Cell tradition
Human umbilical vein ECs (HUVECs) have been bought from Procell Life Science&Know-how Co., Ltd (Wuhan, China). HUVECs have been cultured in EC medium (ScienCell, USA) supplemented with 10% exosome-depleted foetal bovine serum (SBI, USA), 1% EC development complement (ScienCell) and 1% penicillin/streptomycin resolution (ScienCell), and cells have been cultured at 37 ℃ in humidified air containing 5% CO2. HUVECs have been carried out for the experiments in passages 4–6.
Rat SCs (RSC96 cells) have been bought from Procell Life Science&Know-how Co., Ltd. SCs have been cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, USA). Furthermore, the tradition mediums have been supplemented with 10% exosome-depleted FBS (SBI). RSC96 cells have been carried out for the experiments in passages 4–6.
Isolation and characterization of EC-derived exosomes
ECs (Passage 4–6) have been cultured in 10% exosome-depleted FBS DMEM for 48 h to provide sufficient exosomes. Then the media was collected and centrifuged at 800 ×g for 10 min and 5000 ×g for 10 min to take away cells and cell particles. The supernatant was filtered by a 0.22-μM filter (Millipore). The supernatant was centrifuged at 100,000 g for two h at 4 ℃ to pellet exosomes (Micro Ultracentrifuge, Himac, Japan). Subsequent, the exosomes have been washed with PBS and centrifuged at 100,000 ×g for two h. Exosomes have been finally resuspended by 100 μL PBS. Exosomes have been measured with the Bicinchoninic Acid (BCA) Protein Assay Package (Solarbio, China), about 32.1 μg exosomes could possibly be extracted from 1 mL EC tradition medium. Then exosomes used for transmission electron microscopy (TEM) and nanoparticle monitoring evaluation (NTA) have been saved at 4 ℃ and used inside 48 h. Different exosomes have been saved at – 80 ℃ till use.
The exosomal markers TSG101, CD9, CD81 and GAPDH have been analysed by western blot for ECs and exosomes derived from ECs. As well as, NTA measured exosome dimension distribution and zeta potential with the NICOMP Nano-ZLS Z3000 instrument (Beckman Coulter, USA). Exosomes have been fastened with 2% glutaraldehyde stationary liquid. Exosome suspension was dropped onto the copper grid with carbon movie for five min, and a couple of% phosphotungstic acid was dropped on the copper grid to stain for five min at room temperature. The exosomes are noticed beneath TEM (HITACHI, Japan).
Exosomes labeling and in vitro uptake
Exosomes have been labelled with 10 mg/mL 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Beyotime) at a quantity ratio of 1:100 for 30 min at midnight at 37 ℃ in response to the producer’s procedures. Then the labelled exosomes suspension was dialyzed for 12 h in a 100 KD-aperture dialysis bag (Shanghai yuanye Bio-Know-how) to take away the residual fluorescent dye. SCs have been seeded within the 20 mm glass-bottom cell tradition dish (Nest, China) and incubated with DiI-labelled exosomes for 0 h, 2 h, 6 h, 12 h and 24 h. Nuclei have been stained with 4′,6-diamidino-2-phenylindole (DAPI; Servicrbio, China), and cells have been noticed beneath confocal laser scanning microscopy (LSM880, Zeiss, Germany).
