DMEM medium was purchased from Gibco Life Technologies (Grand Island, NY, USA). Fetal bovine serum was purchased from Gibco Life Technologies (Grand Island, NY, USA). Aβ25–35 was purchased from GL Biochem Ltd. (Shanghai, China). Aβ25–35 peptide was dissolved in distilled water at the concentration of 1 mg/ml, filtered to remove bacteria and incubated at 37 °C for 4 days to form aggregated Aβ25–35 peptide Transfection reagent was purchased from Engreen Biotechnology company (Beijing, China). PEI was purchased from Shanghai Qifa Experimental Reagent Co., Ltd. (Shanghai, China). DMTHB was synthesized in our laboratory.
Adult male C57BL/6 mice (8 weeks) were purchased from the College of Veterinary Medicine, Yangzhou University (Yangzhou, China). These mice were housed in temperature- and humidity-controlled rooms. The mice were maintained at a 12-hour light/dark cycle and had free access to rodent chow and tap water. All procedures were approved by the Institutional Animal Care and Use Committee at China Pharmaceutical University and adhered to the Jiangsu Provincial Guidelines for the use of experimental animals.
2.3 Experimental mouse models
The mice were divided into four groups (8 mice in each group): control, Aβ25–35, Aβ25–35 + DMTHB (50 mg/kg) and Aβ25–35 + DMTHB (150 mg/kg) groups. DMTHB was intragastrically administered every day for three weeks. The intracranial injection of Aβ25–35 was conducted at the 4th week after DMTHB treatment. All mice were anesthetized with pentobarbital sodium (50 mg/kg). Then, the anesthetized mice were fixed on the stereotaxic apparatus (RWD Life Science Co., Ltd., Shenzhen, China). Small burr holes were drilled on two sides of the skull (1.0 mm posterior to bregma and 1.5 mm lateral to the midline) to allow intracerebroventricular (icv) injection of Aβ25–35 (2 nmol / 2 μl) / saline at the depth of 2 mm. Control injection speed 1μL/min and leave the syringe for 10 minutes to allow the brain to absorb the injection. Oral medication was continued with DMTHB daily until the end of 6th week of the experiment. The specific operation is shown in Figure 1.
2.4 IL-16 siRNA interference in mice
The mice were recovered for a week after the Aβ25–35 injection. Then, siRNA was injected intracranially every four days for a total of 2 injections. siRNA sequence of IL-16 is 5′-CCU UGG GUU AGA AUU UCC GAC UGC A-3′, The siRNA concentration is 2μg/μl, and the injection volume is 2μl. The specific operation is shown in Figure 2.
2.4 Morris water maze (MWM)
MWM test was carried out at the 6th week after administration of DMTHB. The water temperature of MWM was controlled between 24 and 26 °C during the experiment. The round pool was divided into four quadrants and the target platform. The mice received two consecutive training trials daily for 5 days of the acquisition training session. If a mouse did not find the platform within a period of 60 s, it was gently guided to and remained on the platform for 10 s. The escape latency time and swimming speed were recorded. The probe test was performed on the sixth day. In this test session, the platform was removed from the pool and each rat was allowed to swim for 60 s in the maze. The number of crossing place of the platform and the time spent in the target quadrant were recorded.
2.5 Cell culture
PC12 or BV2 were cultured in Cells DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 5% CO2 incubator at 37°C. Aβ25–35 (0.06 μM) or LPS (200ng/ml) were added to cells for 6h. Then DMTHB was subsequently added to cells at final concentration of 20 μM for 24 h of incubation. For siRNA experiments, siRNA was added to BV-2 cells at final concentration of 100 nM before half hour of LPS addition. For experiment of IL-16, 10 ng IL-16 was added to BV-2 cells and PC12 cells at final concentration of 10 ng/ml.
2.6 pull-down assay and mass spectrometry
The streptavidin magnetic beads were incubated at 30 °C for nearly 30 min with biotinylated DMTHB which was synthesized in our lab. Then, the biotinylated DMTHB-coupled beads complex was incubated with brain tissue lysate at 4°C for nearly 12 h, and separated from the supernatant on at magnetic stand. Subsequently, the samples were sent to the protein mass spectrometry technology platform (Fudan University, Shanghai, China) for testing and analysis.
