C57BL/6J male mice (weighting 22–25 g, 8 weeks old) were purchased from Beijing River Laboratory Animal Corp. Ltd. The mice were raised in specialized boxes that were placed in a quiet room with a light–dark cycle of 12–12 h, a temperature controlled at approximately 22 °C, and free water and food. The animal experiments were approved by the Ethics Committee for Animal Experimentation of Zhengzhou Central Hospital Affiliated to Zhengzhou University (Zhenzhou, China) in accordance with the Animal Research: In vivo Reporting of Experiments (ARRIVE) guidelines. The data were analysed to detect significant differences with PASS (power analysis & sample size) software using a significance level of α = 0.05 with 80% power. All experiments were randomized, and the operator was blinded to the data analysis and experiment.
Reagents and antibodies
Primary antibody: anti-β-actin (sc-47778, 1:1000), anti-HA (sc-7392, 1:1000) and anti-GFAP (sc-33673, 1:200) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); Anti-iNOS (18,985–1-AP, 1:500) was purchased from Proteintech Group (Wuhan, China); anti-CD16/32 (AF1460, 1:500) was purchased from R&D systems (Minneapolis, MN, USA); anti-NF-κB p65 (#8242, 1:1000), anti-IKKα (#11,930, 1:1000), anti-Phospho-NF-κB p65 (#3033, 1:1000), anti-Phospho-IKKα/β (#2697, 1:1000), anti-Phospho-IκBα (#2859, 1:1000), anti-IκBα (#4814, 1:1000), anti-NBR1(#9891, 1:1000), anti-cleaved PARP (5625, 1:1000), anti-cleaved caspase-3 (9664, 1:1000) and anti-cleaved caspase-9 (#20,750, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA); anti-SENP6 (HPA024376, 1:1000) were purchased from Sigma–Aldrich (St. Louis, MO, USA); anti-Iba1 (ab283319, 1:100) was obtained from Abcam (Boston, MA, USA) and anti-NeuN (MAB377, 1:200) was purchased from Millipore Biotechnology (Schwalbach, Germany). Protein A + G agarose beads were obtained from Beyotime Biotechnology (Shanghai, China), and Ni2+-NTA agarose was purchased from QIAGEN (Dusseldorf, Germany). CHX was obtained from Calbiochem (508,739; Darmstadt, Germany).
Transient focal cerebral ischaemia
The intraluminal filament technique was used to induce focal cerebral ischaemia through the transient occlusion of the middle cerebral artery as we reported previously . In brief, mice were anaesthetized using chloral hydrate (350 mg/kg, i.p.). To maintain the rectal temperature at approximately 37 ± 0.5 °C, the mice were placed on a thermostatic blanket (Harvard instrument, Holliston, MA, USA). The left common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were exposed. Ligation of the CCA was performed with surgical nylon monofilament near the distal end of the CCA and ligation of the ECA was conducted at two positions at the end of ECA and near ICA and ECA bifurcations. Then, using ophthalmic scissors, a small incision was made between the nylon filament (0.22 to 0.23 mm in diameter), and the two ECA ligatures of double diameter lengths were gently inserted into the ICA from the ECA stump and advanced to the anterior cerebral artery (ACA) until slight resistance was felt. Successful occlusion was verified by laser Doppler flowmetry (PeriFlux System 5000, PERIMED, Sweden). After 60 min, the filament was withdrawn softly. The sham mice underwent the same surgical operation, but no embolus was inserted.
Cell culture, transfection and OGD/R procedure
Primary microglia were cultured according to our previously reported method . Briefly, mixed glial cells were isolated from the whole brains of C57BL/6 neonatal mice at postnatal days P1 and P2 and then cultured in high-glucose DMEM (Gibco, Gaithersburg, MD, USA) with 1% penicillin–streptomycin and 20% foetal bovine serum (FBS; Gibco) in a 5% CO2 incubator at 37 °C for 7 days. After 7 days, fresh complete medium was used to replace the old medium every 3 days. After 10–14 days post-dissection, primary microglia were gathered from the mixed glial cultures by shaking at 400 rpm for 4 to 6 h at 37 °C in a rotary shaker and seeded into poly-d-lysine (Sigma–Aldrich)-coated six-well plates at a density of 1 × 106 per well for 24 h attachment before further treatment.
