Cell culture
BV2-immortalized murine microglial cells were obtained from the Cell Culture Center of the Chinese Academy of Medical Sciences (China), and cultured in DMEM in a humidified atmosphere of 5% CO2 at 37°C. The BV2 microglia were transfected with siRNA PSMC5 for 24 h, and were then treated with LPS (1.0 µg/ml) for 24 h in serum-free DMEM.
SiRNA PSMC5
The PSMC5 siRNA and its negative control sequence were synthesized by Shanghai Gemar Pharmaceutical Technology Co., Ltd. The site was targeted at 56. It was listed in Table 1.
Table 1
siRNA PSMC5 targeting sequence used
mus | siRNA PSMC5 |
56 | sense: 5ʹ-GCAGUGGACUCCGUCAAUATT-3ʹ |
antisense: 5ʹ-UAUUGACGGAGUCCACUGCTT-3ʹ |
Animals
11- to 12-week-old C57BL/6J mice (from Guangdong Medical Laboratory Animal Center), and TLR4−/− knockout mice (from Model Animal Research Center of Nanjing University) were handled in accordance with the guidelines of the Animal Ethics Committee of Jinan University. All mice were housed in a room maintained on a 12/12-h light/dark cycle. The room temperature was automatically maintained at 21–25°C with a relative humidity of 45%-65%. Chow and water were provided ad libitum.
Intracerebroventricular (i.c.v.) shRNA PSMC5 (lentiviruses encoding mouse shRNA PSMC5; constructed and produced by Obio Technology, Shanghai; 1 × 108 TU/mL) administration was performed using a microsyringe with the stereotaxic coordinates − 0.26 cm dorsal, -0.15 cm lateral, and − 0.02 cm anterior from bregma [20], and was commenced 7 days prior to i.p. injection of LPS (750g/kg). 100 µg/kg VIPER was injected intraperitoneally 2 h before LPS injection. After training, testing was performed every day (day 0 to day 7).
Two-dimensional gel electrophoresis (2-DGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)
Treated cells were collected and lysed, the supernatant was subjected to 2-DGE using an Amersham Biosciences IPGphor IEF System and Hoefer SE 600 (GE healthcare, Uppsala, Sweden) electrophoresis unit (13 cm), according to manufacturer’s instructions. After 2-DGE, the gels were subjected to silver nitrate staining and scanned with an Image Scanner (GE Healthcare, Uppsala, Sweden).
Only protein spots that were consistently different in at least three independent experiments (over two-fold up- or down-regulation) were considered significant for analysis by MALDI-TOF-MS. Molecular mass analysis of the tryptic peptides was performed with an ABI 4800 plus MALDI-TOF-TOF mass spectrometer (Applied Biosystems, Foster City, CA). Spectra were interpreted and processed using the Global Protein Server Workstation (V3.6, Applied Biosystems) with the internal MASCOT search engine (V2.1, Matrix Science, London, UK) to search MS and MS/MS data. MASCOT protein scores (based on combined MS and MS/MS spectra) of > 65 were considered statistically significant (p ≤ 0.05). The individual MS/MS spectrum with the best ion score (based on MS/MS spectra) that was statistically significant (p ≤ 0.05) was also accepted.
Real-Time Quantitative Polymerase Chain Reaction (PCR) Analysis
Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the instructions provided by the manufacturer. Complementary DNA was synthesized from 1 µg of total RNA with EvoScript Universal cDNA Master (Roche).The resulting cDNA was diluted and used for real-time reverse transcription PCR using a BIO-RAD PCR system. The primer sequences for the genes are listed in Table 2.
Table 2
IL-1β | 5ʹ-CTTCCTTGTGCAAGTGTCTG-3ʹ |
5ʹ-CAGGTCATTCTCATCACTGTC-3ʹ |
COX-2 | 5ʹ-GTGCTGGAAAAGGTTCTTCTACG-3ʹ |
5ʹ-GTGAACCCAGGTCCTCGCTTA-3ʹ |
TNF-α | 5ʹ-CCACCACGCTCTTCTGTCTAC-3ʹ |
5ʹ-ATCTGAGTGTGGGGTCTGG-3ʹ |
GAPDH | 5ʹ-TCACCACCATGGAGAAGGC-3ʹ |
5ʹ-GCTAAGCAGTTGGTGGTGCA-3ʹ |
Behavioral tests
The experimental flow chart is as Fig. 1.
Morris water maze (MWM) test
The MWM test is widely used to assess spatial learning and memory in rodents [21]. A circular pool (height: 35 cm, diameter: 120 cm) was filled with water rendered opaque with whole milk and maintained at 23 ± 2°C. An escape platform (height: 14 cm, diameter: 4–5 cm) was submerged in the pool 1 cm below the surface of the water in a specific position. Mice were released into the water facing the pool wall from one of four separate quadrants and were allowed to use visual tips around the pool to find the hidden platforms within 60 s. If a mouse failed to find the platform within 60 s, it was guided to the platform and allowed to stay there for 10 s. The escape latency and swimming pattern of each mouse were recorded. To assess memory consolidation, the probe trial was carried out on the 7th day. The platform was removed from the pool, and the mice were then placed into the water. The time spent in the target quadrant and the number of target-crossings were recorded.
