All the cell culture reagents were purchased from Gibco (USA). CCS, LPS, Aβ1-42 were purchased from Sigma-Aldrich (USA). The IFNγ was purchased from Novus Biologicals (USA). The β-NGF recombinant protein was purchased from DAKEWE (China). The KG501, as a CREB inhibitor, was purchased from MedChemExpress (USA). The anti-IL-1β, TNF-α, iNOS, IL-10, TGF-β, Arg1, CREB, pCREB, NGF, β-Gal, GAP43, Syn, MAP2, NeuN, β-actin, β-tubulin, and GAPDH antibodies were obtained from Cell Signaling Technology (USA). The anti-Iba-1, NGF antibodies, and anti-NGF neutralizing antibodies were obtained from Abcam (USA). The anti-CD86 and CD206 antibodies were obtained from BD (USA). The HRP conjugated Rabbit anti-Goat IgG, Goat anti-Rabbit IgG, Goat anti-Mouse IgG, the FITC conjugated Donkey anti-Rabbit IgG, and the TRITC conjugated Donkey anti-Goat IgG secondary antibodies were purchased from EarthOx Life Science (USA). The plasmids of CREBcDNA, NGF wild type, and NGF mutated type were obtained from Genewiz (China). The primer of IL-1β, TNF-α, iNOS, IL-10, TGF-β, Arg1, NGF, and SanPrep Column Plasmid DNA Extraction kit were obtained from Sangon Biotechnology (China). The Reverse Transcription Master Mix was purchased from TOYOBO (Japan). The TB Green Realtime PCR Master Mix was purchased from TaKaRa (Japan). The polyvinylidene difluoride membrane and enhanced chemiluminescent kit (ECL+) were purchased from Millipore (Germany). The Luciferase Report kit was obtained from Promega (USA). The RIPA buffer, DAPI, BCA, the Chromatin Immunoprecipitation, the Senescence-associated β- Galactosidase Staining, and the Annexin Ⅴ-FITC/Propidium Iodide (PI) Apoptosis kits were purchased from Beyotime Biotechnology (China). The CCK8 kit was purchased from Dojindo (Japan). The IL-1β, IL-10, and NGF activity assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (China). All the other reagents were of analytical grade. All supplies are purchased from Jet (China).
Cell Line and Treatment
Mouse microglial cell line BV2 and rat pheochromocytoma cell line PC-12 was purchased from the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences (China). BV2 cells were grown in Dulbecco’s modified Eagle's media (DMEM), supplemented with 10% Fetal Bovine Serum (FBS) and 100 U mL-1 of penicillin-streptomycin in a 37°C, 5% CO2 incubator. And PC-12 cells were grown in Dulbecco’s modified Eagle's media (DMEM), supplemented with 5% Fetal Bovine Serum (FBS), 10% horse serum (HS), and 100 U mL-1 of penicillin-streptomycin in a 37°C, 5% CO2 incubator. And 293T cells were grown in Dulbecco’s modified Eagle's media (DMEM), supplemented with 10% Fetal Bovine Serum (FBS) and 100 U mL-1 of penicillin-streptomycin in a 37°C, 5% CO2 incubator. Cells at passage 5–20 were used for the experiments. The Aβ1-42 peptide was used after 7 days of incubation at 37°C.
CCS stock solution was prepared in sterile double distilled water at a final concentration of 1 mg·mL-1, and then aseptically dispensed and immediately stored at -20 °C. 24 h after plating the BV2 cells, CCS stock solution was added to the media to a final concentration of 0.1 μg·mL-1, 0.5 μg·mL-1, 1 μg·mL-1 for 1 h in the CCS group. Then, the cells were treated with LPS (1 μg·mL-1) for 1 h, and IFNγ (20 ng·mL-1) for 6 h both in the LPS+IFNγ group and the CCS group, the cells in the control group were treated with cell culture media at the same volume.
BV2 cells were plated in 6-well plates for 24 h. And then, BV2 cells were treated with CCS (1 μg·mL-1), LPS (1 μg·mL-1), IFNγ (20 ng·mL-1), rNGF (50 ng·mL-1) or Neutralizing anti-NGF (0.05 μL·mL-1) in different groups for 24 h. After treating BV2 cells for 24 h, cells were replaced with a complete medium for 24 h. The supernatant of the BV2 cells is used as a conditioned medium for PC-12 cells. Synchronously, PC-12 cells were treated with rNGF (50 ng·mL-1) in 6-well plates for 48 h so that PC-12 cells would be induced into PC-12 neuronal cells. Next, PC-12 neuronal cells as neurons were treated with or without Aβ1-42 for 24 h. Finally, the supernatant of BV2 cells was cultured into PC-12 neuronal cells with or without Aβ1-42 as a conditioned medium and cultured for 24 h to detect.
