2.1 Cell culture
BV-2 microglial cells (CL-0493) (Procell Life Science & Technology Co., Ltd., Wuhan, China) and HT22 cells (337709) (BeNa Culture Collection, Beijing, China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Procell Life Science & Technology Co., Ltd.), 1% 100 µg/mL streptomycin and 100 units/mL penicillin (Gibco, Grand Island, NY, USA) and maintained at 37 °C in a 5% humidified CO2 incubator (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
2.2 Cell viability and apoptosis assay
BV-2 cells seeded into 96-well plates were pretreated with 1 µM and 2.5 µM FTS•B or phosphate buffered saline (PBS) for 3 h, and subsequently co-incubated with 1 µg/mL LPS (Sigma-Aldrich, St. Louis, MO, USA) (dissolved in DMEM) for 24 h. The cell viability was analyzed using 3-(4, 5-dimethyl-2-thiazolyl) 2, 5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma-Aldrich, USA) assay the same as our previous study [22].
In a separate experiment, BV-2 cells were pre-incubated with 1 µM and 2.5 µM FTS•B or PBS at 37 °C for 3 h following with 24-h co-incubation with 1 µg/mL LPS, and then cultured mediated were collected. HT22 cells seeded into 12-well plates were exposed to the collected medium for another 24 h. MTT assay was applied to analyze the HT22 cell viability, and Annexin V/propidium iodide (AV/PI) staining was applied to analyze the HT22 cell apoptosis the same as our previous study [23].
2.3 Experiments performed on AD mice and agent administration protocol
The experimental protocol was carried out under the Ethical Committee of Animal Research of Jilin University (20170301). Twenty-four B6C3-Tg (APPswePSEN1dE9)/Nju double transgenic male mice (Genotype: (Appswe)T, (Psen1) T) (APP/PS1) (8 months, 40–45 g) and eight wild type male mice (Genotype: (Appswe)W, (Psen1) W) (WT) (8 months, 40–45 g) were purchased from Nanjing Biomedical Research Institute of Nanjing University (NBRI), Jiangsu, China (SCXK (SU) 2015-0001), which were maintained at standard conditions (temperature 23 ± 1 °C and humidity 40–60%) with a 12 h light/dark cycle and food available ad libitum.
After a week of adjustable feeding, APP/PS1 mice were randomly divided into three groups (n = 8/gourp) and orally administered with 10 mg/kg and 40 mg/kg FTS•B and 5 mL/kg normal saline (model group) once a day for 4 weeks. A total of 8 WT mice orally administered with 5 mL/kg normal saline for 4 weeks served as the control group. After the last behavioral test, the mice were euthanized (by injection with 150 mg/kg 1.5% pentobarbital). The serum together with tissues including brain, liver, spleen and kidney were collected for biochemical and pathological analysis. The process of the drug administration and behavioral tests was shown in Figure S2.
2.4 Behavioral tests
2.4.1 Morris water maze test
Brain function related to spatial learning and memory of AD mice was assessed by Morris water maze (MT-200, Chengdu, China) through Water Labyrinth Video Tracking Analysis System (S7200, Chengdu Techman Software Co., Ltd., Chengdu, China). A circular pool filled with water containing milk with a platform placed 1 cm below the surface of water was used for training and experiment at 22–24 °C. On the 29th day, mice were trained 5 minutes/day for 4 days. On the 33rd day, mice were placed to the same quadrant of the pool, the escape latency of mice to locate the hidden platform was recorded within 60 s.
2.4.2 Y maze test
The spontaneous alternation test was assessed in Y maze with three symmetrical arms (300 × 200 mm) at 120° angles. The movements of mice were recorded by the video camera mounted above the maze. On the 34th day, mice were put into one distal end of arm to find the food on the other distal end of arm for 5 minutes (set as the maximum time by system). On the 35th day, the time for finding food and the movement locus of mice were recorded by the video camera.
2.4.3 Open field experiment Test
Open field experiment test was performed in a soundproof dark box (bottom area: 30 × 30 cm, central region: 15 × 15 cm) to observe autonomous behaviors of mice in the new environment. On the 36th day, mice were placed in fixed central location and allowed to start free environmental exploration for 5 min. The moving track and the latency time of mice in the center and surrounding areas were recorded by the instrument. The box was wiped off after completing the experiment to avoid the remaining odors and dirt interfere with the results of next test.
2.5 Biochemical criteria detection
BV-2 cells were pretreated with FTS•B at doses of 1 µM and 2.5 µM for 3 h, and then co-exposed to 1 µg/mL LPS for another 24 h. The levels of IL-6 (KT2163-A), tumor necrosis factor-alpha (TNF-α) (KT2132-A), inducible nitric oxide synthase (iNOS) (KT2454-A), nitric oxide (NO) (MM-0658M1) and IL-1β (KT2040-A) in the supernatant were measured by enzyme-linked immunosorbent assay (ELISA) kits (Jiangsu Kete Biotechnology Co., Ltd, Jiangsu, China) according to the manufacturer instructions.
