Loogene Biotechnology Co., Ltd, China) (40 mg/kg, i.p.) and pericardially perfused with heparinized 0.9% saline, prior to removal of the brain. Brains were immersion-fixed in 4% paraformaldehyde (Sun Biomedical Technology Co., Ltd, China) overnight at 4°C and then processed in a phosphate-buffered saline (PBS) solution containing 30% sucrose. Seven days later, brains were embedded in paraffin. Serial coronal sections of the hippocampus were cut at 35-μm intervals for immunohistochemistry (IHC) staining. The brains of three arbitrarily selected mice were separated according to regions and snap frozen for Western blot.
Brain sections were deparaffinized and rehydrated in distilled water. Antigens were then unmasked in 0.01 M citrate buffer (Sun Biomedical Technology Co., Ltd, China) by microwave, and endogenous peroxidase activity was quenched by 0.3% hydrogen peroxide (Sun Biomedical Technology Co., Ltd, China) in methanol (Sinopharm Chemical Reagent Co., Ltd, China) for 20 min at room temperature. The sections were then blocked with 3% bovine serum albumin (Sigma-Aldrich Co. LLC, USA) in PBS for 30 min at 37°C. After excess serum was removed, sections were incubated with primary antibody against SYP (1:400; ab23754, Abcam, USA) in humidified boxes at 4°C overnight. They were then washed again and incubated with biotin-conjugated secondary antibodies (1:300, Fuzhou Maixin Ltd, China) for 30 min at 37°C, then washed again and incubated with Streptavidin-Biotin Complex (SABC) (Wuhan Boster Bioengineering Co., Ltd, China) for 1 h at 37°C. Sections were subsequently developed using the chromogen 3’,3-diaminobenzidine tetrachloride (DAB), after which they were dehydrated and coverslipped. All brain sections chosen for staining were on a similar sagittal plane and contained approximately the same area of hippocampus. SYP average optical density (AOD) was measured in immunostained sections, following the instructions of the Image Pro Plus 6.0 software (Media CY Company, USA). “Nonspecific” IHC staining in sections was chosen as the control area for comparison with the SYP-immunopositive area in the neurons of the dentate gyrus.
Western blots were performed based on a previously described method. Briefly, snap-frozen brain tissues from hippocampus and cortex were weighed and homogenized in brain tissue lysis buffer (Loogene Biotechnology Co., Ltd, China) using a small pestle on ice, at a ratio of 1:10 (w/v) for 2 min, and incubated on ice for 30 min. Homogenates were centrifuged at 4300 x g at 4°C for 30 min, and supernatants were collected. The level of protein in the supernatants was determined by the modified Bradford method using Coomassie Brilliant Blue G-250 (Nanjing Jiancheng Bioengineering Institute, China). Loading buffer was added to samples at a ratio of 4:1, after which samples were placed in boiling water for 5 min and then immediately chilled on ice. Aliquots (10 μL) of each sample and 5 μL of marker (10–170 kDa) were loaded onto 10% acrylamide gels (Sigma-Aldrich Co. LLC, USA) and subjected to SDS-PAGE using the Bio-Rad mini gel system (Bio-Rad, USA). Proteins were then electro-blotted onto polyvinylidine difluoride membranes. Membranes were blocked with 5% milk at 4°C overnight, and then incubated with primary antibody (anti-SYP antibody, ab23754, 1:5000). After three washes with PBS containing 0.5% Tween 20 (PBST) (Sun Biomedical Technology Co., Ltd, China), membranes were incubated at room temperature for 1 h with anti-rabbit IgG (H&L) (Equitech Bio, Inc. USA) horseradish peroxidase-conjugated secondary antibody (Promega Co., USA) at 1:10,000 on a shaker. After three washes with PBST, blots were developed using Luminol reagent (Pierce Biotechnology, USA). Densitometric analysis of the blots was completed using Phoretix 1D software (Total Lab Ltd, UK). Expression of SYP protein is shown as the SYP protein-β actin ratio.
All data were analyzed using SPSS 13.0 software (IBM Software, USA) and are presented as mean ± standard deviation (SD). One-way ANOVA with Tukey’s post-hoc test was used when comparisons were made between two groups. P < 0.05 was considered statistically significant.