Cell culture
Human umbilical vein ECs (HUVECs) were purchased from Procell Life Science&Technology Co., Ltd (Wuhan, China). HUVECs were cultured in EC medium (ScienCell, USA) supplemented with 10% exosome-depleted foetal bovine serum (SBI, USA), 1% EC growth supplement (ScienCell) and 1% penicillin/streptomycin solution (ScienCell), and cells were cultured at 37 ℃ in humidified air containing 5% CO2. HUVECs were performed for the experiments in passages 4–6.
Rat SCs (RSC96 cells) were purchased from Procell Life Science&Technology Co., Ltd. SCs were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, USA). Moreover, the culture mediums were supplemented with 10% exosome-depleted FBS (SBI). RSC96 cells were performed for the experiments in passages 4–6.
Isolation and characterization of EC-derived exosomes
ECs (Passage 4–6) were cultured in 10% exosome-depleted FBS DMEM for 48 h to produce enough exosomes. Then the media was collected and centrifuged at 800 ×g for 10 min and 5000 ×g for 10 min to remove cells and cell debris. The supernatant was filtered by a 0.22-μM filter (Millipore). The supernatant was centrifuged at 100,000 g for 2 h at 4 ℃ to pellet exosomes (Micro Ultracentrifuge, Himac, Japan). Next, the exosomes were washed with PBS and centrifuged at 100,000 ×g for 2 h. Exosomes were eventually resuspended by 100 μL PBS. Exosomes were measured with the Bicinchoninic Acid (BCA) Protein Assay Kit (Solarbio, China), about 32.1 μg exosomes could be extracted from 1 mL EC culture medium. Then exosomes used for transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were stored at 4 ℃ and used within 48 h. Other exosomes were stored at – 80 ℃ until use.
The exosomal markers TSG101, CD9, CD81 and GAPDH were analysed by western blot for ECs and exosomes derived from ECs. In addition, NTA measured exosome size distribution and zeta potential with the NICOMP Nano-ZLS Z3000 instrument (Beckman Coulter, USA). Exosomes were fixed with 2% glutaraldehyde stationary liquid. Exosome suspension was dropped onto the copper grid with carbon film for 5 min, and 2% phosphotungstic acid was dropped on the copper grid to stain for 5 min at room temperature. The exosomes are observed under TEM (HITACHI, Japan).
Exosomes labeling and in vitro uptake
Exosomes were labelled with 10 mg/mL 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Beyotime) at a volume ratio of 1:100 for 30 min in the dark at 37 ℃ according to the manufacturer’s procedures. Then the labelled exosomes suspension was dialyzed for 12 h in a 100 KD-aperture dialysis bag (Shanghai yuanye Bio-Technology) to remove the residual fluorescent dye. SCs were seeded in the 20 mm glass-bottom cell culture dish (Nest, China) and incubated with DiI-labelled exosomes for 0 h, 2 h, 6 h, 12 h and 24 h. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Servicrbio, China), and cells were observed under confocal laser scanning microscopy (LSM880, Zeiss, Germany).
Cell proliferation and colony-forming assay
SCs were seeded on 96-well plates at a density of 2 × 104 cells/mL overnight. EC-EXO of different concentrations (1, 10, 50 and 100 μg/mL) were added to each well and cultured for 24 h and 48 h. Then 10 μL of Cell Counting Kit-8 (Dojindo, Japan) was added into each well and incubated for 2 h at 37 ℃ in humidified air containing 5% CO2. The cell proliferation was determined using a full-wavelength microplate reader at 450 nm. We also used the CCK8 assay to study the growth curve of SCs in different groups. Furthermore, the 5-ethynyl-2ʹ-deoxyuridine (EdU) Cell Proliferation Assay Kit (Ribobio) was also used to measure the cell proliferation of SCs in different groups. EdU-labelled cells were counted manually in three fields of view randomly chosen from each well to calculate the percentages. To study the clonogenic ability of SCs in different groups, cells with different treatments were seeded to 6-well plates (1000 cells/well). SCs were cultured for 10 days and stained with 0.1% Crystal Violet Stain solution (Solarbio). The numbers of colonies were counted manually, and the inverted microscope detected the morphology of the colonies from different groups.