BV2 cells were plated in 6-well plates for 24 h. After 24 h, transient transfection of plasmids into BV2 cells was performed using Lipofectamine 3000 transfection reagent (Invitrogen by Thermo Fisher Sci.) according to the manufacturer’s instructions. Next, the transfection medium was placed in the corresponding group for 24 h and then tested.
Animals and Treatments
The original APP/PS1 double-transgenic mice were obtained from the Jackson Laboratory [B6. Cg-Tg (APPswe, PSEN1dE9) 85Dbo/J]. The C57BL/6 mice were obtained from the Laboratory Animal Center of China Medical University. All animal care and experimental procedures were in line with the Laboratory Animal Ethical Standards of China Medical University and the Standard Medical Laboratory Animals’ Care and Use Protocols.
9-month-old double-transgenic APP/PS1 mice were randomly assigned into two groups: APP/PS1 group (n=8) and CCS group (n=8). Age-matched C56BL/6 littermates were assigned as Wild Type (WT) group (n=8). And we must make sure that each group is half male. CCS (10 mg·kg-1·day-1) was intragastrically received in the CCS group; simultaneously, the same volume of distilled water was intragastrically administered in APP/PS1 group and WT group once daily for 4 weeks.
Morris Water Maze
Mice were given Morris water maze (MWM) for consecutive 6 days, including navigation tests and a probe trial test, as previously described with a few modifications. The Morris water maze is a stainless-steel circular water tank (120 cm diameter × 50 cm height) equipped with a platform (10 cm diameter) placed in the second quadrant and submerged 0.5 – 1 cm below the surface of the water. In brief, mice were allowed to swim freely for 1 min without the platform to adjust themselves to the baseline day’s circumstances (day 0). From the 1st day to the 5th day, the platform was under the water in the tank for navigation tests, and each mouse was subjected to 4 trials per day at an inter-trial interval (ITI) of 1 min for spatial acquisition. Different start locations were used on each trial. If a mouse failed to find the platform within 1 min, it would be picked up and placed on the platform for 1 min. For each trial, the latency and the path length by which the mouse found the hidden platform was recorded. On the 6th day, a probe trial was performed to assess memory consolidation. In the trial, the platform was removed from the tank, and the mice were allowed to swim freely for 1 min to find the place of the original platform. Latency that each mouse spent in the quadrant and the frequency that each mouse crossed the center of the quadrant (where the platform was previously located) were recorded. All the data were obtained by a video tracking system (Chengdu Taimeng Tech. Co. LTD, Chengdu, PR China).
Passive Avoidance test
Mice were subject to the passive avoidance test (PAT) using a PAT apparatus (BA-200, Chengdu Taimeng Tech. Co. LTD, Chengdu, PR China). In the training session, each mouse was put into the light compartment and allowed to explore for 3 min, at which point the guillotine door was raised to allow the mouse to enter the dark compartment. When the mouse entered the dark compartment, an electrical foot shock (voltage 36 V, 5 min) was delivered to make the mouse return to the light compartment. The training session was conducted before the test session. The test session was performed 24 h after the training session. In the test session, each mouse was placed in the light compartment and allowed to explore for 3 min, and then the guillotine door was raised. The latencies (s) and frequencies for mice to enter the dark compartment were recorded during the entire testing period (10 min). The latency of the mice that did not enter the dark compartment within 10 min was recorded as 600 s.
Step-down Avoidance test
The step-down avoidance test was employed to measure the retention of memory. A mouse was placed in a cylindrical insulation platform in a reaction box to adapt to the surrounding environment for 3 min. When the mouse stepped down from the platform, it would immediately receive an electric stimulation (voltage 30 V). In the 5 min training session, the mice would jump on the platform to prevent stimulation, and some mice might jump repeatedly. 24 h after training, the latency period (stepping down from the platform for the first time) and the number of errors (the frequency of jumping off the platform) were recorded.
