Animals

Female C57BL/6 mice (8 weeks old, 20–23 g) were purchased from SJA Laboratory Animal Co., Ltd (Hunan, China). UTXflox/flox and Tek-Cre: UTXflox/flox (UTX−/−) female mice were obtained by hybridization between Tek-Cre male mice and UTXflox/flox female mice, which were purchased from Jackson Lab, the United States. All animals were kept in the laboratory animal department at Central South University in Changsha, China, and had free access to food and water. All experiments were conducted following scientific inspection and received approval from the Medical Ethics Committee of Xiangya Hospital, Central South University.

Establishment of SCI model and treatment

The mice were anesthetized by intraperitoneal injection of 0.3% pentobarbital sodium (75 mg/kg). Using the T10 spinous process as the center, the fur was removed, and a surgical area of about 3 cm × 1.5 cm was exposed. After disinfection, a 1 cm long incision was made in the middle of the dorsal skin of the mouse. The fascia tissue was cut layer by layer with ophthalmic scissors, and the lateral muscles of the spinous process were carefully separated to expose the vertebral body completely. Micro forceps were used to gently exfoliate the T10 spinous process and lamina, clearly exposing the spinal cord. Following the modified Allen’s method, a 10 g impactor rod struck vertically from a height of 2.5 cm. The observation of local hematoma in the spinal cord indicated the successful establishment of the SCI model. Subsequently, 4-0 sutures were used to stitch the muscles, fascia, and skin layer by layer, and the skin was disinfected with iodine. For the Sham group, only laminectomy was performed.

For adeno-associated virus (AAV) intervention, after exposing the spinal cord, 2 μL AAV-CNN1 or negative control AAV (NC-AAV) was locally injected into the T10 segment of the spinal cord using a 5 μL microinjection needle. The injection needle was kept in place for 2 min to promote full infiltration of AAV, then gently pulled out. After 2 weeks, when AAV began to be stably expressed, the SCI model was constructed by referring to the above method.

For dasatinib (D, MCE, United States) and quercetin (Q, MCE, United States) treatment, after establishing the SCI model, 5 μL hydrogel mixed with D + Q at concentrations of 60 μg/mL and 600 μg/mL, respectively, was placed on the surface of the injured spinal cord. Then, the hydrogel mixture was exposed to ultraviolet light for 5–9 s for solidification.

bEnd.3 cell line culture and treatment

The bEnd.3 cell line was purchased from Procell Life Science&Technology Co.,Ltd., and was cultured with high glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. H2O2 was used to induce cell senescence. We used culture medium to configure H2O2 at 0 μmol/L, 50 μmol/L, 100 μmol/L, 200 μmol/L, 400 μmol/L concentrations. Discard the culture medium and wash it with PBS before replacing it with the culture medium containing H2O2. After culturing for 12 h, detect cell senescence markers.

To establish a cell line stably expressing the target gene, lentivirus was constructed and screened with puromycin. UTX knockdown Lentivirus (UTX-KD) and corresponding negative control lentivirus (UTX-NC) were constructed by Shanghai Genechem Co., Ltd. CNN1 overexpression lentivirus (CNN1-OE) and control lentivirus (CNN1-NC) were constructed by OBiO Technology (Shanghai) Corp., Ltd. UTX overexpression lentivirus (UTX-UP) and control lentivirus (UTX-NC) were constructed by Suzhou Haixing Biological Technology Co., Ltd. After the cells reached an appropriate density, they were cultured with culture medium containing lentiviruses (at a MOI of 1:40) and auxiliary transfection reagents. The transfected cells were cultured for 24 h. When the cell growth concentration reached about 90%, stable cell lines were screened using puromycin for a continuous 7 days.

For D + Q treatment in vitro, 200 nmol/L dasatinib (D) and 20 μmol/L quercetin (Q) were added to the culture medium for 2 days, and then changed to normal culture medium until subsequent experiments started.

Isolation and cultivation of BMECs

Mice were euthanized and subjected to disinfection by immersion in a 75% alcohol solution. The brain was extracted, and the brain stem, dura mater, and vascular tissue were removed. The cerebral cortex was then transferred to precooled DMEM medium and dissected using micro-scissors. Subsequently, centrifugation at 300 g for 5 min was performed, and the supernatant was discarded. A solution of 0.1% type II collagenase was introduced into the centrifuge tube. Following agitation and re-suspension, the mixture underwent digestion on a constant temperature shaker (37 °C) at 200 r/min for 1.5 h. The resulting suspension was subjected to centrifugation at 300 g for 5 min, and the supernatant was discarded. A 10 mL filtered solution of 20% BSA was added to the centrifuge tube, and the tissue was blown and resuspended. Subsequent centrifugation at 3 000 g for 20 min yielded the lower microvascular layer. The microvessels were digested with collagenase/dispersing enzyme for 30 min. BMECs were cultured in low-glucose DMEM containing 20% FBS, 0.5% FGF, 0.1% heparin sodium, and 1% penicillin-streptomycin.