Cell cycle and apoptosis analyses
SCs were seeded into 6-well plates (1 × 105 cells/mL) and 50 μg/mL EC-EXO or PBS were added in the corresponding well for 24 h. The cells were collected and resuspended by PBS. Then the Cell Cycle Staining Kit (MultiSciences Biotech, China) was used for cell cycle analyses and the Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme Biotech, China) was used for cell apoptosis analyses according to the manufacturer’s protocol. The cell cycle and apoptosis were detected by flow cytometry analysis. Moreover, data acquisition and analysis were performed using NovoExpress software.
Migration assay
The migration of SCs was evaluated using a transwell with 8 μm pores (Corning, USA). To study the effect of ECs on SC migration and the role of exosomes play in this process, we carried out a coculture of ECs and SCs in the transwell system. First, SCs were seeded at 1 × 104 cells in 100 μL in DMEM supplemented with 1% FBS onto the upper chamber [66]. Then, ECs, ECs with GW4869 (Umibio), the inhibitor of exosome secretion, PBS and PBS with GW4869 were added to the lower chambers. Besides, we also filled the lower chamber of the transwell with a medium including EC-EXO of different concentrations (1, 10, 50, 100 μg/mL) to further estimate the influence of EC-EXO on SC migration more directly. First, cells were incubated for 24 h at 37 ℃ and non-migrated cells were removed using cotton swabs. Next, migrated cells were fixed with 4% paraformaldehyde (Solarbio) for 30 min. Next, PBS washed the fixed cells. Then cells were stained with 0.1% Crystal Violet Stain solution for 30 min. Following 12 h of drying, stained cells were observed with an inverted microscope, and the number of migrated cells was counted using ImageJ software.
Sequencing of miRNAs and data analysis
Total RNA was extracted from SCs treated with EC-EXO or PBS for 24 h by RNAiso Plus (Takara, Japan) and analyzed for RNA integrity and total amount with 2100 bioanalyzer (Agilent, CA, USA). The final ligation PCR products were sequenced using the BGISEQ-500 platform (BGI Group, China). Following acquiring the raw data, the differentially expressed miRNAs were calculated using the t test. The data with ≥ twofold upregulation and a P value < 0.05 were regarded as significantly different.
MiRNA real-time quantitative PCR
Total RNAs in SCs treated with EC-EXO for 24 h were isolated using RNAiso Plus (Takara, Japan) and recerse transcribed using a miRNA first-strand cDNA synthesis kit (Takara, Japan) according to the manufacturer's guidelines. RT–qPCR was performed in a 20 μL reaction system involving forward/reverse primers, cDNA, and NovoStart SYBR qPCR SuperMix Plus (Novoprotein Scientific, China) according to manufacturer’s instructions. We set three replicates in each group and used the 2−ΔΔCT method. The primers were purchased from Ribobio Biotech.
Transfection of miRNA mimic, mimic negative control, miRNA inhibitor and inhibitor negative control
For transfection of the mimic negative control (Mi-NC), miR199-5p mimic, inhibitor negative control (In-NC) and miR199-5p inhibitor (Ribobio, China) at a final concentration of 50 nM using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's instructions. The sequence of miR199-5p is: ACUGGACUUGGAGUCAGAAG.
RNA sequencing
RNA was extracted from SCs treated with or without EC-EXO for 24 h by RNAiso Plus (Takara, Japan) and analyzed for RNA integrity and total amount with 2100 bioanalyzer (Agilent, CA, USA). RNA sequencing library was prepared and sequenced on Illumina HiSeq 6000 (Illumina, CA, USA). The sequencing service was provided by Novogene (Beijing, China). The DESeq2 R package (1.20.0) was used to analyze two groups’ differential expression genes (DEGs). Moreover, the DEGs were screened out on the ground of the threshold of P-value ≤ 0.05 and |log2FoldChange|≥ 1. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Gene set enrichment analysis (GSEA) of DEGs was implemented by the clusterProfiler R package (3.8.1).