Hypothalamus obtained from APP/PS1 mice were homogenized in ice-cold PBS, and the protein concentration was detected via the Pierce™ BCA protein assay kit (23227) (Thermo Fisher Scientific, USA). The levels of toll-like receptor 3 (TLR3; cat. no. KT9375-A), interferon regulating factor 3 (IRF3) (KT9374-A), p-IRF3 (MM-50143M1), interferon-beta (IFN-β) (KT2124-A), JNK-interacting protein 3 (JIP3) (KT9377-A), C-Jun NH2-terminal kinase (JNK) (KT9182-A), p-JNK (KT9372-A), APP (KT2824-A), p-APP (KT9378-A), Aβ (KT9256-A), TNF-α (KT2132-A), IL-1β (KT2040-A), IL-6 (KT2163-A), interleukin-8 (IL-8) (KT2123-A) and interleukin-12 (IL-12) (KT2105-A) in the serum and hypothalamus were detected by ELISA kits according to the manufacturer instructions (Jiangsu Kete Biotechnology Co., Ltd, Jiangsu, China).
2.6 Hematoxylin and eosin (H&E) staining, Thioflavin S staining and Immunohistochemistry examination
The same as our previous study [24], 4% paraformaldehyde-fixed liver, spleen, kidney and brain tissues were dehydrated with alcohol, embedded with paraffin and cut into 5-µm thick standard sections. Specimens were stained with hematoxylin and eosin (H&E) and assessed using inverted microscope CKX41 (Olympus, Japan).
Thioflavin S staining was used to visualize fibrillar Aβ deposits. After permeabilization in xylene for 10 min and rehydration in anhydrous ethanol for 5 min, brain slices were incubated with 1% thioflavin S in dark for 30 min, then differentiated in 70% ethanol and washed with distilled water. After fixing with 50% glycerin in PBS, slices were observed using fluorescent microscope (magnification × 100) (Olympus Corporation, Tokyo, Japan).
The brain sections were prepared as our previous study [25]. The sections were incubated with the primary antibody against Aβ (Dilution ratio 1:1000) (ab2539), p-Tau (phospho S396) (Dilution ratio 1:4000) (ab109390) and 4-hydroxynonenal (4-HNE) (Dilution ratio 1:200) (ab46545) (Abcam, Cambridge, MA, USA), glial fibrillary acidic protein (GFAP) (Dilution ratio 1:200) (bs-0199R) and ionized calcium binding adapter molecule 1 (Iba1) (Dilution ratio 1:200) (bs-1363R) (Bioss Inc., Beijing, China) at 4 °C overnight. The following day, sections were washed with PBS, incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (sc-3836) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 25 °C for 1 h, and incubated with streptavidin-organism horseradish peroxidase (HRP) complex (Shanghai BestBio Science, Shanghai, China) 25 °C for another 1 h. 5% diaminobenzidine tetrahydrochloride solution and hematoxylin were added to slices to counterstain (25 °C for 5 min). Photomicrographs were obtained via inverted fluorescent microscope (magnification × 40 and × 200) (Olympus Corporation, Tokyo, Japan).
2.7 Proteomics
2.7.1 Protein extraction and digestion
Samples were homogenized in radio immunoprecipitation assay (RIPA) buffer containing 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and 2% phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, St. Louis, MO, USA) on ice for 30 min. After centrifugation, BCA protein assay kit was used to quantify protein concentrations of the supernate. Subsequent to acetone precipitation, re-suspend protein for tryptic digest, and cleaning up of sodium deoxycholate (SDC), peptide samples were obtained. Peptides were desalted by reversed phase chromatography (RPC-HPLC) using C18 column. After desalination, peptides were dried and resuspended in buffer containing 0.1% formic acid (FA) and 2% acetonitrile (ACN).
2.7.2 Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)
1 µg of peptide were separated and analyzed via an Easy-nLC1200 nano-UPLC (Thermo Scientific,Waltham, MA) connected to a Q-Exactive mass spectrometry (Thermo Scientific, Waltham, MA). Separation was performed using a reversed phase column (100 µm, ID × 15 cm, Reprosil-Pur 120 C18-AQ, 1.9 µm, Dr. Math). A non-linear gradient was established using phase A (0.1% FA and 2% ACN in water) and phase B (0.1% FA and 80% ACN in water) to elute the peptides with a 120 min gradient at 300 nL/min flow rate. The gradient started with 8% phase B and increased to 35% during 92 min. In the next 20 min, phase B increased to 45% and finally, it augmented to 100% in 2 min and it was kept at 100% for 2 min. Data dependent acquisition was performed in profile and positive mode with Orbitrap analyzer at a resolution of 70,000 (@200 m/z) and m/z range of 350–1600 for MS1; For MS2, the resolution was set to 17,500 with a dynamic first mass. The automatic gain control (AGC) target for MS1 was set to 3.0 E+ 6 with max IT 50 ms, and for MS2 was set to 5.0 E+ 4 with max IT 100 ms. The top 20 most intense ions were fragmented by Higher Energy Collisional Dissociation (HCD) with normalized collision energy (NCE) of 27%, and isolation window of 2 m/z. The dynamic exclusion time window was 30 s.