Cell proliferation and colony-forming assay
SCs have been seeded on 96-well plates at a density of two × 104 cells/mL in a single day. EC-EXO of various concentrations (1, 10, 50 and 100 μg/mL) have been added to every effectively and cultured for twenty-four h and 48 h. Then 10 μL of Cell Counting Package-8 (Dojindo, Japan) was added into every effectively and incubated for two h at 37 ℃ in humidified air containing 5% CO2. The cell proliferation was decided utilizing a full-wavelength microplate reader at 450 nm. We additionally used the CCK8 assay to check the expansion curve of SCs in numerous teams. Moreover, the 5-ethynyl-2ʹ-deoxyuridine (EdU) Cell Proliferation Assay Package (Ribobio) was additionally used to measure the cell proliferation of SCs in numerous teams. EdU-labelled cells have been counted manually in three fields of view randomly chosen from every effectively to calculate the chances. To check the clonogenic potential of SCs in numerous teams, cells with totally different therapies have been seeded to 6-well plates (1000 cells/effectively). SCs have been cultured for 10 days and stained with 0.1% Crystal Violet Stain resolution (Solarbio). The numbers of colonies have been counted manually, and the inverted microscope detected the morphology of the colonies from totally different teams.
Cell cycle and apoptosis analyses
SCs have been seeded into 6-well plates (1 × 105 cells/mL) and 50 μg/mL EC-EXO or PBS have been added within the corresponding effectively for twenty-four h. The cells have been collected and resuspended by PBS. Then the Cell Cycle Staining Package (MultiSciences Biotech, China) was used for cell cycle analyses and the Annexin V-FITC/PI Apoptosis Detection Package (Vazyme Biotech, China) was used for cell apoptosis analyses in response to the producer’s protocol. The cell cycle and apoptosis have been detected by move cytometry evaluation. Furthermore, information acquisition and evaluation have been carried out utilizing NovoExpress software program.
Migration assay
The migration of SCs was evaluated utilizing a transwell with 8 μm pores (Corning, USA). To check the impact of ECs on SC migration and the function of exosomes play on this course of, we carried out a coculture of ECs and SCs within the transwell system. First, SCs have been seeded at 1 × 104 cells in 100 μL in DMEM supplemented with 1% FBS onto the higher chamber [66]. Then, ECs, ECs with GW4869 (Umibio), the inhibitor of exosome secretion, PBS and PBS with GW4869 have been added to the decrease chambers. Moreover, we additionally crammed the decrease chamber of the transwell with a medium together with EC-EXO of various concentrations (1, 10, 50, 100 μg/mL) to additional estimate the affect of EC-EXO on SC migration extra immediately. First, cells have been incubated for twenty-four h at 37 ℃ and non-migrated cells have been eliminated utilizing cotton swabs. Subsequent, migrated cells have been fastened with 4% paraformaldehyde (Solarbio) for 30 min. Subsequent, PBS washed the fastened cells. Then cells have been stained with 0.1% Crystal Violet Stain resolution for 30 min. Following 12 h of drying, stained cells have been noticed with an inverted microscope, and the variety of migrated cells was counted utilizing ImageJ software program.
Sequencing of miRNAs and information evaluation
Whole RNA was extracted from SCs handled with EC-EXO or PBS for twenty-four h by RNAiso Plus (Takara, Japan) and analyzed for RNA integrity and complete quantity with 2100 bioanalyzer (Agilent, CA, USA). The ultimate ligation PCR merchandise have been sequenced utilizing the BGISEQ-500 platform (BGI Group, China). Following buying the uncooked information, the differentially expressed miRNAs have been calculated utilizing the t check. The information with ≥ twofold upregulation and a P worth < 0.05 have been considered considerably totally different.
MiRNA real-time quantitative PCR
Whole RNAs in SCs handled with EC-EXO for twenty-four h have been remoted utilizing RNAiso Plus (Takara, Japan) and recerse transcribed utilizing a miRNA first-strand cDNA synthesis equipment (Takara, Japan) in response to the producer’s pointers. RT–qPCR was carried out in a 20 μL response system involving ahead/reverse primers, cDNA, and NovoStart SYBR qPCR SuperMix Plus (Novoprotein Scientific, China) in response to producer’s directions. We set three replicates in every group and used the two−ΔΔCT methodology. The primers have been bought from Ribobio Biotech.
