Adult male Sprague Dawley (SD) rats (250 ± 20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (certificate number: SCXK (Beijing) 2016-0006). The animals were housed with temperature (22 ± 1°C), humidity (50 ± 5%), and light (12-h light/dark cycle) control with free access to water and food. All experiments were conducted after being approved by the Institutional Animal Ethical Care and Use Committee of the Beijing University of Chinese Medicine (Approval ID: BUCM-4-2017121725-4025).
Animal surgical procedure
VD model was established by permanent bilateral common carotid artery occlusion (2VO) as described before . Briefly, rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (45 mg/kg). The neck of the rats was disinfected, and an incision was made to expose and separate the bilateral common carotid arteries along the cervical anterior median. Then, the bilateral blood vessels were ligated with 5-0 silk thread. In the sham-surgery group, the same surgery was performed with the exception of arterial ligation. The rats’ body temperature was maintained at about 37 ± 1°C during surgery.
SZJN formula is composed of 3g Panax ginseng C.A.Mey., 9g Anemarrhena asphodeloides Bunge, and 9g Paeonia anomala subsp. veitchii (Lynch) D.Y.Hong & K.Y.Pan. All herbs were pharmacopoeia-grade and prepared into granules by Beijing Tcmages Pharmaceutical Co., Ltd. (Beijing, China). Details of herbal materials are listed in Supplementary Fig. 1. The material basis of SZJN formula was determined using high-performance liquid chromatography (HPLC) and infrared spectrum (IS) analysis for monitoring the quality control purposes (Supplementary Fig. 2). Ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1, Mangiferin, and paeoniflorin (the purities of all standards were higher than 98% by high-performance liquid chromatography analysis) were purchased from Beijing Solarbio Biotech Co., Ltd. (Beijing, China). The granules were suspended with distilled water to a final concentration of 38 g/L.
Rats were randomly divided into control group, sham-surgery group, VD group, SZJN group, and memantine hydrochloride (MH, H.Lundbeck A/S, Denmark) group. Rats in the SZJN group and MH group were intragastrically administered with SZJN formula (dissolved with distilled water, 0.2 g/kg), and memantine hydrochloride (dissolved with distilled water, 2.1 mg/kg) one day after surgery for 14 days, respectively . In the control group, sham-surgery group and VD group, rats were administered with equivalent distilled water intragastrically once per day for 14 days. Fig. 1A presents a schematic timeline of experiments.
PC12 cell culture and experimental design
Differentiated PC12 cells (Procell Life Science & Technology Co., Ltd., Wuhan, China) were maintained in RPMI 1640 (Procell) supplemented with 10% (v/v) fetal bovine serum (Procell)) and an antibiotic mixture of penicillin and streptomycin (1%, Procell) in a humidified incubator at 37°C and 5% CO2. When these cells reached 70%–80% confluence, they were trypsinized and subcultured. After seeding onto poly-L-lysine-coated 96- or 6-well plates at 3 cells/well for 24 h, the cells were exposed to glutamate (final concentration: 22.5 mM, Sigma, USA) and incubated in the presence or absence of various concentrations of SZJN formula (final concentrations: 0.05, 0.1 and 0.2 mg/mL) and memantine hydrochloride (MH, 10 μM) for 24 h. The control cells were not administered any test agent or glutamate as the vehicle control. The glutamate-exposed cells were treated with glutamate for 24 h alone. The concentrations of SZJN formula in this experiment were selected according to our preliminary experiment (Supplementary Fig. 3). All experiments were replicated three times.
Cell death and morphology changes
To observe cell death and morphology changes during intervention process, the live-cell Essen Bioscience IncuCyte imaging system was used. Briefly, PC12 cells cultured by different concentrations of test agents were exposed to 5% YOYOTM-1 lodide solution, a cell impermeable dye that only enters cells with compromised membranes. The samples were observed under the IncuCyte imaging system at 10× magnification, which records both phase-contrast as well as fluorescent images over time.
Novel object recognition (NOR) test
NOR test was performed on the 8th day of treatment as previously described . During the training period, the exploration time for two identical objects was recorded within 5 min. One day later, one object was changed to a different color and shape but with the same size. The exploration time for the new object and the familiar one was recorded within 5 min. Rats were evaluated for their memory by expressing a preference for exploring the novel object . Preference for the novel object was expressed as a discrimination index (DI, equation below).