Real-time quantitative polymerase chain reaction (RT–qPCR)
SCs in each group were used for RNA extration with RNAiso Plus. Total RNA was transcribed into complementary DNAs (cDNAs) by the NovoScriptPlus All-in-one 1st Strand cDNA Synthesis SuperMix (gDNA Purge; Novoprotein Scientific, China). RT–qPCR was performed in a 20 μL reaction system involving forward/reverse primers, cDNA, and NovoStart SYBR qPCR SuperMix Plus according to manufacturer's instructions. We set three replicates in each group and used the 2−ΔΔCT method. All primers used in this study were listed in (Additional file 1: Table S1).
Western blot analysis
Exosomes lysate and cell lysate were prepared by RIPA Lysis Buffer (Yazyme, China) added with Protease Inhibitor Cocktail (Yazyme) and Phosphatase Inhibitor Cocktail (Yazyme). Total proteins were separated in SDS-polyacryla-mide gel (Yazyme) and transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime). The membranes were blocked using Protein Free Rapid Blocking Buffer (Yazyme) for 30 min at room temperature. The blocked membranes were incubated overnight at 4 ℃ with antibodies specific for the TSG101 (Abcam, 1:1000), CD9 (Abcam, 1:1000), CD81 (Abcam, 1:1000), GAPDH (Abcam, 1:1000), BAX (Abcam, 1:1000), Bcl2 (Abcam, 1:1000), PCNA (CST, 1:1000), c-Jun (Abcam, 1:1000), STAT3 (CST, 1:1000), p-STAT3 (CST, 1:1000), PI3K (Abcam, 1:1000), p-PI3K(Affinity, China, 1:1000), PTEN (Abcam, 1:1000), AKT (Abcam, 1:10,000), p-AKT (Abcam, 1:5000), NGF (Abcam, 1:1000), VEGFA (Abcam, 1:1000), CNTF (Abcam, 1:1000), BDNF (Abcam, 1:5000) and GDNF (Abcam, 1:1000) and β-actin (Solarbio, 1:1000). Then the membranes were washed and incubated with horseradish peroxidase (HRP)-coupled secondary antibodies (Solarbio). The blots were detected using Amersham Imager 600. GAPDH or β-actin was used as the loading control, and the interested protein's relative intensity was normalized to that of the control group. LY294002, a PI3K inhibitor (PI3Ki, GLPBIO) was also used as a well-known PI3K signaling pathway inhibitor.
Animal model and EC-EXO delivery
Adult male Sprague–Dawley rats (300–400 g) were obtained from Beijing Vital River Laboratory Animal Technology Co.,Ltd (China, Beijing). The living conditions and experimental procedures conformed to the National Institutes of Health (NIH) Guide Concerning the Cre and Use of Laboratory Animals. In addition, the whole animal experiment was approved by the Animal Experimentation Ethics Committee of Zhengzhou University. Five rats per cage were kept in the specific-pathogen-free (SPF) room with constant temperature (23–24 ℃), humidity (55 ± 5%), and light (12 h light–dark cycle). All rats had free access to food and water.
The rats were randomly divided into three experimental groups (n = 5 for each group): a sham group, a PNI group, and an exosome treatment (EXO) group. Following an effective inhalation of ether, the rat was intraperitoneally injected with 2.0 mL per kg body weight of 2% pentobarbital sodium. Then the right nerve of the rat was exposed using the gluteal muscle dissection method. First, the PNI model was established at a location 5 mm away from the sciatic notch using Dumont No. Five forceps three times (10 s each time, 10 s intervals). Subsequently, the PNI group received multi-site injections of 20 μL of PBS without EXO under the epineurium of the sciatic nerve by using a micro syringe (Hamilton, USA). After each injection, the needle was indwelled for 30 s to prevent leaking out. Then a 2-mm-long translucent band was formed at the injury site, which was marked with a 10–0 nylon epineural suture for later identification. Next, the EXO group received multi-site injections of 20 μL of 50 μg/mL EC-EXO under the epineurium of the sciatic nerve by using a micro syringe [16]. Finally, the rats in the sham group underwent the same procedure without suffering any sciatic nerve damage.
