The double transgenic 5XFAD mouse strain (Tg6799), which co-expresses mutant human APP and PS1 in neural cells, was previously described  and purchased from Nanjing Biomedical Research Institute of Nanjing University. The 5XFAD mice were maintained as hemizygotes in B6/SJL background. Mice were kept in SPF facility at a 12-hour light/dark cycle with free access to food and water. The primers used for genotyping the human-derived double transgen fragments are hAPP-F 5’-AGGACTGACCACTCGACCAG-3’, hAPP-R 5’- CGGGGGTCTAGTTCTGCA T-3’; hPS1-F 5’- AATAGAGAACGGCAGGAGCA-3’ and hPS1-R 5’- GCCATGAGGGCACTAATCAT-3’. All animal experiments were approved and complied with corresponding regulations issued by the Animal Experimental Ethic Committee of Southwest Medical University.
To generate hemizygous 5XFAD mice for downstream experiments, transgenic mice were crossed with wild type B6/SJL mice. In the offspring, the double transgene-positive mice which were supposed to be hemizygous and the non-transgenic littermates were enrolled for in vivo animal experiment. For tetrandrine treatment, the heterozygous 5XFAD mice were allocated into 4 groups. 3 groups of 5XFAD mice were intraperitoneally injected with 10 (n = 9), 20 (n = 12), 40 (n = 6) mg/kg of tetrandrine (tetrandrine hydrochloride, cat# H20053840, Yintao Pharma. China) respectively every 2 days from 5-month old to 7-month old. The left one group (n = 6) of age-matched 5XFAD mice received vehicle (0.9% NaCl) injection. Additional group (n = 8) of age-matched non-transgenic littermates were also injected with saline and serve as healthy control. At the end of the experiment, cognitive behavior of the mice was assessed by Morris water maze test as described below. Next day, mice were sacrificed by cervical dislocation. The brain was harvested and cut into halves along the sagittal axis. Half of the brain was fixed with 4% paraformaldehyde (in PBS) for 24 h followed by standard paraffin embedding. The left half of brain was snap frozen in liquid nitrogen and stored at -80 oC for downstream RNA analysis.
Morris Water Maze Test
The Morris water maze test was performed with 5 consecutive days of memory training before the probe trial in a circular swimming pool (120 cm in diameter, 50 cm in depth, Shanghai Xinxin Information Tech. Inc. China) filled with water mixed with milk powder. The pool was divided into 4 equal fan-shaped quadrants with one quadrant containing a circular translucent platform (8 cm in diameter) submerged 1.5 cm below the water surface. Four plates with different geometric shape of different color were hanged around to serve as spatial cues. For training, the mice were placed into the water at the edge of one quadrant with face towards the wall. The mice were allowed to swim to find the platform until 90 seconds elapsed. Mice which found the platform were allowed to stay on it for 10 s. Mice which failed to find the platform in 90 seconds were guided to the platform by experimenter and also allowed to stay for 10 s. The time for mice to find the platform was recorded as escape latent time. Each mouse was trained 4 times with the entrance to the water from different quadrants per day. For the probe trial session on the 6th day, the platform was removed. Mice were placed into the water from the opposite quadrant to the quadrant previously containing the platform and allowed to swim for 90 s. The length of time that the mice spent in target quadrant (previously with platform) and number of the times the that mice crossed the site of removed platform were recorded for downstream analysis.
High Performance Liquid Chromatography (HPLC)
C57BL/6 mice were injected with 40 mg/ml tetrandrine or saline. Mice were sacrificed at indicated time points by cervical dislocation. The brains were harvested and homogenized in 1 ml of methanol. After clearing by centrifugation, the supernatant was dried under nitrogen and reconstituted in 0.2 ml of methanol/0.3% triethylamine (98/2, v/v). 20 µl of the solution was loaded onto the LC1260 HPLC system equipped with an Innoval ODS-2 column (4.6 × 250 mm, 5 µm) settled at 30 oC. The mobile phase is methanol/0.3% triethylamine (98/2, v/v, 1 ml/min). The detection wavelength is 282 nm.
