Animals
Lewis male rats (SPF grade, 4 weeks) were purchased from Dashuo Animal Experiment Co., Ltd. (Chengdu, Sichuan). The rats were housed in a controlled environment with a temperature of 25 ± 1°C, relative humidity ranging from 50–60%, and a light/dark cycle of 12 hours. They were provided ad libitum access to food and water. The study protocols were approved by Animal Experimental Ethical Inspection Form of Guizhou Medical University.
Animal modeling and therapy
EAMG models were established according to previously described protocols. The synthetic murine AChRα subunit R97-116 peptide 50 µg, 100 µL complete Freund's adjuvant (CFA), 1 mg H37Ra and 100 µL PBS were fully mixed on ice into antigen emulsion. The rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg). The foot pad, base of the tail, and both sides of the back of the spine of the rats were selected as the immunogen injection sites, and 40 µL of antigen emulsion was injected into each site with microsyringes. On the 30th day after immunization, the antigen emulsion containing AChRα subunit R97-116 peptide 50 µg, 100 µL IFA, 100 µL PBS was injected into the subcutaneous area on both sides of the spine and at the base of the tail. The control group was injected with CFA emulsion. During the whole immunization process, local reactions such as redness, swelling, and ulcers were checked closely. Starting on the 18th-day post-modeling, a total of 25 µL of BMSCs-derived exosomes (BMSCs-Exo, 100 µg) or BMSCs-EXO- NC agomir (BMSCs-Exo, 100 µg) were intravenously injected via the tail vein. Alternatively, BMSCs-Exo-NC agomir (BMSCs-Exo, 100 µg) was administered once weekly for six weeks.
Animal behavior testing
The hanging ability of the entire body was assessed by conducting a hanging test on rats. During this test, the rats were suspended from a cage lid and inverted for a maximum duration of 60 seconds, while the time taken to fall was recorded. Each rat underwent three repetitions of this experiment, with a 5-minute interval between each trial to allow for recovery. The forced swimming test was used to evaluate the immobility time as previously described (Katrancha et al., 2019).
A forced swimming test was used to evaluate the immobility time. The rats were placed in a transparent glass cylinder (14 cm in diameter) filled with water at a depth of 15 cm and maintained at a temperature of 24 ± 1°C. The behavior of the rats was recorded for 15 minutes using video technology. Before the swimming test, all animals underwent a swimming session lasting for 15 minutes, which took place exactly 24 hours earlier. Immobility time, defined as the period during which the rats floated without exhibiting any signs of struggle or making minimal movements necessary to keep their heads above water level, was assessed by an experimenter who remained unaware of the experimental conditions.
Cell culture and transfection
Rat BMSCs were isolated and cultured as previously described. Briefly, the Lewis rats were anesthetized with 1% sodium pentobarbital (50 mg/kg) and euthanized. The femur and tibia of the rat were excised and immersed in a phosphate-buffered saline (PBS, Invitrogen, Carlsbad, CA, USA) solution to facilitate the detachment of muscular tissue to remove the muscle attached to the bone. The bone tissue was transferred into a petri dish containing 2% α-MEM medium, both ends of the leg bone were cut off, and the culture medium was extracted with a 1ml syringe to repeatedly flush the bone marrow cavity until the bone turned white, and the flush solution was collected. The rinsed solution was centrifuged at 1000rpm for 5min, the supernatant was discarded, the cells were re-suspended withα-MEM medium containing 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA), the cell density was adjusted to 5.0 × 105 cells /ml, and the cells were inoculated in a 25 cm2 culture bottle or petri dish, and placed in the cell incubator. The cells were cultured at 37℃ for 24 h and then replaced with fresh medium. After that, the fluid was changed once every 2 ~ 3 days, and the morphology of the cells was observed. Lipofectamine 3000 transfection reagent was used for the transient transfection of NC-agomir/miR-29a-3p-agomir into BMSCs (1.0 × 105 cells/well) according to the manufacturer's instructions.
Identification of BMSCs
The phenotypic characteristics of BMSCs were assessed using flow cytometry. BMSCs were cultured until reaching 80% ~ 90% confluency, trypsinized, and resuspended in PBS (Invitrogen, Carlsbad, CA, USA) at a cell concentration of 1.0 × 106 cells/ml. Subsequently, the BMSCs were stained with specific antibodies against CD34 and CD90. Analysis of the stained cells was performed using a Beckman-Coulter Epics XL flow cytometer (Beckman-Coulter, Miami, FL), and the results were analyzed using Flow Jo software (Ashland, OR, USA).