Transfection of miRNA mimic, mimic unfavorable management, miRNA inhibitor and inhibitor unfavorable management
For transfection of the mimic unfavorable management (Mi-NC), miR199-5p mimic, inhibitor unfavorable management (In-NC) and miR199-5p inhibitor (Ribobio, China) at a ultimate focus of fifty nM utilizing Lipofectamine 3000 (Invitrogen, USA) in response to the producer’s directions. The sequence of miR199-5p is: ACUGGACUUGGAGUCAGAAG.
RNA sequencing
RNA was extracted from SCs handled with or with out EC-EXO for twenty-four h by RNAiso Plus (Takara, Japan) and analyzed for RNA integrity and complete quantity with 2100 bioanalyzer (Agilent, CA, USA). RNA sequencing library was ready and sequenced on Illumina HiSeq 6000 (Illumina, CA, USA). The sequencing service was supplied by Novogene (Beijing, China). The DESeq2 R package deal (1.20.0) was used to investigate two teams’ differential expression genes (DEGs). Furthermore, the DEGs have been screened out on the bottom of the edge of P-value ≤ 0.05 and |log2FoldChange|≥ 1. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment evaluation and Gene set enrichment evaluation (GSEA) of DEGs was applied by the clusterProfiler R package deal (3.8.1).
Actual-time quantitative polymerase chain response (RT–qPCR)
SCs in every group have been used for RNA extration with RNAiso Plus. Whole 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 carried out in a 20 μL response system involving ahead/reverse primers, cDNA, and NovoStart SYBR qPCR SuperMix Plus in response to producer’s directions. We set three replicates in every group and used the two−ΔΔCT methodology. All primers used on this research have been listed in (Extra file 1: Desk S1).
Western blot evaluation
Exosomes lysate and cell lysate have been ready by RIPA Lysis Buffer (Yazyme, China) added with Protease Inhibitor Cocktail (Yazyme) and Phosphatase Inhibitor Cocktail (Yazyme). Whole proteins have been separated in SDS-polyacryla-mide gel (Yazyme) and transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime). The membranes have been blocked utilizing Protein Free Speedy Blocking Buffer (Yazyme) for 30 min at room temperature. The blocked membranes have been incubated in a single day at 4 ℃ with antibodies particular 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 have been washed and incubated with horseradish peroxidase (HRP)-coupled secondary antibodies (Solarbio). The blots have been detected utilizing Amersham Imager 600. GAPDH or β-actin was used because the loading management, and the protein’s relative depth was normalized to that of the management group. LY294002, a PI3K inhibitor (PI3Ki, GLPBIO) was additionally used as a well known PI3K signaling pathway inhibitor.
Animal mannequin and EC-EXO supply
Grownup male Sprague–Dawley rats (300–400 g) have been obtained from Beijing Very important River Laboratory Animal Know-how Co.,Ltd (China, Beijing). The dwelling situations and experimental procedures conformed to the Nationwide Institutes of Well being (NIH) Information Regarding the Cre and Use of Laboratory Animals. As well as, the entire animal experiment was accepted by the Animal Experimentation Ethics Committee of Zhengzhou College. 5 rats per cage have been saved within the specific-pathogen-free (SPF) room with fixed temperature (23–24 ℃), humidity (55 ± 5%), and lightweight (12 h gentle–darkish cycle). All rats had free entry to meals and water.
The rats have been randomly divided into three experimental teams (n = 5 for every group): a sham group, a PNI group, and an exosome remedy (EXO) group. Following an efficient inhalation of ether, the rat was intraperitoneally injected with 2.0 mL per kg physique weight of two% pentobarbital sodium. Then the fitting nerve of the rat was uncovered utilizing the gluteal muscle dissection methodology. First, the PNI mannequin was established at a location 5 mm away from the sciatic notch utilizing Dumont No. 5 forceps thrice (10 s every time, 10 s intervals). Subsequently, the PNI group acquired multi-site injections of 20 μL of PBS with out EXO beneath the epineurium of the sciatic nerve by utilizing a micro syringe (Hamilton, USA). After every injection, the needle was indwelled for 30 s to stop leaking out. Then a 2-mm-long translucent band was shaped on the damage web site, which was marked with a ten–0 nylon epineural suture for later identification. Subsequent, the EXO group acquired multi-site injections of 20 μL of fifty μg/mL EC-EXO beneath the epineurium of the sciatic nerve by utilizing a micro syringe [16]. Lastly, the rats within the sham group underwent the identical process with out struggling any sciatic nerve harm.