The paraffin embedded brain tissue was sectioned at 4 µm. The sections were dewaxed and rehydrated in gradual ethanol. Antigen retrieval was performed by boiling in 0.01M citrate buffer (pH 6.0) for 10 min. The tissue sections were treated with 3% H2O2 for 15 min to inactivate endogenous peroxidase and blocked by 10% goat serum for 1 h. Primary antibodies were applied overnight at 4 oC with indicated dilution ratio. After washing with PBS, the sections were incubated with HRP-conjugated secondary antibody solution for 30 min at 37 oC. Again, the sections were washed with PBS. The signal was developed in DAB solution and monitored under microscope. The chromogenesis was stopped by washing in distilled H2O. Then, the sections were counter-stained with hematoxylin and dehydrated. At last, the sections were cleared in xylene and mounted with neutral balsam mounting medium. Photos were captured with a light microscope (Nikon Eclipse 80i, Japan). Antibodies used in IHC are rabbit anti Aβ 1–42 antibody (Abcam, cat# ab10148, USA) and HRP-conjugated goat anti rabbit antibody (Abcam, cat# ab6721, USA).
TUNEL was performed with the peroxidase (POD)-based In Situ Cell Death Detection Kit following the manufacturer’s instruction (Roche, cat# 11684817910, Switzerland). DAB was used for signal development. The section was counter-stained with hematoxylin before mounting. Images were captured as described above. The percentage of apoptotic cell in hippocampus was calculated with Image J software (NIH, USA).
The brain tissue was milled into powder in liquid nitrogen. About 0.05 g of powder was used for RNA isolation with Trizol (Beyotime, cat# R0016, China) according to the standard instruction. 1 µg of RNA was reverse-transcripted into cDNA with PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, cat# 6210B, Japan). RT-PCR was performed with UltraSYBR Mixture (CwBio, cat# CW0957, China). Primers used in this study included TNFα forward 5’- CCCTCCAGAAAAGACACCATG-3’, reverse 5’- CACCCCGAAGTTCAGTAGACAG-3’; IL-1β forward 5’- GCTTCAGGCAGGCAGTATCA-3’, reverse 5’- TGCAGTTGTCTAATGGGAACG-3’; IL6 forward 5’- GGGACTGATGCTGGTGACAAC-3’, reverse 5’-CAACTCTTTTCTCATTTCCACGA-3’; COX-2 forward 5’- GGGGTGATGAGCAACTATTCC-3’, reverse 5’- GAGGCAATGCGGTTCTGATAC-3’; iNOS forward 5’- TTGGAGCGAGTTGTGGATTG-3’, reverse 5’- GGTCGTAATGTCCAGGAAGTAGG-3’; NF-κB forward 5’- CTGGAGCAAGCCATTAGCC-3’, reverse 5’- GGTTATCAAAAATCGGATGTGAG-3’ and β-actin forward 5’- GAGACCTTCAACACC CCAGC-3’, reverse 5’- ATGTCACGCACGATTTCCC-3’. The data was analyzed with the Delta-Delta Ct method.
Drugs And Cell Culture
To prepare the aggregated Aβ1–42, powder Aβ peptides (Sigma, cat# A9810, USA) were dissolved in MiniQ H2O to get a final stock concentration of 1 mg/ml (0.22 mM) followed by incubation at 37 °C for 7 days and further stored at 4 °C. Tetrandrine was the same to that used in the in vivo animal experiment. Microglial BV2 cells were cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS, Gibco, cat# 10500064, USA), 100 U/ml of penicillin and 100 µg/ml streptomycins. In BV2 cells, tetrandrine pre-treatment was performed for 1 h and Aβ1–42 stimulation lasted for 12 h. Cell pellet or conditional medium was collected for indicated experiment. PC12 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum (Gibco, cat# 26050070, USA), 100 U/ml of penicillin and 100 µg/ml streptomycin. Preceding treatment, PC12 cells were firstly differentiated into neuron-like cells in medium containing 50 ng/ml nerve growth factor (NGF) for 7 days. Then, the cells were stimulated for 12 h with conditional medium from BV2 cells which was diluted at a ratio of 1:1 with cultural medium of PC12. All cells were maintained in incubator set at 37 °C with 5% CO2 and 100% humidity. Cellular images were taken with an inverted microscope (Nikon, ECLIPSE TS100, Japan).