Immunofluorescent staining

To obtain spinal cord tissue, mice were anesthetized through an intraperitoneal injection of 0.3% sodium pentobarbital (75 mg/kg). Following the full exposure of the heart, a perfusion needle was inserted into the left ventricle, and 20 mL of physiological saline followed by 4% paraformaldehyde were sequentially infused at a rate of 4–5 mL/min. A 1.5 cm length of spinal segment was excised and dehydrated consecutively using 20% and 30% sucrose solutions. Subsequently, it was embedded in optimal cutting temperature (OCT) agent (Sakura, United States) for sectioning at a thickness of 16 μm.

After permeabilization and blocking, the sections were incubated with diluted primary antibodies at 4 °C overnight. On the following day, the primary antibodies were removed, and the sections were rinsed three times with a PBS solution. The corresponding diluted secondary antibodies were then added to the sections and incubated at room temperature for 1 h. Subsequently, the secondary antibodies were removed, and the sections were washed three times with PBS. 4,6-diaminyl-2-phenylindoles (DAPI, GeneTex, United States) were used to label cell nuclei. The images were captured using an APTOME Fluorescence Microscope (Zeiss, Germany). The antibodies used are listed in Table S1.

When cells reached a confluence of 85% in a 24-well plate, the culture medium was discarded, and the cells were washed with PBS three times. Subsequently, 500 μL of 4% paraformaldehyde was added to each well for fixation for 12 min. The paraformaldehyde was then washed away with PBS. Next, 200 μL of PBST containing 0.1% Triton-X100 was added to each well for permeabilization, and the cells were incubated for 20 min. Following permeabilization, the cells were blocked with a 3% BSA solution for 30 min. Subsequently, 200 μL of diluted primary antibodies were added to each well for overnight incubation at 4 °C. On the following day, the primary antibodies were removed, and the cells were washed with PBS. Diluted secondary antibodies were then added, and the cells were incubated at room temperature for 1 h. The cell nucleus was stained with DAPI. Images were captured using an inverted fluorescence microscope (Thermo Fisher, United States).

SA-β-gal staining

The SA-β-gal kit (CST, United States) was thawed in advance. Initially, 200 μL of fixative was added to the specimen at room temperature for 15 min. Subsequently, the SA-β-gal working solution was prepared according to the manufacturer’s instructions, consisting of 10 μL of β-galactosidase solution A, 10 μL of β-galactosidase solution B, 930 μL of β-galactosidase solution C, and 50 μL of X-Gal solution. The pH value of the solution was adjusted to 6.8 using a pH meter. After washing with PBS twice, each specimen was treated with 200 μL of the working solution for incubation in a 37 °C oven overnight. On the following day, the working solution was removed, and the slices were photographed using an optical microscope (Leica, Germany).

For SA-β-gal staining of cells, when cell proliferation reached 75% confluence, the culture medium was removed, and 1 mL of 4% paraformaldehyde solution was added to each well to fix the cells for 15 min. The subsequent SA-β-gal staining process was identical to the procedure described above.

Enzyme-linked immunosorbent assay (ELISA)

ELISA was employed to determine the concentration of senescence-associated secretory phenotype (SASP) factors (TNF-α, PAI-1, and CCL-5) in spinal cord tissue (Elabscience, China). Following euthanasia of the mouse, the spinal cord tissue containing the injured segment was obtained and rinsed with pre-cooled PBS solution. After homogenizing the tissue using a grinder, centrifugation at 4 °C, 5 000 g for 8 min was performed, and the supernatant was collected. The standard solution was prepared according to the instructions for constructing the standard curve. Subsequently, the sample was added to the ELISA plate and incubated at 37 °C for 90 min. Following this, biotinylated antibody diluent was added, and the incubation continued at 37 °C for 1 h. After terminating the reaction, a microplate reader (Thermo Fisher, United States) was utilized to measure absorbance at a wavelength of 450 nm.

Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR)

Total RNA of spinal cord tissue or bEnd.3 cells were extracted using Trizol agent (Sigma, United States). Add chloroform in the ratio of Trizol: chloroform (5:1). Leave the mixture on ice for 10 min after shaking. Then centrifuge at 4 °C at 12 000 r/min for 15 min. Carefully absorb the top supernatant and transfer it to a new 1.5 mL EP tube. Add an equal amount of isopropanol to the EP tube, turn it upside down 10 times. Then centrifuge at 12 000 r/min for 15 min. Discard the supernatant and wash the RNA with 75% alcohol. Then 20 μL enzyme-free water was added and gently blown to fully dissolve the RNA. After detecting the RNA concentration using a Nanodrop (Thermo Fisher, United States), a Reverse Transcription System (Promega, United States) was used to reverse transcription of RNA into cDNA. GoTaq® qPCR Master Mix (Promega, United States) was used for real-time quantitative PCR. PCR results were analyzed by 2−ΔΔCt method. GAPDH was used as internal reference. Primers used were listed in Table S2.

Western blot (WB)

Total proteins from spinal cord tissue or bEnd.3 cells were extracted using RIPA lysis (Sigma, United States) and protease inhibitor (Sigma, United States). The tissue or cells were fully lysed after ultrasonic treatment for 120 s. Subsequently, the lysate was centrifuged at 4 °C and 12 000  r/min for 15 min, and the supernatant was collected. The protein concentration was determined using the BCA protein detection kit and a microplate reader (Thermo Fisher, United States). A 10% gel was utilized to separate proteins with different molecular weights. After adding samples and markers, the voltage was adjusted to 90 V for electrophoresis until the protein bands were completely separated. The proteins were then electro-transferred to a polyvinylidene fluoride (PVDF) membrane at 300 mA for 90 min. The PVDF membrane was blocked with 5% non-fat milk for 1.5 h and incubated with corresponding primary antibodies at 4 °C overnight. On the following day, the primary antibodies were removed, and the membrane was rinsed with TBST solution three times. Subsequently, secondary antibodies were added for incubation at room temperature for 1 h. Bands were covered with ECL chemiluminescence (Millipore, United States) and visualized using a chemiluminescence instrument (Bio-Rad, United States). Actin was used as an internal reference. Antibodies used are listed in Table S1.

Tube formation and transwell migration

bEnd.3 cells were subjected to serum starvation by replacing the culture medium with FBS-free medium. Following trypsin digestion and centrifugation, bEnd.3 cells were adjusted to a concentration of 80 000 cells/mL with FBS-free culture medium. Matrigel (Corning, United States) was evenly applied to cover a 96-well plate, and the plate was then incubated in a constant temperature incubator for 30 min. Subsequently, 100 μL of the cell suspension was added to each well of the 96-well plate and cultured in a constant temperature incubator for 6 h. The canalization of cells was observed using an optical microscope (Leica, Germany) and quantified using Image J software.

For the assessment of the vertical migration capacity of bEnd.3 cells, Transwell plates (Corning, United States) were employed. The lower chamber was filled with 600 μL of culture medium containing 10% FBS, and 100 μL of the cell suspension was added to the upper chamber. After incubating in a constant temperature incubator for 12 h, the transwell chambers were removed, fixed with 4% paraformaldehyde for 15 min, and washed three times. Following staining with 1% crystal violet solution for 10 min, the number of cells that migrated from the upper chamber to the lower chamber was observed using a microscope (Leica, Germany), and Image J software was used for counting.

Scratch wound healing assay

bEnd.3 cells were seeded in six-well plates, with approximately 200 000 cells in each well. Once the cells completely covered the surface of the six-well plate, they were subjected to a 6 h starvation period. Using a 200 μL tip, lines perpendicular to the cell surface were drawn, and the detached cells were washed off with PBS. Subsequently, the cells were replenished with FBS-free culture medium and cultured for an additional 12 h in a constant temperature incubator. Cell migration was observed using optical microscopy at 0 and 12 h after creating the scratches, and Image J software was employed to calculate the migration distance.

PCR-array

PCR-array kits were customized by Wcgene Biotech, Shanghai, China. bEnd.3 cells were used to detect SASP expression after H2O2 treatment. BMECs were used to screen differentially expressed epigenetic factors after H2O2 treatment. cDNA mix solution was prepared according to the manufacturer’s instructions. The PCR-array plate was rapidly centrifuged for 20 s, and 9 μL cDNA mix solution was added to each well. The PCR process was set according to the following procedures: activation of polymerase at 95 °C for 2 min, denaturation at 95 °C for 15 s and annealing extension at 60 °C for 1 min (40 cycles).