Morris water maze (MWM) test
To assess spatial learning and memory, MWM test was performed on the 10th day of treatment according to a procedure described with some modifications . The circular water maze pool was 120 cm in diameter and divided into 4 quadrants. Each rat was allowed to search for the platform within 60 s. When the rats failed to locate the submerged platform, they were manually directed toward the platform and allowed to remain on it for 10 s. Each rat was trained 4 times a day for 4 consecutive days. On the 5th day, a spatial probe trail was conducted by removing the platform from the pool. The rats were monitored to swim freely for 60 s. The escape latency, total swimming distance, and percentage time spent in the target quadrant were recorded. All rats were kept from a motor impairment.
Histological and pathological assessment
Rats were anesthetized using 2% sodium pentobarbital administered intraperitoneally (45 mg/kg) and transcardially perfused with normal saline (50-100 mL) at 37°C and then perfused with 4% paraformaldehyde (100 mL) for 30 min. The brain was immersion fixed in 4% paraformaldehyde for 48 h after which the brains were embedded in paraffin. A series of adjacent 3μm thick coronal sections were cut and employed for various staining. Additional hippocampal tissues were frozen in liquid nitrogen and stored at −80°C for further analysis. For hematoxylin-eosin (H&E) staining and Nissl staining, slices were dehydrated with EtOH (70%, 95%, 100%, 5-min each), until xylene (5-min), followed by staining using hematoxylin-eosin as well as toluidine blue, respectively. Histopathological changes in the hippocampi were observed using an Olympus microscope (BX53, Japan).
Brain coronal sections were incubated for 1 h at room temperature with 10% goat serum then overnight at 4°C with primary antibodies to clathrin, RAB5B and NMDAR1 (Supplementary Table 1). After incubation with secondary antibodies for 50 min, immunostaining was detected with 3,3′-diaminobenzidine kit (DAB, G1211, Servicebio). Image pro plus 6.0 image analysis software (Media Cybernetics, Rockville, MD, USA) was used for data analysis. The Area and Integral Optical Density (IOD) of the positive regions were imaged and the relative protein expression levels were calculated as the mean optical density (IOD/Area).
PC12 cells were rinsed with PBS and fixed with 95% ethanol or 4% paraformaldehyde. After blocked with 3% BSA for 1 h at 37°C, cells were incubated with primary antibodies (Supplementary Table 1) for 2 h at 37°C. Subsequently, cells were washed and incubated with secondary antibodies in the dark for 1 h at 37°C, followed by staining with 20μg/ mL Hoechst 33342 (Sigma-Aldrich) for 15 min. A IN Cell Analyzer high connotation cell imaging analysis system (2500HS, GE, USA) was used the observation.
Western blot analysis
Plasma membrane proteins were separated based on the Plasma Membrane Protein Isolation Kit (SM-005, Invent) protocol. The extracted protein of cells or tissues was determined for concentration with a BCA kit (Servicebio). Equal amounts of protein were electrophoresed on 10% polyacrylamide gels and then transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% skim milk for 1 h, the membranes were incubated overnight at 4oC with primary antibodies directed against clathrin, RAB5B, NMDAR1, and β-actin (Supplementary Table 1). The membranes were incubated with secondary antibodies at room temperature for 1 h and visualized using an ECL substrate (WBKLS0100, Millipore). FlourChem software (Alpha Innotech, FlourChem) was used to analyze the ratio of the gray value of each band to the β-actin band for relative expression levels. All samples were analyzed independently and in triplicate.
Quantitative real-time PCR
Total RNA was extracted from tissue samples and cells using a HiPure Total RNA Mini Kit (R4111-02, Magen) following the manufacturer instructions. After reverse transcription using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA), cDNA was used as the template for quantitative RT-PCR and performed using the real-time fluorescence quantitative PCR instrument (CFX96, Bio-Rad). The PCR cycle was as follows: 95oC pre-denaturation for 10 min, 95oC denaturation for 15 s, 60oC annealing for 1 min, with a total of 40 cycles. β-actin was designated as the internal reference gene and the relative expression level of target genes was calculated by the method. All samples and assays were performed in triplicate. Primers (Sangon Biotech, Shanghai, China) for clathrin, NMDAR1, RAB5B, and β-actin are listed in Supplementary Table 2.
SPSS 25.0 software was used for data analysis. Data were expressed as mean ± SD. MWM data were analyzed using two-way repeated measure ANOVA. For the others were analyzed by one-way ANOVA followed by post hoc test (Tukey's tests). If the variance of statistics was uneven, a Nonparametric test was performed. P values < 0.05 was considered statistically significant.