Isolation of BMSC-derived Exosomes
The BMSC-Exos were isolated through differential ultracentrifugation. Briefly, passage 3 BMSCs were cultured in complete DMEM supplemented with 10% FBS for 2 days to collect exosomes by ultracentrifugation. The cell supernatants were centrifuged at 300 × g for 10 min at 4 ℃, followed by centrifugation at 1200×g for 20 min at 4°C and subsequently at 10,000 × g for 30 min at 4°C to remove non-viable cells and large cellular debris. II The supernatants were collected and subjected to ultracentrifugation (Hitachi, CP100NX, Japan) at 100,000 × g for 70 min at 4°C. Subsequently, they were washed with PBS and further ultracentrifuged at 100,000 × g for 70 min at 4°C to eliminate any potential protein contaminants. The resulting pellets were resuspended in sterile PBS (100 µl) and characterized using a Particle Metrix ZetaView® instrument (Particle Metrix GmbH, Inning, Germany), transmission electron microscopy (Hitachi H-7100; Hitachi High Technologies Corp), and Western blot.
Transmission electron microscopy (TEM)
The morphology of BMSC-Exos in purified samples was examined using transmission electron microscopy (TEM). Enriched exosomes (15 µl) were applied onto carbon-coated copper TEM grids and incubated for 10 minutes. After removing excess fluid, the samples were stained with 2% phosphotungstic acid (pH = 6.5) for 1–2 minutes at room temperature before being subjected to TEM analysis.
Hematoxylin and eosin (H&E) stain
The gastrocnemius tissue of rats was collected and fixed in 4% paraformaldehyde overnight, processed, and embedded in paraffin. The tissue sections (5 µm) were placed in a water bath at 49°C and pasted on a slide coated with polylysine. The slides were dewaxed twice with toluene, dehydrated with ethanol, washed with distilled water, and then stained with hematoxylin. After washing, the slices were separated with 1% hydrochloric acid alcohol, dyed with eosin, and washed with distilled water for 10 min. Finally, the slices are dehydrated with xylene and sealed with a neutral glue. The tissue sections were observed under 100× and 400× times magnification digital trinocular camera microscope (BA210Digital, Motic).
ELISA
The levels of IFN-γ, IL-4, IL-10, and AchR-Ab in serum were detected by ELISA. The procedure concerned the instructions of the kit. In brief, the serum (25 µL per well) was added to the ELISA plate, followed by the addition of sample diluent (225 µL). The plate was then incubated at 37℃ for 90 min. Subsequently, a biotin-labeled antibody (100 µL) was added to each well and incubated at 37℃ for 60 min. Afterward, the plate underwent three washes using an automatic washing machine. Following this, a color-developing solution consisting of 3,3,5,5-tetramethylbenzidine (TMB) (90 µL) was added and protected from light at 37℃ for 15 min. Finally, a termination solution (100 µL) was added to stop the reaction. Absorbance was measured at 450nm optical density using a microplate reader.
Measurement of proteins’ RNA levels
The total RNAs of gastrocnemius were isolated employing TRIzol® reagent (Thermo Fisher, Massachusetts, USA). cDNA was gained by reverse transcription kit (Invitrogen, Carlsbad, CA, US). The relative levels of target gene RNA transcriptome were obtained by RT-qPCR by using a SYBR Pemix Ex Taq kit (Bao Biological Engineering, Dalian, China). The reverse transcriptional reaction conditions were as follows: 95˚C for 30 s, 40 cycles of 95˚C for 5 s, and 60˚C for 30 s. The relative gene expression level was determined by applying the 2−△△Ct method on ABI software, Foster City, CA. The sequences used in this study were as follows: miR-29a-3p, forward, 5′-TGC GGA CTG ATT TCT TTT GG-3′, reverse, 5′- CCA GTG CAG GGT CCG AGG T-3′. U6, 5′-TGC GGG TGC TCG CTT CGG CAG C-3′, reverse, 5′- CCA GTG CAG GGT CCG AGG T -3′.
Western blot analysis
The RIPA kit (Signaling Technologies, Inc.) was used to extract all proteins from gastrocnemius tissues, and the BCA kit (Biyuntian Biotechnology Co., Ltd., P0009) was utilized for protein quantification. Based on the quantitative results, a loading volume of 20 µg per well was utilized for spot sampling, and the total protein was separated through 10% SDS-PAGE electrophoresis. After that, the membrane was transferred to Immobilon-PSQ PVDF membrane (Sigma-Aldrich, ISEQ00010), blocked with 5% skim milk, and incubated with the appropriate amount of primary antibody at 4℃ overnight. After washing with Tris-buffered saline/0.1% Tween (TBST), the goat anti-rabbit IgG (H + L) HRP (Affbiotech, S0001; 1/5000) was incubated at room temperature for 2 h. Finally, the ECL color development solution was utilized for chromogenic detection.
Statistical analysis
The experimental data were analyzed utilizing the advanced SPSS 22.0 software (IBM Corp., Armonk, NY, USA). Data are expressed in terms of methods and standard deviation. The data were analyzed by the method of single one-way ANOVA and compared between groups by the method of least variance. The statistical significance was determined for P < 0.05.