HEK293T cells were purchased from the American Type Culture Collection and grown in DMEM supplemented with 1% penicillin–streptomycin and 10% FBS at 37 ℃ in a 5% CO2-containing atmosphere. Following the manufacturer’s instructions, approximately 80%-90% confluent cell layers were transfected with plasmids by using Lipofectamine 3000 (Invitrogen, NY, USA). OGD/R was carried out as we described before. Typically, primary microglial cells and HEK293T cells were taken from a CO2 incubator, and glucose-free DMEM (Gibco) preheated to 37 ℃ was used to replace the cell culture medium. Next, the cells were placed in an oxygen-deprived (94%N2/5%CO2/1%O2) incubator at 37 °C for 1 h to establish OGD conditions. Finally, glucose-free DMEM was discarded, and then high-glucose DMEM was added to the cell plates to reoxygenate the cells under normoxic conditions for 24 h for subsequent assays.
Protein extraction and preparation, immunoprecipitation and immunoblot analysis
Protein extraction and subcellular separation were carried out using the method reported previously . Whole-cell lysates were prepared in radioimmune-precipitation assay (RIPA) lysate (Beyotime Biotechnology) supplemented with cOmplete™ protease inhibitor cocktail tablets (5 mg/ml; Roche Diagnostics, Rotkreuz, Switzerland). The NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA) was chosen to extract nuclear and cytoplasmic fractions according to the manufacturer’s instructions. Then, a BCA protein assay kit (Beyotime Biotechnology) was applied to determine the protein concentrations of the above extracts. For immunoprecipitation, cells were lysed in immunoprecipitation (IP) buffer supplemented with NEM (N-ethylmaleimide, Sigma–Aldrich) or a protease inhibitor mixture. Then, anti-HA, anti-His, anti-NBR1 and anti-IKKα antibodies were added to suitable cell lysates, followed by incubation for 12 h using a shaker at 4 °C. After that, cell lysates with antibodies were incubated for 4 h at 4 °C with protein A/G plus agarose (Santa Cruz) and washed three times with precooled PBS buffer. Then, 2 × SDS–PAGE loading buffer was added to the samples followed by boiling at 95 °C for 5 min, and immunoblot analysis was performed to detect the protein expression. For immunoblotting, the protein samples were separated using SDS–PAGE. Then, the proteins were transferred to a polyvinylidene difluoride membrane (Roche Diagnostics) and blocked with 5% bovine serum albumin (BSA) for 60 min at room temperature. Finally, the membranes were probed using primary antibodies overnight at 4 °C. Horseradish peroxidase–conjugated secondary antibodies (1:20,000) were used. A chemiluminescence substrate kit (Thermo Pierce, Rockford, IL, USA) was used to perform immunodetection.
Ni2+-NTA affinity purification
As reported previously, SUMOylated ANXA1 under the condition of denaturation was affinity-purified by Ni2+-NTA pull down . In brief, plasmids expressing HA-tagged ANXA1, Myc-tagged SENP6 and His-tagged SUMO2 were transfected into HEK293T cells. After 24 h, the cells were taken from the incubator, washed twice with cold PBS buffer, and then treated with 800 mL Ni2+-NTA denatured buffer (10 mM Tris; 20 mM NEM; 6 M Gu-HCl and 100 mM NaH2PO4, pH 8.0). The samples were treated with ultrasound (2 × 20 s) to cut the DNA and then cleared via centrifugation (15,000×g, 10 min, 4 °C). The supernatant was mixed with 50 mL prewashed Ni2+-NTA agarose (Qiagen, Dusseldorf, Germany) and rotated at 4 °C to incubate for 3 h. Finally, 1 mL Ni2+-NTA washing buffer (0.1% Triton X-100; 100 mM NaH2PO4; 10 mM Tris/HCl, pH 6.3; 8 M urea) was added to wash the beads, and lastly eluted by boiling the beads in 50 μL 2 × SDS–PAGE loading buffer mixed with 200 mM imidazole for 5 min.