Passive-avoidance test
The passive-avoidance test (PAT) is a common method for evaluating memory in mice [22]. The PAT, using a “step-through” apparatus (Cheng Du Technology & Market Co, LTD.) was divided by a retractable door into two compartments: a bright compartment and a dark compartment. When the mice entered the dark compartment, they immediately received an electric shock (39 V, 3-s duration). The latency to enter the dark compartment and the number of electrical shocks (error times) within 5 min were recorded. Mice were placed in the illuminated compartment facing away from the dark compartment during the training trials for the first 3 days. A retention test was conducted again 24 h later for 7 days.
Pole climbing test and Traction test
Mouse motor behavior is evaluated using the pole test and traction test [23]. For the pole test, we conducted 5 days of training on a rough-surfaced pole (1 cm diameter and 60 cm height). After training, the pole tests were performed 7 days after LPS injection. The time taken for mice to climb down was evaluated (reaching the first half and second half of the pole and total length of the pole). The following standards were used for scoring: crossing the three parts within 3 s was scored as 3 points, within 6 s was scored as 2 points, and more than 6 s was scored as 1 point. Results were expressed as total scores.
The traction test evaluates the muscle strength of mice [24]. For this test, mice were trained to hang from a horizontal wire by their forepaws and observed for 30s for 5 days before LPS injection. If the mouse used both hind paws to catch the wire, they scored 3 points. If they used only one hind paw, they scored 2 points. If they used their forepaws, they scored 1 point, and if they dropped from the wire, they scored 0 points.
Transmission electron microscopy (TEM)
Brain tissues were extracted in cold 4% paraformaldehyde and 2.5% glutaraldehyde solution, rinsed with 0.1M phosphoric acid rinse solution, and fixed with 1% osmium acid fixative for 2–3 h. After fixation, brain tissues were dehydrated with ethanol and acetone. Following dehydration and embedding, brain tissues were cut using an LKB-1 ultrathin slicing machine. Finally, the samples were imaged under the transmission electron microscope (JEM, Tokyo).
Hematoxylin and eosin (HE) staining
After 7 days of behavior tests, mice were killed and perfused using an ice-cold saline (0.9%) solution until the liver turned white. The brains were then fixed in 4% paraformaldehyde (PFA), washed 3 times with PBS for 1 h, gradient eluted using graded ethanol, and routinely processed for embedding in paraffin wax. Paraffin sections (5 µm) were then subjected to hematoxylin and eosin (HE) staining. The hippocampal tissue morphology was observed under a light microscope (Leica DMLS; Leica Microsystems Inc., Depew, New York, USA).
Immunofluorescence staining
Immunofluorescence analyses were carried out on 10 µm-thick brain slice sequential sections prepared on a microtome (Leica CM 1850; Leica Microsystems, Seoul, Korea). The brain sections were transferred to 30% sucrose solution and permeabilized with Triton X-100 (0.3% in TBST) at room temperature for 10 min followed by three washes in 0.025% TBST, and blocked with 1% bovine serum albumin (BSA) in TBST for 1 h. After blocking, the sections were incubated at 4°C overnight with primary antibodies against TNF-α (Abcam, Inc., Cambridge, MA, USA), chitinase 3 like protein 3 (YM-1; Abcam, Inc., Cambridge, MA, USA), or ionized calcium-binding adapter protein 1 (IBA1; Millipore Corp., Billerica, MA, USA). After washing three times with TBST, the sections were incubated with the appropriate TRIC-conjugated and DyLight 488- or 546-conjugated secondary antibodies (Invitrogen-Molecular Probes, Carlsbad, CA) at room temperature in the dark for 60 min. Nuclear staining was performed with a 4′, 6-diamidino-2- phenylindole (DAPI) staining solution for 10 min at room temperature. Finally, fluorescence images were obtained using fluorescence microscopy (Leica Microsystems, Wetzlar, Germany).
Griess reaction and ELISA assay
Nitrite is a stable oxidative product of NO and is indirectly determined by a Griess reaction. NO production was assessed by measuring nitrite levels in the cell supernatant or serum or brain and calculated by reference to a standard curve. For the ELISA assay, serum or brain tissue was collected after treatment. IL-1 and TNF- were measured using an ELISA kit from eBioscience (Vienna, Austria), and PGE2, IL-4, and IL-10 were measured using an ELISA kit from R&D Systems (Minneapolis, MN) according to manufacturers’ instructions.
