SCI model

Eight-week-old female Sprague–Dawley rats were purchased from the Animal Center of Jiangsu University, China. All rats were housed and handled in compliance with the regulations of the Animal Committee at Jiangsu University. Rats were anesthetized via isoflurane inhalation and subjected to laminectomy to expose the spinal cord at the level of the T9–T10 vertebrae. Then, an impactor (weighing 5 g, 2 mm in diameter, 150 mm in length) was vertically dropped onto the exposed spinal cord; the wound was then sutured. SCI was considered to be successfully induced when it resulted in spinal cord compression, swaying legs, tail swing reflexes, and slow paralysis. All rats were housed after surgery in a separate environment at 24 °C with adequate water, food, and clean bedding, and assistance for urination was provided three times daily. Rats were randomly assigned to four groups and, immediately after laminectomy and SCI operation, received an intralesional injection of normally conditioned sEVs (CON-sEVs), EF-sEVs, or an equal volume of PBS using a microneedle. Specifically, when the spinal cord was contused, immediately after hemostasis, 80 µL of either PBS or PBS containing CON-sEVs or EF-sEVs (2.4 mg total protein, Bicinchoninic acid assay) were slowly injected into the upper and lower sides of the spinal cord lesion site with a depth of 0.9 mm using a pulled-glass micropipette. After the solution was absorbed, the musculature and skin were sutured sequentially. Rats receiving laminectomy but no spinal cord compression were prepared as a sham group.

Functional behavioral assessments

Neurological function was evaluated using the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale at 1, 7, 14, 21, and 28 days after surgery. In brief, BBB scores range from 0 points (complete paralysis) to 21 points (normal locomotion). Footprint analysis was used to evaluate the recovery of hindlimb muscle strength and motor coordination. The fore- and hindlimbs of the rats were dipped into black and red ink, and the rats walked on a narrow passage covered with paper. The distance between the left and right rear paws was measured and considered the base of support. Stride length was measured as the perpendicular distance between the fore and hind limbs to assess the coordination ability.

Histological analysis

Rats were deep euthanized using 0.6% sodium pentobarbital (10 g/0.1 mL) 4 weeks after SCI. Animals were then intracardially perfused with PBS followed by 4% paraformaldehyde. The spinal cord containing the injury site was dissected, fixed in 4% paraformaldehyde for 24 h, and embedded in paraffin. Samples were then cut into 20 μm sections. A general review and lesion cavity assessment was performed using hematoxylin and eosin (HE) staining in samples from each spinal cord treatment group. Nissl staining was used to estimate the number of neurons at the lesion sites.

Immunofluorescence staining assays

Rat spinal cords (postoperative week 4) were embedded in paraffin and sectioned, and the sections were dehydrated and treated to inhibit peroxidase activity. Next, the sections were blocked using 5% bovine serum albumin and 0.3% Triton X-100 and incubated overnight at 4 °C with primary antibodies. The primary antibodies were anti-neurofilament (NF;Abcam, Cambridge, UK), anti-choline acetyltransferase (ChAT; Omnimabs, Alambra, CA, USA), and anti-glial fibrillary acidic protein (GFAP; Boster Bio, Pleasanton, CA, USA). After standard washing procedures, samples were incubated with secondary fluorescence-conjugated antibodies (Invitrogen, Waltham, MA, USA), and Hoechst (1:300; Sigma-Aldrich, St. Louis, MO, USA) was used to label cell nuclei. Images were captured using a microscope (Nikon, Tokyo, Japan).

Cell culture and induction of EF

hucMSCs were obtained and cultured as previously reported [27]. In brief, fresh umbilical cord tissue was acquired from patients in the Affiliated Hospital of Jiangsu University after written informed consent was obtained. The use of human umbilical cord samples was approved by the ethics committee of Jiangsu University. hucMSCs were maintained in 10% fetal bovine serum (FBS; ExCell Bio, Shanghai, China) and Dulbecco’s Modified Eagle Medium. hucMSCs from passages 3–5 were used for further experiments. We designed an novel EF device in vitro for delivering direct current electrical stimulation to cultivated cells (Additional file 1: Fig. S1) and was manufactured by Changzhou Ruishen’an Medical Equipment Co., LTD. The hucMSCs were cultured under a 100 mV/mm electric field intensity 1 h per day for three consecutive days. Our previous study have determined that the optimal stimulus parameter was 100 mV/mm 1 h per day. PC12 cells were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and maintained in 10% FBS (Excell Bio, China) and RPMI 1640 medium (Invitrogen, USA).

sEVs isolation and identification

sEVs were extracted using ultracentrifugation as previously described [27]. The culture medium of the hucMSCs was replaced with sEVs-depleted 10% FBS, with or without a 100 mV/mm microcurrent, for an additional 3 days. When the confluency of the hucMSCs reached 90–100%, the two supernatants were collected and the both types of sEVs were extracted using standard methods, labeled as CON-sEVs and EF-sEVs, respectively. The morphology and size of the CON-sEVs and EF-sEVs were identified using transmission electron microscopy (FEI Tecnai 12; Philips, Amsterdam, Netherlands) and nanosight tracking analysis (NTA; Particle Metrix, Ammersee, Germany). The protein content in sEVs was measured using a Pierce Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific). Western blot analysis was used to identify sEVs surface markers including Alix, CD81, CD63, TSG101, and calnexin. For sEVs uptake experiments, CON-sEVs and EF-sEVs were incubated with DiI dye solution for 30–60 min at 37 °C in darkness. Labeled CON-sEVs and EF-sEVs were incubated separately with PC12 cells and imaged using confocal microscopy (Beckman Coulter, USA).