2.7.3 MaxQuant analysis and LFQ quantification
Raw MS files generated on the mass spectrometer were processed with MaxQuant (Version 1.5.6.0). The protein sequence database (Uniprot_organism_2016_09) was downloaded from Uniprot. This database and its reverse decoy were then searched against via MaxQuant software. The quantification type was label-free quantification (LFQ) with match between run and intensity-based absolute quantification (iBAQ); Trypsin was set as specific enzyme with up to 3 miss cleavage; Oxidation [M] and Acetyl [protein N-term] were considered as variable modification (max number of modifications per peptide is 3), Carbamidomethyl [C] was set as fixed modification; Both peptide and protein FDR should be less than 0.01. Only unique and razor peptides were used for quantification. All the other parameters were reserved as default.
2.7.4 Bioinformatics and statistical analysis
Statistical analysis was performed on the standardized quantitative results to obtain the corresponding differentially expressed proteins. The experiment contained two biological replicates, and the arithmetic mean was used to calculate the fold change. The proteins with fold change expression (ratio A/B > 1.5 or ratio A/B < 0.66) were defined as significant differences, and subsequent GO, KEGG pathway, and protein interaction analysis were performed.
2.8 Western Blot
One part of the hippocampus tissues obtained from the experimental mice were lysed with RIPA buffer containing 1% protease inhibitor cocktail and 2% PMSF. BCA protein assay kit was used to quantify protein concentration. Subsequent to the separation of 40 µg lysates by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred to polyvinylidene difluoride membrane (PVDF) (0.45 µm; Merck Millipore, Billerica, MA, USA). 5% bovine serum albumin (BSA) was used to block the membranes at 4 °C for 2 h. The membranes subsequently incubated with primary antibodies including JIP3 (Dilution ratio 1:500) (147 kDa) (bs-13648R), JNK (Dilution ratio 1:1000) (48 kDa) (bs-2592R), Amyloid Precursor Protein (APP) (Dilution ratio 1:1000) (86 kDa) (bs-0112R), inhibitor of nuclear factor kappa-B kinase alpha + beta (IKKα + β) (Dilution ratio 1:500) (85 kDa) (bs-7557R), Interferon regulatory Factor 3 (IRF3) (Dilution ratio 1:1000) (47 kDa) (bs-52116R), p-IRF3 (Ser396) (Dilution ratio 1:500) (47 kDa) (bs-3195R), Iba1 (Dilution ratio 1:500) (16 kDa) (bs-1363R) and GFAP (Dilution ratio 1:500) (48 kDa) (bs-0199R) (Bioss Inc., Beijing, China), p-JNK1 + 2 + 3 (phosphor Y185 + Y185 + Y223) (Dilution ratio 1:10000) (48 kDa) (ab76572), p-IKK(α + β) (phosphor S176 + S177) (Dilution ratio 1:500) (85 kDa) (ab194528), p-APP (phosphor T743) (Dilution ratio 1:1000) (86 kDa) (ab206297), ELKS (Dilution ratio 1:5000) (128 kDa) (ab180507), inhibitor of nuclear factor kappa-B alpha (IκBα) (Dilution ratio 1:10000) (35 kDa) (ab32518), p-IκBα (phosphor S36) (Dilution ratio 1:10000) (35 kDa) (ab133462), nuclear factor kappa-B p65 (NF-κB p65) (Dilution ratio 1:500) (65 kDa) (ab7970), and p-P65NF-κB (phosphor S536) (Dilution ratio 1:10000) (65 kDa) (ab76302), WD-repeat and FYVE-domain-containing protein 1 (WDFY1) (Dilution ratio 1:1000) (46 kDa) (ab125329), and Toll-like receptor 3 (TLR3) (Dilution ratio 1:500) (108 kDa) (ab13915), and the reference protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Dilution ratio 1:500) (36 kDa) (ab181602) (Abcam, Cambridge, MA, USA) at 4 °C overnight. After washing with TBST (composes of 500 ml t-butyldimethylsilyl and 0.5 ml Tween-20), the membranes then incubated with HRP-conjugated secondary antibody (SH-0032; Bejing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) at a dilution of 1:2000 at 4 °C for 4 h. An enhanced chemiluminescence (ECL) kit (Merck Millipore, Billerica, MA) and imaging system (Bio Spectrum 600, UVP company, Upland, CA, USA) were used to visualize bands, and ImageJ software (National Institutes of Health, Bethesda, MD, USA) was applied to quantify the blots.
2.9 Statistical Analysis
All data were presented as the mean ± S.D. One-way analysis of variance (ANOVA) followed by post-hoc multiple comparisons (Holm-Sidak test) was used to analyze differences and significance via SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). The statistically significant difference was declared for p < 0.05.