Western blot analysis
Cells or brain tissue was lysed in ice-cold radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Beyotime Institute of Biotechnology, China) and centrifuged at 12,000g rpm for 20 min at 4°C. The supernatant was collected and quantified using a BCA kit (Beyotime Institute of Biotechnology) following the manufacturer’s instructions. Cytoplasmic and nuclear p65 detection was performed using a NE-PER® kit according to manufacturer’s instructions (Thermo Scientific, Rockford, IL, USA). Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto poly vinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk at room temperature, the membranes were incubated overnight at 4°C with primary antibodies against PSMC5, SYP, PSD95, COX-2, iNOS, IKK-α/β, phospho-IKK-α/β (p-IKK α/β), t-IκBα, phospho-IκBα (p-IκBα), MyD88, TRIF, TLR4, GAPDH, tubulin, and lamin-B1 (Cell Signaling Technology Inc, MA, USA), and then incubated with the secondary antibody for 1 h at room temperature. The results were quantified using scanning densitometry.
Immunoprecipitation
The BV2 microglia were washed twice with ice-cold PBS and incubated on ice for 30 min with 1 ml lysis buffer (50 mM Tris-Cl, 150 mM NaCl, 0.5 mM MgCl2, 10% glycerol, and 0.5% TritonX-100, pH 8) supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland). Cell lysates were centrifuged for 10 min at 12,000 g. The supernatant was preabsorbed for 1 h at 4°C with 50 ml of protein G agarose (Thermo Fisher Scientific). The recovered supernatant was incubated with PSMC5 antibody and TLR4 antibody (all from Santa Cruz Bio-technology) at 4°C overnight. 50 ml protein G agarose was added to the lysate-antibody mixture and incubated at 4°C on a roller for 2 h. Agarose beads were washed 4 times with lysis buffer and boiled in 30 ml of SDS-PAGE loading buffer. Samples were resolved on 10% SDS–polyacrylamide gels and subjected to western blot analysis.
Screening of amino acid binding sites by molecular docking and molecular simulation
Homologous models of protein TLR4 and protein PSMC5 were constructed using the software MOE 2015. 10: We used the Moe Homology Model module for homologous modeling, and Amber12 to optimize the structure of homologous models. The TLR4 and PSMC5 protein optimized homology models were used to investigate their interactions through the protein-protein docking function module in MOE 2015.10. In the functional module of MOE protein-protein interaction, the docking results were further analyzed and visualized. The sequences of protein TLR4 and protein PSMC5 were downloaded from GenBank. The template of homology modeling was identified by sequence comparison in the MOE-Search PDB module. The homology structures of TLR4 and PSMC5 proteins were constructed by software MOE 2015.10 and optimized by Molecular dynamics simulation (MD Simulation). In this study, Amber12 was used in a molecular dynamics simulation study. Adopt Amber FF03. The R1 force field uses the LEAP program to generate Molecular dynamics topology and parameter files. The TIP3P water model was used to fill the water molecules in the homologous model, which made the protein system solvable, and added ions to the system to make the system charge balance. The protein simulation system was optimized for energy, then slowly heated to 300 K, and then simulated for 300PS until the system reached thermodynamic equilibrium. Finally, the stable protein system was simulated by 10 ns long molecular dynamics, and the last 1 ns stable conformation was taken as protein-protein docking conformation.
GST-pull down
The PSMC5 and PSMC5 mutants with GST tag were transformed into E. coli Rosetta (DE3) competent cells (Millipore-Sigma), which were cultured at 37°C to an optical density at 600 nm of 0.5–1.0, followed by induction induced with 0.1 mM isopropyl b- D -1-thiogalactopyranoside for 16–18 h at 18°C. The bacteria were collected and crude bacterial lysates were prepared by sonication in lysis buffer (50 mM Tris-Cl, 150 mM NaCl, and 1% Triton X-100,1mM PMSF, pH8) in the presence of a protease inhibitor cocktail. Bacterial lysates were centrifuged for 20 min at 12,000 g and the supernatants were used for fusion-protein purification with glutathione S-transferase (GST)–bind beads (Millipore-Sigma) according to the manufacturer’s protocol. HeLa cells overexpressing TLR4 were washed twice with cold PBS, lysed in lysis buffer, shaken for 30 min on ice. The lysate was cleared by 10 min of centrifugation at 12,000 g rpm in a microcentrifuge. For pull-down, 10 mg GST or GST- PSMC5 and 30 ml GST-bind beads were incubated for 30 min on ice followed by washing 3 times with PBS. Cell lysates were then added and incubated at 4°C overnight. The beads were washed 3 times with lysis buffer, resuspended in 23 SDS-PAGE loading buffer at 98°C for 5 min, and resolved on 10% SDS–polyacrylamide gels for western blotting.
Statistical analysis
Data were analyzed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and presented as the mean ± standard error of the mean (S.E.M.). Comparisons between two groups were made using Student’s t-test. Comparisons among multiple groups were made using one-way ANOVA followed by Bonferroni post hoc pairwise comparisons. Repeated-measures ANOVA with Bonferroni post hoc test was used to analyze latencies in the MWM. Differences were deemed statistically significant if P < 0.05.