2.7 Histological examination and immunohistochemistry
The brain tissues were collected and fixed in 10% formalin, embedded in paraffin, cuted into 5μm section and were stained with hematoxylin and eosin (H&E), p-tau and Aβ in brain tissue was stained with immunohistochemistry detection (Servicebio Technology Co., Ltd., Wuhan, China). These stained sections were observed by a technician blinded to the experimental groups. Tissues were visualized in a Nikon Eclipse Ni-U microscope.
Total RNA was extracted from freshly isolated brain tissues and BV-12 macrophage cells using Trizol (10604ES60, Yeasen, China) following the kit’s instructions. Isolated RNA was reverse-transcribed into cDNA with HiFiScript cDNA Synthesis Kit (1708891 Bio-rad, USA). The mRNA levels of CD86, MHC II, CD11b, CD16 and GAPDH were examined by qRT-PCR. The mRNA expression levels were displayed as fold change over that of the internal-control GAPDH. The following primer pairs were used: CD86, 5’- ACG ATG GAC CCC AGA TGC ACC A-3’ and 5’- GCG TCT CCA CGG AAA CAG CA-3’, MHC II, 5’- ACA GGA ATT GTG TCC ACG GG -3’ and 5’- AAG GCC TGG GTC AGG GAT AA -3’, CD11b, 5’- GAG CAG CAC TGA GAT CCT GTT TAA -3’ and 5’- ATA CGA CTC CTG CCC TGG AA -3’, CD16, 5’- TGT GTG TCG TCG TAG ACG GT -3’ and 5’- TTC GCA CAT CAG TGT CAC CA -3’, GAPDH, 5’- AGG TCG GTG TGA ACG GAT TTG -3’ and 5’- TGT AGA CCA TGT AGT TGA GGT CA -3’,
2.9 Western blotting analysis
The mice brain tissue and BV-2 cells were washed three times with pre-cooled PBS. The total proteins of brain tissue were extracted using RIPA Lysis Buffer (20μL/mg tissue, Yeasen, China) following the manufacturer’s recommendations. Protein concentration was measured by BCA protein assay kit (B18020, Yeasen, China). Protein samples were separated by 10 % SDS-PAGE electrophoresis gels, then transferred to PVDF membranes, and incubated with 3% BSA for 90 minutes at room temperature. The membranes were washed in TBST buffer (0.1 % of Tween) for three times and incubated overnight at 4℃ with primary antibodies. IL‐1β (ab200478, 1:1000), GSK 3β (ab32391, 1:1000) and AKT (ab38449, 1:1000) were purchased from Abcam, Bcl-2 (15071, 1:1000), IL-6 (12912, 1:1000), P-PI3K (17366, 1:1000), PI3K (4257, 1:1000), P-AKT (4060, 1:1000) and P-GSK (5558, 1:1000) were purchased from Cell Signaling Technology, Bax (WL01637 1:1000), Iba-1 (WL02406, 1:1000), Tau (WL03184, 1000), P-Tau (WL03540, 1000) and TNF-α (WL01581, 1:1000) were purchased from Wan Lei Biological Technology Co., Ltd. (Shenyang, China). GAPDH (60004, 1:10000) was purchased from proteintech (Chicago, USA). IL-16 (DF6600, 1:1000) was purchased from affinity. Before incubated with the second antibodies, these membranes should be washed in TBST buffer three times and fifteen minutes each time. Then the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) for 2h at room temperature. Proteins were visualized with normal or enhanced chemiluminescence detection kit (Tanon, China) and analyzed with Gel-pro Analyzer.
2.10 Statistical analysis
All data were represented as mean ± standard error of the mean (SEM). Student’s t-test and one-way ANOVA were applied to compare difference between two groups and multiple groups. Analyses were implemented by Graph Pad prism software (version5.0).