Bioluminescence imaging
Exosomes were stained with the liposomal dye DiR (US Everbright, China) according to the manufacturer's instructions to visualize their distribution in vivo. Then the labelled exosomes suspension was dialyzed in a 100 KD-aperture dialysis bag for 12 h to remove the residual fluorescent dye. As for the control group, the DiR dye was diluted in PBS, then the solution was also dialyzed in a 100 KD-aperture dialysis bag for 12 h to verify the interference of vestigital DiR to this experiment. Injection of DiR-labelled exosomes and control solution under the epineurium of the sciatic nerve was performed in the rats by using a micro syringe (dosage per rat: 1 μg of DiR-labelled exosomes, in 20 μL of PBS), and 20 μL of DiR solution was used as the control (n = 3 for each group). An IVIS imaging system (PerkinElmer, USA) was used to perform living and sciatic nerve tissue imaging 1 day, 3 day, 7 day, 14 day and 28 day after the injection.
Exosomes labeling and in vivo uptake
To further visualize the distribution of exosomes in sciatic nerve, exosomes were prestained with the DiI according to the manufacturer's instructions. Then the labelled exosomes suspension was dialyzed in a 100 KD-aperture dialysis bag to remove the residual fluorescent dye. For the control group, the DiI dye was diluted in PBS, then the solution was also dialyzed in a 100 KD-aperture dialysis bag for 12 h as above. As stated above,we used a micro syringe to inject DiI-labelled exosomes locally under the epineurium of the sciatic nerve (dosage per rat: 1 μg of DiI-labelled exosomes, in 20 μL of PBS), and 20 μL of DiI solution was used as the control. After 1 day, 3 day, 7 day, 14 day and 28 day, the rats were sacrificed and the sciatic nerves were embedded in Tissue-Tek O.C.T. (Leica, Wetzlar, Germany) to make frozen blocks for fluorescent staining. Nuclei were stained with DAPI, and the sections were observed under confocal laser scanning microscopy.
TUNEL staining
The apoptosis rate in each group of rat sciatic nerve was detected using a TUNEL staining kit (Vazyme) according to the manufacturer’s instructions. Nuclei were stained with DAPI. TUNEL-labelled cells were stained with green fluorescence and counted manually in three fields of view randomly chosen from each well to calculate the percentages.
Walking track analysis
To evaluate the motion function following nerve injury, walking track analysis was used on the injury model rats postoperatively at 3 days before operation, 7, 14, and 28 days following operation. In this trial, the plantar surfaces of both hind paws were painted with Eosin Y solution (Solarbio), and the rat was allowed to walk along a narrow corridor with white paper on the base towards a dark compartment at the end. Paw length (PL), the toe-spread distance between toes 1 and 5 (TS), and toe-spread distance (IT) between toes 2 and 4 were recorded from the normal (N) and experimental (E) hind limbs. Sciatic Functional Index (SFI) was calculated as the following formula [67].
$${\text{SFI}} =\, \frac{{ - 38.3 \times \left( {{\text{EPL}} - {\text{NPL}}} \right)}}{{{\text{NPL}}}} + \frac{{109.5 \times \left( {{\text{ETS}} - {\text{NTS}}} \right)}}{{{\text{NTS}}}} + \frac{{13.3 \times \left( {{\text{EIT}} - {\text{NIT}}} \right)}}{{{\text{NIT}}}} - 8.8$$
The rat footprint measurements could evaluate the functional muscle status of the hind limbs according to the walking track analysis [68]. In general, a SFI value of 0 indicates normal neurological function, while a SFI value of -100 indicates complete loss of motor function.