Basso mouse scale (BMS) scores

BMS scores were performed to explore motor function of mice, as previously described.55 Prior to the test, mice were placed on the experimental table to acclimate to the environment. Two well-trained observers simultaneously observed hind limb movements and trunk stability for each mouse over a 4-minute period. The final score at each time point was determined by calculating the average score from the two observers (0 indicated complete paralysis, and 9 indicated normal motor function). BMS scores were assessed before the injury, and at 1 day, 3 days, 7 days, 2 weeks, 3 weeks, and 4 weeks post-injury.

Neuroelectrophysiological evaluation

The Neuroelectrophysiological evaluation was used to detect the neural conductivity in mice, and performed as previously described.56 Briefly, mice were anesthetized via intraperitoneal injection of 0.3% sodium pentobarbital (75 mg/kg). A stereotaxic device was utilized to determine the skull location, with bregma set as the origin. Two stimulation electrodes were punctured into the skin and positioned on the skull surface (1 mm on the caudal side and 0.5 mm on the lateral side, −4 mm on the caudal side and 0.6 mm on the lateral side). Recording electrodes were inserted into the tibialis anterior muscle of the contralateral posterior limb, and a reference electrode was placed in the subcutaneous tissue between the stimulation and recording electrodes. Electrical stimulation was applied at a voltage of 3 V and a frequency of 333 Hz, repeated every 5 s. The evoked potential was determined by the peak and trough voltage, while the latent period was defined as the time from the initial stimulation to the occurrence of a dramatic change in the waveform.

Evaluation of neurogenic bladder

Bladder function was assessed by measuring the bladder diameter and detrusor muscle thickness through HE staining. Following anesthesia, mice were perfused with physiological saline and 4% paraformaldehyde. The bladder was photographed, and the diameter at its widest part was measured. For detrusor muscle thickness measurement, the bladder underwent gradient dehydration with varying concentrations of anhydrous ethanol. Subsequently, the bladder tissue was embedded in paraffin and sectioned into 8 μm slices. Paraffin sections were dewaxed, hydrated, and stained with hematoxylin for 5 min. Excess hematoxylin solution was washed off, followed by staining with eosin solution for 1 min. After removing excess eosin solution, the slices were dehydrated and sealed with neutral resin.

RNA-seq

RNA-seq analysis was conducted by Seqhealth Technology (Wuhan, China) with four biological replicate samples per group. Primary brain microvascular endothelial cells (BMECs) were isolated from 8-week-old UTX−/− and UTXflox/flox female mice by flow cytometry. Following treatment with 200 μmol/L H2O2 for 12 h to induce senescence, BMECs underwent RNA extraction. Oligo (dT) was then employed to enrich mRNA, and the enriched mRNA underwent fragmentation, reverse transcription, and PCR amplification to generate a sequencing library. Subsequently, Illumina Hiseq was utilized for RNA sequencing. After preprocessing, quality control, and sequence alignment of the data, protein-coding genes annotated by the genome were obtained. Differentially expressed genes were identified using the criteria of an absolute logFC > 1 and P < 0.05. These genes were selected for subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The top 10 down-regulated genes were listed in Table S3.

ChIP-qPCR

To investigate the targeted binding relationship between UTX and H3K27 of the CNN1 promoter, ChIP-qPCR detection was conducted. bEnd.3 cells with stable UTX overexpression were obtained through Lentivirus transfection. Formaldehyde was added to the cell culture medium, followed by incubation at room temperature for 10 min, and glycine was added to terminate crosslinking. After discarding the culture medium and washing with precooled PBS, digestive fluid was added to the cells for ultrasonic lysis. Ten microliters of supernatant were taken as input, and the remaining supernatant was used for IP. The IP group received either H3K27me3 antibody or IgG antibody, while no antibody was added to the input group. After eluting DNA from the antibodies, a DNA purification kit (Magen, China) was used to recover the eluted DNA. The primers for ChIP-qPCR are listed in Table S4.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8 software. A student’s t test was utilized for comparisons between two groups, while differences among multiple groups were assessed using one-way ANOVA. Tukey post-test was employed for pairwise comparisons in the case of multiple groups. BMS scores at different time points within each group were analyzed using repeated-measure two-way ANOVA. Results were presented as mean ± standard deviation (Mean ± SD), and when P < 0.05, it is considered statistically significant.