After indicated treatment, the culture medium of BV2 cells was collected, clarified by centrifugation and stored at -80 °C. Concentration of secreted TNFα and IL-1β were determined with mouse TNFα ELISA kit (BeJing LiKe Tech. cat# DRE30030 ,China) and mouse IL-1β ELISA kit (BeJing LiKe Tech. cat# DRE30027 ,China) following the manufacturer’s instruction.
0.1 g brain tissue was lysed in RIPA buffer. Protein concentration was determined by Pierce BCA protein assay kit (Thermo, cat# 23227, USA). 20 µg of total protein was used for Western blot as previously described . Primary antibodies employed in this study were rabbit anti-NF-κB (Abcam, cat# ab32536, USA), rabbit anti-TLR4 (Abcam, cat# ab13867, USA), rabbit anti-COX-2 (Abcam, cat# ab15191, USA), rabbit anti-iNOS (Abcam, cat# ab15323, USA) and mouse anti-GAPDH (Abcam, cat# ab8245, USA). Secondary antibodies were HRP-conjugated goat anti-mouse IgG (Sigma, cat#A3682, USA) and HRP-conjugated goat anti-rabbit IgG (Sigma, cat# A6154, USA).
1 × 104 PC12 cells were seeded into the well of 96-well plate. After adhesion, the cells were differentiated for 7 days followed by tetrandrine and Aβ1–42 treatment as described above. Then, CCK8 reagent was added into the medium to a final concentration of 10% (v/v). After incubation for 3 h, optical density at 450 nm wave length was recorded with spectrophotometer. Each treatment was performed in triplicate.
0.5 × 106 PC12 cells were seeded into 6-well plate. After differentiation and treatment with tetrandrine and Aβ1–42, cells were collected by standard typsin digestion. Then the cells were washed with PBS and subjected to Annexin V/PI staining according to the manufacturer’s instruction (BD Bioscience, cat# 556547, USA) and analyzed with Aquios CL Flow Cytometry System (Beckman, USA). Three samples were included for each treatment.
PC12 cells were seeded onto round sterile glass coverslip and subjected to differentiation and treatment as described above. For immunofluorescent staining, cells were fixed in cold 4% paraformaldehyde for 10 min. After washing, the cells were permeabilized with 2.5% triton X-100 for 10 min and blocked with 10% goat serum for 1 h at RT. Primary antibody was applied at 4 °C overnight. After washing with PBS, fluorescent secondary antibody incubation was performed at RT for 1 h. Next, the cells were washed with PBS and counter-stained with DPAI and mounted on glass slide. Antibodies used included rabbit anti-cleaved Caspase-3 (Abcam, cat# ab2302, USA), rabbit anti-Bcl-2 (Abcam, cat# ab182858, USA) and Alexs Fluor 488-conjugated goat anti-rabbit IgG (Abcam, cat# ab150077, USA). Photos were taken under a fluorescent microscope (Nikon, Eclipse Ci-S, Japan).
The quantitative data were presented as mean ± SEM. Two-way analysis of variance (ANOVA) for repeated measures was used to analyze the escape latency data in Morris water maze test followed by Tukey’s multiple comparison test. For additional multiple groups of quantitative data, one-way ANOVA followed by Tukey’s post hoc test was used. p < 0.05 was regarded as statistical significance.