Electrophysiological assessment
The electrophysiological assessment was conducted using previously developed methods [69]. MD3000-C multichannel physiological signal acquisition and processing system (Anhui Zhenghua Biological Instrument, China) was used to evaluate functional recovery 28 days after the operation [70]. First, the rats were anesthetized, and the sciatic nerve tissues were exposed. Bipolar electrodes were placed at the proximal end of the crushed site to send single electrical stimulation. In the meantime, a recording electrode was inserted into the homolateral gastrocnemius muscle. The recorded nerve’s compound muscle action potentials (CMAPs) were obtained to perform a comparative analysis of the three groups.
Histological and morphological analysis of regenerated nerve
At 28 days after the operation, the sciatic nerves were removed and fixed overnight in 4% paraformaldehyde (PFA), then dehydrated in gradient grade ethanol, and embedded in paraffin. Longitudinal and transverse sections (5 μm) were de-waxed and hydrated after paraffin embedding. The nerve sections were stained with Hematoxylin–eosin (HE) and Masson staining according to manufacturer's instructions. At last, slides were fixed with neutral resin and capped. Images of stained sections were acquired with a light microscope (Olympus, Japan).
Electron microscopy and TB staining assessments
The sciatic nerves 3–5 mm distal to the injury site were harvested and put in 2.5% glutaraldehyde overnight. The tissues were immersed in 1% osmic acid for 2 h and dehydrated with acetone. Then the samples were encapsulated in epoxy resin and oven-dried. The tissues were sectioned into 0.5 μm semi-thin cross-sections and 70.0 nm ultrathin cross-sections. The semi-thin sections were stained with toluidine blue (TB) and observed using a light microscope. The ultrathin cross-sections were examined using a projection electron microscope (Hitachi). ImageJ software measured myelinated axon number, G-ratio (inner axonal diameter to fiber diameter ratio), and myelin sheath thickness. We randomly selected 3 representative TEM pictures and counted the mean thickness and G-ratio of all the myelin sheath in each picture.
Histological assessment of muscle
At 28 days after the operation, bilateral gastrocnemius muscles were harvested from rats and weighed promptly to acquire the muscle relative wet weight ratio by calculating the ratio of ipsilateral muscle weight. Then the experimental gastrocnemius muscle belly was fixed, embedded in paraffin, and stained with HE and Masson staining. Finally, the representative images of stained sections were observed with a light microscope. We randomly selected 3 representative HE staining pictures and counted the mean muscle fiber diameter in each picture.
Immunofluorescence staining and immunofluorescence evaluation
At 28 days following the operation, the sciatic nerve tissues containing the area of crush injury site (5 mm away from the sciatic notch) were harvested and fixed with 4% paraformaldehyde. Then the longitudinal sections and transverse sections of the nerve tissue were prepared. Moreover, the sections were stained with NF200 (CST, 1:200), S100β (Abcam, 1:200), TuJ1 (Abcam, 1:200), MBP (Abcam, 1:200), CD31 (Abcam, 1:200), CD34 (Abcam, 1:200), VEGFR (Abcam, 1:200), GAPDH (Abcam, 1:200), Akt (Abcam, 1:500), p-AKT (Abcam, 1:500), PI3K (Abcam, 1:500), p-PI3K (ThermoFisher, 1:500) and PTEN (Abcam, 1:500). Secondary antibodies were as follows:Alexa Fluor568–conjugated Goat Anti-Rabbit IgG (Abcam), CoraLite594-conjugated Goat Anti-Mouse IgG (Proteintech, China), CoraLite488-conjugated Goat Anti-Rabbit IgG (Proteintech), CY3-labeled goat anti-rabbit (Servicebio) and AlexaFluor594-labeled goat anti-rabbit IgG (Abcam). Besides, we also used FITC-Tyramide (Servicebio) and CY3-Tyramide (Servicebio) to amplify fluorescence intensity. Nuclei were stained with DAPI, and the sections were observed under confocal laser scanning microscopy. The percentages of the markers positive areas were calculated by dividing integrated option density by selected region area, then multiplied by 100%. All parameters were measured using ImageJ.
Statistical analysis
Statistical analysis was conducted using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). The results were presented as mean ± SD. One-way ANOVA was used for comparisons within multiple groups, and a two-tailed unpaired Student's test was used for comparisons between two groups. P values < 0.05 were considered statistically significant.