LDH release and cell survival assays
According to the manufacturer’s instructions, an LDH Cytotoxicity Assay Kit (Beyotime Biotechnology) was employed to determine LDH release from primary microglial cells. For the survival assays, cells were fixed with 4% formaldehyde for 30 min and then permeabilized with 0.1% Triton X-100 for 15 min. Next, the primary neurons were incubated using a 50 μL /well /24-well plate TUNEL reaction mixture at 37 °C for 1 h and with 50 μl /well /24-well plate 1 × DAPI for 10 min at room temperature. Finally, the cells were examined, and TUNEL-positive cells were counted using fluorescence microscopy (IX73, Olympus, Tokyo, Japan).
RNA extraction, reverse transcription, and quantitative real-time PCR (RT–qPCR)
Total RNA was extracted from primary microglial cells with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions, and the RNA concentration was measured using spectrophotometry (Thermo Fisher Scientific). Next, the reverse transcription of the complementary DNA (cDNA) from 1 μg total RNA was performed by the ReverTra Ace-α-First Strand cDNA Synthesis Kit (Toyobo, Osaka, Japan). According to the manufacturer’s instructions, quantitative real-time PCR was conducted using a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and SYBR Green PCR Master Mix (Applied Biosystems). The relative gene expression was normalized to β-actin mRNA levels, and the gene expression was evaluated using the 2−ΔΔCt method. The primer sequences are listed in Additional file 5: Table S1.
Luciferase reporter assay
HEK293T cells plated in 96-well plates were transfected with NF-κB luciferase reporter and plasmids carrying vector, SENP6, sh. NC or sh. SENP6 with pRL-Renilla as a control. The cell lysate was collected after transfection for 48 h, and the Dual-Luciferase kit (Promega, Madison, WI, USA) was used according to the manufacturer’s instructions to detect the luciferase activity. Then, firefly luciferase activity was normalized to Renilla luciferase using the internal transfection efficiency control. The experiments were conducted in triplicate.
Plasmids were constructed as we previously described . PCR technology was used to amplify the full-length DNA segment ANXA1 and SUMO2 coding sequences, which were cloned into HA-tagged pcDNA 3.0 (HA-ANXA1) and Myc-pcDNA 3.0 (Myc-SUMO2), respectively. Similarly, His-tagged SENP6 was constructed by cloning the full-length DNA segment coding sequence into His-pcDNA 3.0 (His-SENP6). Wild-type SENP6 and shRNAs against SENP6 plasmids were purchased from Sangon Biotechnology Company (Shanghai, China). The plasmids were all transfected into HEK293T cells with a Lipofectamine 3000 reagent kit according to the manufacturer’s instructions.
Viral vector production and transduction
Adenoviruses encompassing vector, wild-type SENP6, scramble control and shRNAs against SENP6 purchased from Vigene Biosciences (Jinan, China) were employed to infect primary cultured microglia. The sequences of shRNA were designed and verified as follows: mSENP6, 5′-GGG CAA ATC TAC TCA GTG TAG-3′. Primary cells were infected using diluted recombinant adenovirus at an optimal multiplicity of infection of approximately 50:1 to 100:1 according to our preliminary tests. After 48 h of transfection, the cells were subjected to subsequent experiments.
The adeno-associated virus expressing SENP6 shRNA (AAV2/6-CMV-sh. SENP6) and control adeno-associated virus (AAV2/6-CMV-sh. NC) were purchased from Genechem (Shanghai, China). As we performed previously, mice were anaesthetized with an intraperitoneal injection of 5% 350 mg/kg chloral hydrate and then fixed on a stereotaxic apparatus. A drill was used to perforate burr holes into the skull, and a stepper-motorized microsyringe (Hamilton, Reno, NV, USA) was used to inject 500 nl of virus solution into the injection sites in the area of the hippocampal CA1 region, cerebral cortex and striatum of the left hemisphere. After injection, the needle was kept in place for 5 min and then removed slowly. Four weeks later, mice that underwent virus injection were subjected to MCAO/R surgery for subsequent experiments. Injection into the hippocampus CA1, cerebral cortex and striatum were, respectively at coordinates anteroposterior (AP) − 2.00 mm, lateral (L) − 1.55 mm and dorsoventral (DV) − 1.55 mm; anteroposterior (AP) 0.00 mm, lateral (L) − 2.05 mm and dorsoventral (DV) − 1.50 mm; anteroposterior (AP) 0.14 mm, lateral (L) − 2.28 mm and dorsoventral (DV) − 3.50 mm.