Bioluminescence imaging
Exosomes have been stained with the liposomal dye DiR (US Everbright, China) in response to the producer’s directions to visualise their distribution in vivo. Then the labelled exosomes suspension was dialyzed in a 100 KD-aperture dialysis bag for 12 h to take away the residual fluorescent dye. As for the management group, the DiR dye was diluted in PBS, then the answer was additionally dialyzed in a 100 KD-aperture dialysis bag for 12 h to confirm the interference of vestigital DiR to this experiment. Injection of DiR-labelled exosomes and management resolution beneath the epineurium of the sciatic nerve was carried out within the rats by utilizing a micro syringe (dosage per rat: 1 μg of DiR-labelled exosomes, in 20 μL of PBS), and 20 μL of DiR resolution was used because the management (n = 3 for every group). An IVIS imaging system (PerkinElmer, USA) was used to carry out dwelling 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 additional visualize the distribution of exosomes in sciatic nerve, exosomes have been prestained with the DiI in response to the producer’s directions. Then the labelled exosomes suspension was dialyzed in a 100 KD-aperture dialysis bag to take away the residual fluorescent dye. For the management group, the DiI dye was diluted in PBS, then the answer was additionally dialyzed in a 100 KD-aperture dialysis bag for 12 h as above. As acknowledged above,we used a micro syringe to inject DiI-labelled exosomes regionally beneath 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 resolution was used because the management. After 1 day, 3 day, 7 day, 14 day and 28 day, the rats have been sacrificed and the sciatic nerves have been embedded in Tissue-Tek O.C.T. (Leica, Wetzlar, Germany) to make frozen blocks for fluorescent staining. Nuclei have been stained with DAPI, and the sections have been noticed beneath confocal laser scanning microscopy.
TUNEL staining
The apoptosis fee in every group of rat sciatic nerve was detected utilizing a TUNEL staining equipment (Vazyme) in response to the producer’s directions. Nuclei have been stained with DAPI. TUNEL-labelled cells have been stained with inexperienced fluorescence and counted manually in three fields of view randomly chosen from every effectively to calculate the chances.
Strolling monitor evaluation
To judge the movement operate following nerve damage, strolling monitor evaluation was used on the damage mannequin rats postoperatively at 3 days earlier than operation, 7, 14, and 28 days following operation. On this trial, the plantar surfaces of each hind paws have been painted with Eosin Y resolution (Solarbio), and the rat was allowed to stroll alongside a slender hall with white paper on the bottom in direction of a darkish compartment on the finish. Paw size (PL), the toe-spread distance between toes 1 and 5 (TS), and toe-spread distance (IT) between toes 2 and 4 have been recorded from the traditional (N) and experimental (E) hind limbs. Sciatic Useful Index (SFI) was calculated as the next method [67].
$${textual content{SFI}} =, frac{{ – 38.3 occasions left( {{textual content{EPL}} – {textual content{NPL}}} proper)}}{{{textual content{NPL}}}} + frac{{109.5 occasions left( {{textual content{ETS}} – {textual content{NTS}}} proper)}}{{{textual content{NTS}}}} + frac{{13.3 occasions left( {{textual content{EIT}} – {textual content{NIT}}} proper)}}{{{textual content{NIT}}}} – 8.8$$
The rat footprint measurements might consider the useful muscle standing of the hind limbs in response to the strolling monitor evaluation [68]. On the whole, a SFI worth of 0 signifies regular neurological operate, whereas a SFI worth of –100 signifies full lack of motor operate.
