ADSC isolation
Adipose tissue were harvested from normal mice, which was cleaned utilizing phosphate-buffered saline (PBS) before mechanical chopping and digestion with 0.2% collagenase I (Sigma–Aldrich, WI, USA) for 1 h at 37 °C applying intermittent shaking. Our team cleaned tissue that digested with Dulbecco's modified Eagle's medium (DMEM; Sigma–Aldrich) such like 15% fetal bovine serum (FBS) to be centrifuged at 1000g for ten minutes to eliminate mature adipocytes. We resuspended cell pellets in DMEM supplemented with 100 U/mL penicillin, 15% FBS and 100 μg/mL streptomycin in 37°C incubator with 5% CO2. We detached ADSCs achieving 80%~90% confluence with 0.02% EDTA/0.25% trypsin (Sigma–Aldrich) for five minutes under room temperature and replated them. For phenotypic analyses, we used phycoerythrin. The CD29, CD105, CD90, CD44, CD34 and CD34 expressions were investigated using flow cytometry. We cultured ADSCs under normoxic conditions in 95% air with 5% CO2 and 20% O2. For hypoxic pretreatment, we cultured ADSCs under hypoxic conditions in 93% N2, 5% CO2, and 2% O2.
ADSC multilineage differentiation
To identify the multilineage differentiation capacity of ADSCs, our lab cultivated third-passage mouse ADSCs with adipocyte differentiation and osteoblast differentiation for 14 and 21 days, respectively. Alkaline phosphatase (ALP) staining and Oil Red O staining were conducted to capture adipocyte differentiation and osteogenic differentiation.
ADSC-Exo identification and isolation
Upon achieving 80%~90% confluence, we rinsed ADSCs using PBS, which were cultivated in EGM-2MV media with no FBS and supplemented with 1× serum replacement solution (PeproTech, NJ, USA) for additional two days. We obtained conditioned medium from ADSCs, which we centrifuged at 300g for ten minutes and 2000g for another 10 minutes to remove cellular debris and dead cells. Finally, after centrifugation at 12,000g for 30 min, our team conducted all processes at 4°C. Our group defined Exo protein content using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). Technician maintained ADSC-Exos at –80 °C, which were applied for downstream experiments. Transmission electron microscopy, NTA assay were utilized to characterize the selected Exos.
Strand-specific NGS RNA-Seq library construction
We extracted total RNA from ADSCs-Exo and ADSCs-HExo applying TRIzol reagent (Invitrogen, CA, USA). Our team utilized 3 μg RNA from every sample with VAHTS. Total RNA-Seq (H/M/R) Library Prep kits from Illumina (Vazyme Biotech Co., Ltd, Nanjing, China) were used to eliminate ribosomal RNA; RNA classes, such like mRNAs and ncRNAs, were retained. Technician dealt with RNA using 40 U RNase R (Epicenter) at 37°C for 3 h, followed by TRIzol purification. Our team made RNA-Seq library through KAPA Stranded RNA-Seq Library Prep kits (Roche, Basel, Switzerland), which we employed for NGS (Illumina HiSeq 4000 at Aksomics, Inc., Shanghai, China).
Cell culture and cell transfection
BV2 were obtained from ScienCell, which we cultivated in DMEM (HyClone, UT, USA) and 10% FBS. For OGD induce, BV2 were cultured for 3 h in oxygen-glucose deprivation (OGD) condition before for the following experiment. BV2 were stored in a humidified incubator at 37 °C with 5% CO2. circ-Astn1 overexpression vector were made by putting circ-Astn1 cDNA into pcDNA3.1 vector. miR-138-5p mimics and Atg7 siRNA were synthesized via Genepharma (Suzhou, China). We performed cell transfection applying Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol.
ELISA for soluble inflammatory cytokines
The expression of inflammatory factors IL-6, IL-1β and TNF-α in BV2 cell or spinal cord tissue were measured using commercially available ELISA kits (Sen-Xiong Company). The optical density (OD)450 was calculated by subtracting the background, and standard curves were plotted.
Bioinformatics analysis
We predicted correlations among miRNA, mRNA, and circRNA applying online website http://starbase.sysu.edu.cn/.
RNA isolation and real-time polymerase chain reaction
Total RNA were extracted using TRIzol reagent (Invitrogen), followed by cDNA synthesis applying TransScript all-in-one First-Strand cDNA Synthesis SuperMix (Transgen Biotech, Beijing, China). We made polymerase chain reaction (PCR) utilizing Bio-Rad PCR instrument (Bio-Rad, CA, USA) with 2× Taq PCR Master Mix (Solarbio, Beijing, China) following the protocols. The fold changes were calculated using the 2−△△Ct approach.