RNA sequencing

For lncRNA-seq experiments, total RNA was extracted from sEVs using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions and RNA integrity numbers were analyzed using an Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Qualified total RNA was further purified using the RNA Clean XP Kit (Cat#A63987; Beckman Coulter, Inc., Brea, CA, USA) and RNase-Free DNase Set (Cat#79254, Qiagen). Purified total RNA was subjected to rRNA removal, fragmentation, first-strand cDNA synthesis, second-strand cDNA synthesis, end repair, 3′ end addition, ligation, and enrichment to complete the library construction of sequencing samples. The Illumina NovaSeq6000 sequencer was used, and the PE150 mode was selected for sequencing.

Quantitative real-time PCR

Total RNA was extracted from cells and sEVs using TRIzol (Invitrogen, USA), and cDNA was synthesized using a reverse transcription kit (Nanjing Vazyme Biotech Co, Nanjing, China) with total RNA for mixing with SYBR-Green reagents to perform qRT-PCR experiments. Expression levels were evaluated using the 2−ΔΔCT method. The sequences of primers were listed in Additional file 2: Table S1.

Oligonucleotide transfection and lentivirus transduction

MiR-22-3p mimics, mimics NC, miR-22-3p inhibitor, and inhibitor NC were synthesized and purified by GenePharma (Suzhou, China). Transfection was performed using Lipofectamine 2000 (Invitrogen, USA). After transfection for 30 h, total RNA was isolated from PC12 cells. Lentiviral constructs for short hairpin MALAT1 (shMALAT1) were generated by Hanbio (Shanghai, China). Scrambled lentiviral constructs were used as negative controls. The sequences of the shRNA were listed in Additional file 2: Table S2.

Establishment of an in vitro model of SCI

The PC12 cell line, derived from pheochromocytoma of Rattus norvegicus, is commonly used to study nervous system diseases. PC12 cells have strong viability, and it has been previously confirmed that PC12 cells exposed to hydrogen peroxide (H2O2) can effectively replicate the effects of SCI in vitro [28]. This cellular SCI model is often used in in vitro studies of SCI. Therefore, the PC12 cells were exposed to 200µM H2O2 solution in Dulbecco’s Modified Eagle Medium without FBS for 24 h to develop the H2O2-induced model of oxidative stress.

Flow cytometry

The rate of apoptosis was examined using flow cytometry. H2O2-induced PC12 cells were co-cultured with sEVs for 24 h, and cells were stained using the Annexin V-Fluorescein Isothiocyanate and Propidium Iodide Apoptosis Kit (Nanjing Vazyme Biotech Co, China). Cellular apoptosis rates were then estimated using flow cytometry (BD, FACSCalibur, USA).

Reactive oxygen species (ROS) measurement

To evaluate the protective function of sEVs for PC12 cells against oxidative stress, we performed a range of experiments. PC12 cells were seeded on a 12well plate and co-cultured with different sEVs. Subsequently, the culture medium was replaced with peroxidative medium containing 200 µM H2O2 for 24 h. Following the peroxidative culture, reactive oxygen species (ROS) levels were measured using an ROS assay kit (Beijing Solarbio Science & Technology Co., Ltd.) in accordance with the manufacturer’s protocols. Samples were imaged using fluorescence microscopy (Olympus, Tokyo, Japan), and relative fluorescence was calculated using ImageJ and GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) software.

Western blot

Cells and spinal cord tissue were lysed using radio immunoprecipitation assay buffer (Thermo Fisher Scientific). Equal amounts of proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and then transferred to polyvinylidene difluoride membranes, blocked with 5% bovine serum albumin, and incubated overnight at 4 °C with primary antibodies against p-AMPK (1:1000; Bioworld, USA), cleaved caspase 3 (1:1000; Cell Signal Technology, Danvers, MA, USA), Bcl-2 (1:1000; Abcam, UK), Bax (1:1000; Abcam, UK), beclin-1 (1:1000; Abcam, UK), LC3B (1:1000; Abcam, UK), P62 (1:1000; Abcam, UK), SIRT1 (1:1000; Abcam, UK), TSG101 (1:1000; Abcam, UK), CD63 (1:1000; Abcam, UK), CD81 (1:1000; Abcam, UK), β-actin (1:1000; Abcam, UK). The next day, the membranes were incubated with secondary antibodies (1:10,000; Jackson ImmunoResearch Labs, West Grove, PA, USA). Protein bands were detected using an ImageQuant LAS4000 mini chemiluminescence imager (GE Healthcare, Chicago, IL, USA).

Luciferase assay

The MALAT1 mimic or MALAT1 inhibitor luciferase reporter plasmids and miR-22-3p mimics or miR-22-3p inhibitors were co-transfected into HEK293T cells together with the Renilla luciferase gene. Six hours following transfection, cells were cultured in complete medium for 18 h. Cells were then lysed, and luciferase activity was assessed using an enhanced luciferase assay kit (Nanjing Vazyme Biotech Co) following the manufacturer’s instructions. Luciferase activity levels were normalized to Renilla activity levels. Then, SIRT1 mimic or SIRT1 inhibitor luciferase reporter plasmids and miR-22-3p mimics or miR-22-3p inhibitors were also detected as described above. All plasmids and oligonucleotides were synthesized by Genepharma, and their sequences and modifications are shown in Additional file 2: Table S1.

Statistical analysis

All data represent at least three independent replicates. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software). Data are presented as mean ± standard deviation (SD). The independent samples Student’s t-test was used to analyze differences between two unpaired groups, and differences between multiple groups were analyzed using one-way analysis of variance. P-values < 0.05 were considered statistically significant.