The locomotivity test was conducted after other behavioral tests using a locomotivity testing paradigm (ZZ-6 system for mice, Chengdu Taimeng Tech. Co. LTD, Chengdu, PR, China). Briefly, mice were placed in the system, and the exploration was assessed for 5 min. Cages were routinely cleaned with ethanol following each session. The locomotivity and the frequency of stand-up for each mouse were recorded.
Transmission Electron Microscope (TEM)
After the animals’ behavioral tests, the hippocampus tissues were separated immediately, and immersion fixation was completed at around 1 mm3. Samples were rinsed in cold phosphate-buffered saline (PBS) and placed in 2.5% glutaraldehyde at 4 °C for 2 h. The tissues were rinsed in buffer and post-fixed with 1% osmium tetroxide for 2 h. The tissues were then rinsed with distilled water before undergoing a graded ethanol dehydration series and were infiltrated using a mixture of half acetone and half resin overnight at 4 °C. The tissues were embedded 24 h later in resin and cured fully, in turn, as follows: 37°C overnight, 45 °C for 12 h, and 60 °C for 24 h. After that, 70-nm sections were cut and stained with 3% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Ultrastructure changes of synapses in the hippocampus were observed under TEM (HITACHI H-7650).
Quantitative real-time PCR and RT-PCR
Total RNA from BV2 cells was extracted with TRIzon Reagent. RNA was reverse-transcribed to complementary DNA (cDNA) using the RT-PCR Quick Master Mix. And quantitative real-time PCR was performed according to the manufacturer’s directions. After amplification, the PCR products were resolved by 2% agarose gel electrophoresis. For murine BV2 cells, primer sequences were listed as follows. The GAPDH gene was used as an internal control for IL-1β, TNF-α, iNOS, IL-10, TGF-β, Arg1, and NGF mRNA expressions analysis. And the results were analyzed using the 2-ΔΔCT method for quantification.
GAPDH, forward: 5’-AGCCTCGTCCCGTAGACAAAA-3’,
IL-1β, forward: 5’-CCTGCAGCTGGAGAGTGTGGAT-3’,
TNF-α, forward: 5’-AGCCCACGTCGTAGCAAACCAC-3’,
iNOS, forward: 5’-CCTTGGTGAAGGGACTGAGC-3’,
IL-10, forward: 5’-GGTTGCCAAGCCTTATCGGAA-3’,
TGF-β, forward: 5’-GCAACAATTCCTGGCGTTACCTTG-3’,
Arg1, forward: 5’-TGGGAATCTGCATGGGCAAC-3’,
NGF, forward: 5’-GCAGTGAGGTGCATAGCGTAA-3’,
BV2 cells, PC-12 cells, and the frozen hippocampus were homogenized, and the proteins were extracted and quantified with a BCA kit. For Western Blotting, the protein extracts of cells or tissues were separated on SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. Next, the membrane was incubated with TBS containing 0.1% Tween-20 (TBST) with 5% bovine serum albumin for 1 h and then probed overnight at 4 °C with the following primary antibodies at the appropriate dilution: IL-1β, TNF-α, iNOS, IL-10, TGF-β, Arg1, NGF, pCREB, CREB, MAP2, NeuN, Syn, GAP43, β-Gal, β-tubulin, β-actin, GAPDH. After washing with TBST, the membranes were incubated with the corresponding secondary HRP-labeled antibody for 1 h at room temperature. After another TBST washing, the immunoreactive bands were visualized using the ECL+ kit. The immunoblots were quantified by measuring the density of each band with Image-J Software. The target protein expression was normalized to β-tubulin as a protein loading control.
BV2 cells or PC-12 cells were fixed for 2 h at room temperature using 4% paraformaldehyde. After washing with PBS, cells needed to be permeabilized with 0.5% Triton X-100 for 10 min. Then, the frozen-sectioned brains and the permeabilized cells were washed with PBS and incubated with anti-Iba-1 or MAP2 antibody overnight in a wet chamber at 4 °C. Afterward, the brain tissues and the cells were washed with PBS, incubated with FITC-conjugated Donkey anti-Rabbit IgG in the dark for 1 h at 37°C. Next, the frozen-sectioned brains and the permeabilized cells were washed with PBS and incubated with anti-CD86, CD206, CREB, NGF, or β-Gal, GAP43 antibody overnight in a wet chamber at 4 °C. The brain tissues and the cells were washed with PBS, incubated with TRITC-conjugated Donkey anti-Rabbit IgG in the dark for 1 h at 37°C, followed by incubation with DAPI for 10 min at room temperature. Finally, immunofluorescence images were acquired by Confocal Laser Scanning Microscopy (C2, Nikon, Japan).
