Cell Culture and Treatment
CHME-5 cells were cultured in a 25-cm2 culture flask with High Glucose DMEM (Gibco/Thermo-Fisher, Waltham, MA, USA) supplemented with 5% fetal bovine serum (Hyclone/GE, Logan, UT). at 37 °C in a humidified atmosphere containing 5% CO2. cells were pretreated with 20 nM rapamycin (Sigma, USA) for 1 h and then co-treated with HIV-1 gp120V3loop (Shanghai, China; sequence: NNTRKSIRIQRGPGRAFVTIGKIG; molecular formula:C114H199N41O31;molecular weight: 2640.06, 1 μg/mL) another 24 h, the RP group were pretreated with random peptide (Shanghai, China; sequence: KCSEYKKWIDLKKSEYKVDKYKK; molecular weight: 3066.64 kD, 1 μg/mL)
Western Blot Analysis
The cells were treated according to the study design and harvested, The brain samples were suspended in buffer solution and then centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was separated and collected for protein analysis. Protein was extracted using a protein extraction kit (Beyotime, China), and the concentration was determined using a Bio-Rad Protein Assay. The cytosolic and nuclear fractions were separated using a nuclear and cytoplasmic protein extraction kit, according to the manufacturer’s instructions (WLA020a, Wanleibio Co., Ltd. Shenyang China). SDS–polyacrylamide gels were used for separation via electrophoresis and then transferred to polyvinylidene fluoride membranes at 120 V for 30 min and 80 V for 2 h. The transferred membranes were blocked using fresh 5% nonfat dry milk dissolved in Tris-buffered saline Tween-20 (TBST) at room temperature for 1 h. The immunoblots were then probed with the appropriate antibodies. Primary antibodies used were: anti-GAPDH (5174s), anti-p62 (23214s), beclin-1 (3495s), anti-LC3 (3868s) anti-Keap1 (4678s) were purchased from Cell Signaling Technology, and anti-Nrf2 (WL02135, 1:1000, Wanleibio, Shenyang, China), anti-Lamin B (WL01775,1:1000, Wanleibio, Shenyang, China), anti-HO-1 (ab223349, 1:1000, Abcam), anti-iNOS (ab178945, 1:1000, Abcam). Each primary antibody was individually incubated with the membrane at 4°C for 12 h. Following incubation with primary antibodies and washing three times with TBST for 10 min each, the membranes were incubated for 1 h with the appropriate secondary antibody (diluted 1:5000 in TBST, 5% skim milk) and subsequently washed an additional three times with TBST for 10 min each. The detection of specific proteins was performed with an enhanced chemiluminescent detection system. The bands were quantified by densitometry using Image-ProPlus 6.0.
TNF-a, MCP-1 and IL-1β kits (USCN Business Co., Ltd, China) were used to determine the cytokine levels in mouse serum according to the manufacturer's instructions.
RFP‑GFP‑LC3 Adenovirus Transfection
To analyse autophagic flux, CHME-5 cells were transfected with mRFP-GFP-LC3-expressing adenovirus (Genechem Co.,Ltd. China), according to the manufacturer’s instructions. The procedure was performed as follows: the CHME-5 cells were plated on a confocal dish and treated with 250 μL of infection enhancement medium containing 1×105 PFU RFP-GFP-LC3-expressing adenovirus. After 2 h of incubation, the medium was replaced by the adenovirus-free medium. Later, at 24 h, the treatment solutions were then applied to the plate, followed by incubation for 24 h. The images were acquired with an Leica fluorescence microscope with the cells fixed.
CHME-5 cells were transfected either with p62, Nrf2 or HO-1 siRNAs. Briefly, mPMs were seeded into 6-well plate at a density of 3×105 cells/well and cultured in a humidified, 5% CO2 incubator at 37°C. On the next day, the culture medium was replaced with Opti-MEM® I Reduced Serum Medium. Meanwhile, the individual targeted siRNA and scrambled siRNA (500 pmol/ml) and Lipofectamine® 2000 (2 μl/ml) were incubated separately with Opti-MEM® I Reduced Serum Medium for 5 min at room temperature. After incubation, both individually targeted siRNAs and Lipofectamine® 2000 mix were mixed and subsequently, incubated at room temperature for another 15 min, mixed liquids were gently added to the culture medium. After 6 to 8 h the culture medium was replaced with DMEM supplemented with 10% heat-inactivated FBS. The transfected mouse primary microglia cells were then exposed to HIV-1 gp120V3 loop for 24 h, and transfection efficiency was analyzed by western blotting. All experiments were repeated at least 6 times.
CHME-5 cells were lysed and analyzed using previously established protocols in Western Blot Analysis. Lysates were centrifuged at 12,000 g for 15 min at 4 °C. Cleared lysates were incubated overnight with the indicated antibodies and for 1 h with protein A/G beads (Med Chem express, USA). The pellets were then washed four times with ice-cold lysis buffer. The immunoprecipitation samples were eluted from the beads by heating with SDS sample buffer. Eluted immunoprecipitates or whole cell lysates were separated by SDS-PAGE and analyzed by western blot.