Cell apoptosis assay
Flow cytometry was used to determine the percentage of apoptotic cells. Apoptotic cells were differentiated from viable or necrotic cells by the combined application of annexin V (AV)-FITC (Roche, Indianapolis, IN, USA) and propidium iodide (PI) (Roche). Cells were washed twice and adjusted to a concentration of 1×106 cells/mL with cold D-Hanks buffer. AV-FITC (10 μL) and PI (10 μL) were added to 100 μL of cell suspension and incubated for 15 min at room temperature in the dark. Finally, 400 μL of binding buffer was added to each sample without washing and analyzed with flow cytometry. Each experiment was performed at least in triplicate.
Dual-luciferase reporter assay
Atg7 gene 3′-UTR and circ-Astn1, including miR-138-5p binding sites, were amplified applying PCR. We put fragments into multiple cloning sites in pMIR-REPORT luciferase miRNA expression reporter vector (Ambion, Austin, USA). We co-transfected BV2 cells with 0.1 μg luciferase reporter vectors comprising wild-type (WT) or mutant-type (MUT) Atg7 or circ-Astn1 3′-UTR and miR-138-5p mimic or miR-control utilizing Lipofectamine 2000 (Invitrogen, CA, USA). We computed relative luciferase activity via normalizing firefly luminescence to Renilla luminescence through a dual-luciferase reporter assay system (Promega, WI, USA) following the manufacturer’s protocols 2 days after transfection.
Preparation of contusive spinal cord injury mouse model and experimental groups
The animal protocols were approved by the Animal Committee of the Huashan Hospital. An SCI model of male mice (C57BL/6, 6–8 weeks old) was established, as previously described. After anesthetizing the animal, a laminectomy was used to expose the spinal cord at T10, and a spinal cord impactor (68,097, RWD, CA, USA) was used to create injury by dropping a rod (weighing 5 g) onto the spinal cord from a height of 6.5 cm. Muscles were sutured immediately after administration, and the skin was closed. Bladders of animals were manually voided three times per day until reflexive control of bladder function was restored.
Mice were randomly assigned into several groups (n = 6/group for each time point). Mice were subjected to SCI, followed by tail vein injection of Exos, HExos, circ-Astn1-Exos (200 μg of total protein of exosomes precipitated in 200 μL PBS), or an equal volume of PBS (200 μL) immediately following SCI.
Exosome uptake by spinal cord tissue
Fluorescent labeling of Exos and HExos was carried out according to the manufacturer’s instructions. Briefly, 4 mg/mL PKH26 solution (Molecular Probes, Eugene, OR, USA) was added to PBS containing exosomes and incubated. Excessive dye from labeled exosomes was removed by ultracentrifugation at 100,000×g for 1 h at 4 °C. Exosome pellets were then washed three times by re-suspending the pellet in PBS with a final wash and resuspension in PBS. These PKH26-labeled exosomes were by tail vein injection for 24 h, and the tissues were collected and fixed in 4% paraformaldehyde. The uptake of PKH26-labeled Exos and HExos by spinal cord tissue was then observed by laser confocal microscopy and the fluorescence intensity of PKH26 was measured with ZEN lite software.
Behavioral testing of locomotor function
The Basso, Beatlie and Bresnahan (BBB) functional score was used to quantify locomotor function. The test was performed before the spinal cord surgery and weekly thereafter at the same time of day and were graded by the three blinded observers. Functional restoration was assessed as per BBB locomotor scores. The BBB test score evaluated hindlimb locomotor function on a scale from 0 to 21. Scoring was based on spontaneous hindlimb movement during a 5-min observation period in the open field after the animals already habituated to the arena. The animals (n = 6) were placed on a circular platform with a diameter of 2 m, and the walking and limb activities of the hind limbs were observed.
Immunohistochemical analyses
We fixed tissue samples using 4% paraformaldehyde solution to embed them in paraffin. Technician cultured sections overnight applying primary antibodies against LC3 at 4 °C and then with secondary antibodies (Abcam) for 1 h at 37 °C. Cell nucleus counterstained using 3,3-diaminobenzidine. We conducted TUNEL staining and examined sections applying Axiophot light microscope (Zeiss, Oberkochen, Germany).
Statistical analyses
The continuous variables were expressed by means ± SD. Our team conducted one-way variance analyses for comparisons with GraphPad Prism (GraphPad, CA, USA). P value ≤ 0.05 indicated a statistical significance.