The neurological score was determined as we have previously described . After 24 h of MCAO and reperfusion, the modified neurological severity score (mNSS) was selected to assess neurological dysfunction, which includes beam balance tests (scored 0 to 6), reflexes absent & abnormal movements (scored 0 to 2) and motor tests (including flexion of forelimb, flexion of hindlimb and head movement, scored 0 to 6). Accumulative scores of 1 to 4, 5 to 9, and 10 to 14 indicated slight, moderate, and serious damage, respectively. Neurological performance was evaluated by independent blinded researchers.
TTC (2,3,5-triphenyltetrazolium chloride) staining
Approximately 24 h after MCAO surgery, the mice were euthanized. Their brains were extracted carefully and then frozen for 5 min at − 20 °C. The whole brain was cut into 6 consecutive approximately 2-mm-thick coronal slices from the frontal tips using a mouse brain matrix. Then, the cells were dyed using 2% TTC (Sigma–Aldrich). The brain slices were fixed using 4% paraformaldehyde after incubating for 20 min at 37 °C. ImageJ analysis software (NIH, Baltimore, MD, USA) was selected to measure the infarct area, which was the white region, and the normal tissue, which was the dark red region. The infarct size (%) was calculated using the equation: infarct size (%) = (contralateral area − ipsilateral noninfarct area)/contralateral area × 100%.
Adhesive-removal somatosensory test
Mice were subjected to the adhesive removal test to evaluate somatosensory and motor deficits. In the process of the experiment, sensory deficits were detected in the latency for animals to become aware of the sticker on their paws, while motor deficits were detected in the latency for animals to remove the stickers. Briefly, animals in the experiment were placed in an empty cage and given 1 min to adjust to the tests. Adhesive tape was cut into small squares and then pasted on the animals’ paws. Next, the time of the mouse to first notice the stickers or remove the stickers was observed and recorded. A time limit of 120 s was enforced. The experiments were repeated continuously four times with a 10-min break after each trial, and the data were statistically analysed.
Morris Water Maze (MWM) test
During this study, we conducted MWM tests to measure spatial learning and memory as reported previously . Briefly, the water maze contained a circular pool 60 cm high and 120 cm in diameter and a round platform 6 cm in diameter and was filled with water submerging the platform 1 cm at 22 ± 2 °C. Different shapes as spatial reference clues were placed around the tank. Above the maze, a digital tracking device was used to record mouse swimming traces. Before the tests started, the mice were habituated to the testing room for 24 h. In the first 6 consecutive days, each group performed four sessions in turn to search for the submerged platform within 60 s. In the condition of a mouse not finding the platform in the allowed time, we guided it to the platform and allowed it to stay there for 10 s. Finally, the platform was removed after completing the training on the seventh day, and the animal traces in 60 s were recorded by a digital tracking device (Xinruan Information Technology, Shanghai, China), which automatically analysed the time to cross the platform area, the time the mice spent in the target quadrant and the latency time to reach the platform.
The rotarod test
To evaluate motor function, a rotarod containing a 6-cm-diameter rotating cylinder with a coarse surface was used. Beforehand, the mice were placed in the testing environment for 30 min to acclimate. Then, the mice were placed in the centre and subjected to a training session at a speed of 5 to 10 rpm on the rotarod. Next, the speed of the rotarod was increased to 40 rpm within 5 min, during which time the mice fell off the rotarod. The experiments were repeated continuously four times with a break of 30 min after each trial, and the data were analysed statistically.
Data from at least three repeated experiments are shown as the mean ± S.E.M. and the corresponding analysis was conducted using GraphPad Prism software (version 8.0.1, GraphPad Software, Inc.). The comparisons for more than two groups were performed using one-way or two-way analysis of variance (ANOVA) and repeated-measures ANOVA tests, which are shown in the figure legends. In addition, as indicated, Dunnett’s or Tukey’s post-hoc multiple comparison measurements were used for significant effects in variance analysis. The number of mice crossing over the platform location on Day 7 in the MWM test was analysed using the Kruskal–Wallis nonparametric test, followed by Dunnett’s post-hoc test. A P value lower than 0.05 was considered statistically significant.