In vitro: After incubation according to the study design, the CHME-5 cells were fixed with 4% PFA in PBS for 30 min, washed with PBS three times and then permeabilized with Triton-X-100 for 15 min. After washing with PBS three times, the cells were blocked with 5% BSA for 1 h at room temperature and then incubated with anti-HO-1 (ab223349, 1:100, Abcam), anti-iNOS (ab178945, 1:100, Abcam) in 5% BSA overnight at 4 °C. Subsequently, the cells were washed in PBS three times and incubated with fluorescein isothiocyanate-labeled secondary antibody for 1 h at 37 °C. The cells were then washed and incubated with DAPI for 15 min and then washed again and mounted. The cells were then imaged using Leica fluorescence microscope.
In vivo: After irrigation with 4% PFA, the brain tissue was removed and fixed in PFA for 24h. After washing with PBS for three times, 10% sucrose →20% sucrose →30% sucrose was used for gradient dehydration, and then oct-embedded and frozen at -80°C for about 40min before sectioning. The brain was cut into 20 m thick coronal sections with a frozen slicer, and baked for 2h at 38-40°C on a sheet baking machine. After drying to room temperature, the tissue slices could be stained with immunofluorescence. Sections (10 μm thick) were cut and mounted on microscope slides. Staining of p62 and Iba-1 using anti- p62 (1:100, 23214s, Cell Signaling Technology), anti- Iba-1 (1:100, ab5076, Abcam) primary antibodies were performed in mouse hippocampus and cortex.
Animals and intracerebroventricular injection
Male C57BL6J mice (8 weeks old) were procured from Shandong Experimental Animal Center (37009200014313). All animals were kept under a 12-hour light/12-hour dark cycle and allowed free access to water and food. mice were housed in wire cages at 20–22℃ and 50 ± 5% humidity. All experiments were approved by the Laboratory Animal Ethics Committee of Jinan University.
Intracerebroventricular injection: mice were anesthetized with 1.2% tribromoethanol and placed in a stereotaxic apparatus (RWD Life Science, Shenzhen China). A burr hole was drilled 0.2-0.5 mm posterior to bregma and 1.0 mm lateral to the midline, and the lateral ventricle was injected with a micro syringe at 2.5-3.0mm below the skull. The sham group was given random peptides and gp120V3 loop group was given 100ng/5μL/d, which was administered continuously for 3 days, once a day. After lateral ventricle injection, the brusatol group and gp120V3 loop+brusatol group were intraperitoneally injected with 2 mg/kg of brusatol, once every other day, until sampling. The control group and gp120 group were intraperitoneally injected with the same amount of normal saline. The random peptide and gp120 were dissolved with artificial cerebrospinal fluid (ACSF: NaCl 124.0 mM, KCl 3.0 mM, CaCl2 2.0 mM, MgCl2 2.0 mM, NaHCO3 26 mM, NaH2PO4 1.25 mM, Glucose 10.0 mM, pH 7.3-7.4).
Morris water maze
The Morris water maze test is used to assess memory. We performed this test as described by Morris et al . A circular plastic pool (height: 60 cm, diameter: 120 cm) was filled with milk kept at 22–24°C. An escape platform (height: 30 cm, diameter: 10 cm) was submerged 1-1.5 cm below the surface of the water in position. A camera was fixed to the ceiling above the water maze and connected to a computer-based program (Ethovision XT, Noldus Netherlands). mice were tested over 5 days (four trials/day) to find the hidden escape platform in the bottom left quadrant. They had a maximum of 60 s to find the platform. If animals were unable to locate the platform, they were gently guided to the platform and required to stay on the platform for 30 s before being removed. Mice were subjected to a probe trial by removing the platform during 60s. The number of rats crossing the target quadrant and time spent in the target quadrant were used as measures of spatial memory.
Step-Through Passive Avoidance test
Step-Through Passive Avoidance test is a behavioral experiment designed to observe the short-term memory reproduction ability of mice. The experimental device (Chengdu tmin life technology co., LTD) is half dark room and half-light room, connected with adjustable small holes, and the bottom of the dark room is equipped with an electric shock device. If mice escape from the dark room to the dark room, they will be shocked immediately. The experiment was divided into two days of Training and Test at an interval of 24 h.
Training: close the hole between the bright room and the hint before starting, open the boot cover and p, At first, lace the mouse in the bright room for 5 min to adapt to the environment. Then turn on the power (the sustained electric shock starts from the bottom of the dark chamber), open the hole at the same time, observe and record the latency of the corresponding mice, which is the time when the mice enter the dark chamber from the open chamber for the first time. The Number of errors was the Number of times that the mice entered the dark room from the open room. The incubation period and Error times of the mice within 300s were observed and recorded.
Test: clean the experimental equipment and remove the smell to ensure that the environment is basically consistent with the training. Test was performed on the 24th h after the training of mice, the hole was closed before startup, the hole was opened immediately after the mice were put into the open chamber and the electric power was switched on. Latency and error times of the corresponding mice were observed and recorded.
Data are presented as mean ± SEM. The data are from at least three different experiments, with each data point within an individual experiment representing triplicate measurements. Statistical analyses were performed using SPSS 13.0. Values were compared using the Nonparametric Kruskal – Wallis One-way ANOVA followed by the Dunn post hoc test was used to determine the statistical significance between multiple groups and the Wilcoxon test was used to compare between 2 groups .The results were considered statistically significant when P < 0.05.