Electrophysiological evaluation
The electrophysiological evaluation was performed utilizing beforehand developed strategies [69]. MD3000-C multichannel physiological sign acquisition and processing system (Anhui Zhenghua Organic Instrument, China) was used to guage useful restoration 28 days after the operation [70]. First, the rats have been anesthetized, and the sciatic nerve tissues have been uncovered. Bipolar electrodes have been positioned on the proximal finish of the crushed web site to ship single electrical stimulation. Within the meantime, a recording electrode was inserted into the homolateral gastrocnemius muscle. The recorded nerve’s compound muscle motion potentials (CMAPs) have been obtained to carry out a comparative evaluation of the three teams.
Histological and morphological evaluation of regenerated nerve
At 28 days after the operation, the sciatic nerves have been eliminated and stuck in a single day in 4% paraformaldehyde (PFA), then dehydrated in gradient grade ethanol, and embedded in paraffin. Longitudinal and transverse sections (5 μm) have been de-waxed and hydrated after paraffin embedding. The nerve sections have been stained with Hematoxylin–eosin (HE) and Masson staining in response to producer’s directions. Ultimately, slides have been fastened with impartial resin and capped. Pictures of stained sections have been acquired with a lightweight microscope (Olympus, Japan).
Electron microscopy and TB staining assessments
The sciatic nerves 3–5 mm distal to the damage web site have been harvested and put in 2.5% glutaraldehyde in a single day. The tissues have been immersed in 1% osmic acid for two h and dehydrated with acetone. Then the samples have been encapsulated in epoxy resin and oven-dried. The tissues have been sectioned into 0.5 μm semi-thin cross-sections and 70.0 nm ultrathin cross-sections. The semi-thin sections have been stained with toluidine blue (TB) and noticed utilizing a lightweight microscope. The ultrathin cross-sections have been examined utilizing a projection electron microscope (Hitachi). ImageJ software program measured myelinated axon quantity, G-ratio (interior axonal diameter to fiber diameter ratio), and myelin sheath thickness. We randomly chosen 3 consultant TEM footage and counted the imply thickness and G-ratio of all of the myelin sheath in every image.
Histological evaluation of muscle
At 28 days after the operation, bilateral gastrocnemius muscle tissue have been harvested from rats and weighed promptly to accumulate the muscle relative moist weight ratio by calculating the ratio of ipsilateral muscle weight. Then the experimental gastrocnemius muscle stomach was fastened, embedded in paraffin, and stained with HE and Masson staining. Lastly, the consultant photos of stained sections have been noticed with a lightweight microscope. We randomly chosen 3 consultant HE staining footage and counted the imply muscle fiber diameter in every image.
Immunofluorescence staining and immunofluorescence analysis
At 28 days following the operation, the sciatic nerve tissues containing the realm of crush damage web site (5 mm away from the sciatic notch) have been harvested and stuck with 4% paraformaldehyde. Then the longitudinal sections and transverse sections of the nerve tissue have been ready. Furthermore, the sections have been 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 have been 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). Moreover, we additionally used FITC-Tyramide (Servicebio) and CY3-Tyramide (Servicebio) to amplify fluorescence depth. Nuclei have been stained with DAPI, and the sections have been noticed beneath confocal laser scanning microscopy. The chances of the markers constructive areas have been calculated by dividing built-in choice density by chosen area space, then multiplied by 100%. All parameters have been measured utilizing ImageJ.
Statistical evaluation
Statistical evaluation was performed utilizing GraphPad Prism 8.0 (GraphPad Software program, La Jolla, CA, USA). The outcomes have been offered as imply ± SD. One-way ANOVA was used for comparisons inside a number of teams, and a two-tailed unpaired Scholar’s check was used for comparisons between two teams. P values < 0.05 have been thought of statistically important.