Enzyme-linked Immunoabsorbent Assay (ELISA)
The supernatants of BV2 cells were harvested. The blood of mice was collected from the eyeballs and centrifuged to collect serum. According to the manufacturer's protocol, the NGF, IL-1β, and IL-10 protein concentrations in BV2 cells and serum were determined by ELISA. Briefly, samples were assayed at 450 nm, and the concentrations were calculated from respective standard curves.
Flow Cytometric Analysis
BV2 cells were harvested by centrifuging after washing the BV2 cells with cold PBS. Next, BV2 cells were stained with anti-mouse CD86-FITC or anti-mouse CD206-APC followed standard protocols and manufacturer’s instructions. Finally, the stained cells were immediately analyzed by flow cytometry (BD, USA).
PC-12 cells were harvested and washed with cold PBS, followed by the addition of 195 μL binding buffers. Then incubate the cells with 5 μL Annexin V-FITC for 10 min and 10 μL PI for 5 min at room temperature in the dark. Finally, the stained cells were immediately analyzed by flow cytometry (BD, USA).
PC-12 cells with or without Aβ1-42 were cultured with the supernatant of different groups of BV2 cells in 96-well plates. Then, 10 μL CCK-8 reagents per well were added to the cells at 37°C for 2 h, and the absorbance at 450 nm was measured to calculate the cell viability using a multi-mode reader (LD942, Beijing, China).
Luciferase Reporter Assay
Plasmids of pcDNA3.1, pGL3, and CREBcDNA were constructed by Genewiz. The plasmid of the NGF promoter region containing five predicted sites to bind CREB would be constructed into WT and MUT plasmids. 293T cells and BV2 cells were seeded in 96-well plates. Then, according to the following group, to transfect the cells: pcDNA3.1 + pGL3, CREBcDNA + pGL3, CREBcDNA + NGF WT, CREBcDNA + NGF MUT. According to the manufacturer, transient transfection of plasmids into 293T cells and BV2 cells was performed using Lipofectamine 3000 transfection reagent’s instructions (Invitrogen by Thermo Fisher Sci.). After 24 h post-transfection, cells were washed in PBS and lysed, and luminescence was measured following instructions of Nano-Glo ® Reporter Assay System Dual-Luciferase (Promega, USA).
Chromatin Immunoprecipitation (ChIP) Assay
ChIP assays were carried out according to the manufacturer’s protocol (P2078, Beyotime Co.) with slight modifications. Chromatin solutions were sonicated and incubated with anti-CREB or with control IgG and rotated overnight at 4 °C. DNA-protein cross-links were reversed, and chromatin DNA was purified and subjected to PCR analysis. Forward and reverse primers were designed to amplify the NGF promoter region containing one predicted site to bind CREB to five predicted sites. After amplification, PCR products were resolved on a 2% agarose gel and visualized by ethidium bromide staining.
Senescence-associated β- Galactosidase Staining (SA-β-Gal)
Briefly, PC-12 cells in conditional culture were cultured in 6-well plates for 24 h, fixed with the fixative solution. Then senescence-associated β-galactosidase (SA-β-Gal) staining was performed according to the SA-β-Gal kit’s instructions (Beyotime) for 12 h. The cells were then photographed under a microscope for the qualitative detection of SA-β-Gal activity.
Microarray datasets GSE48350 were downloaded from Gene Expression Omnibus and collected using the following platform: GPL570 [HG-U133 Plus 2]. The raw data was converted to a recognizable format by GEO2R. GEO2R performs comparisons on original submitter-supplied processed data tables using the GEOquery package, which parses GEO data into R data structures used by other R packages and limma (Linear Models for Microarray Analysis) R package from the Bioconductor project. Each chip group was compared with AD and NC, respectively. Difference analysis was performed by GEO2R analysis with P < 0.05 and log |FC| > 1 as the cutoff value for screening differentially expressed genes (DEGs). We used the gplots R package to draw the heat map. Next, we calculated the Pearson correlation coefficients of related genes to calculate whether they were linearly related.
All data were presented as mean ± standard deviation (SD). Differences between groups were assessed by one-way ANOVA, followed by Turkey’s test using SPSS22.0. All assays were performed in triplicate. P < 0.05 was considered statistically significant.