Animal studies

Ethical approval for the animal experiments outlined in this study was granted by the Institutional Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine. The manuscript adheres to the ARRIVE guidelines for the reporting of animal experiments. Ninety-six male Sprague–Dawley rats, approximately 60 d old and weighing approximately 250 ± 10 g, were obtained from SLAC Laboratory Animal Co., Ltd. located in Shanghai, China. Animals were randomly distributed in the cages of the different groups. The rodents were kept in a regulated setting with a temperature of 22 ± 1 °C, while the air moisture was maintained at 60 ± 5%. Additionally, they were exposed to a 12-h cycle alternating between light and darkness. The animals were given unlimited access to food and water to ensure that they had unrestricted availability.

The ACS model was created as we previously outlined [17]. Briefly, the rats were anesthetized through an intraperitoneal injection of 3% pentobarbital sodium. After the rats were unconscious, a 24-gauge angiocatheter was inserted into the anterior compartment of the left hindlimb in the experimental group to increase the pressure. The pressure was raised to 80 mmHg and kept at 80 ± 10 mmHg for 2 h. A single-incision fasciotomy was performed at the end of the experiment to relieve the pressure in the compartment. In the control group, the rats underwent all procedures except the pressure was maintained at the baseline level (0 mmHg).

We utilized the NC3Rs website to ascertain the optimal and adequate number of rats per group required to attain statistically meaningful outcomes. During the reception of the animals, the attainment of traceability and individual identification was established through the utilization of ear pins. The researchers possessed awareness throughout the process of allocation, the execution of the experiment, the assessment of outcomes, and the analysis of data according to the ARRIVE guidelines. To evaluate the treatment using ESC–MSC-EVs, the experimental groups were then divided into four groups of six rats each: Sham + vehicle group, Sham + EVs group, ACS + vehicle group, and ACS + EVs group by means of the random number method. To treat the rats, either ESC–MSC-EVs (100 μg) or a control vehicle (PBS) were given through the dorsal penis vein. At the conclusion of the trial, the rodents were sacrificed by administering an excessive amount of 3% pentobarbital sodium through an intra-arterial injection. Every effort was made to minimize suffering by adding enrichment such as excessive anesthesia and rapid execution. Samples of blood and muscle from the tibialis anterior (TA) were gathered for additional examination.

ESC–MSCs preparation and identification

A two-step process was used to differentiate ESCs into ESC–MSCs as we have previously described [17]. Flow cytometry was utilized to phenotypically characterize ESC–MSCs by employing specific antibodies obtained from BD Pharmingen (USA). The antibodies used for this analysis included anti-CD31, anti-CD90, anti-CD105, anti-CD19, anti-CD45, anti-CD14, anti-CD34, anti-CD11b, anti-CD79a, and anti-HLA-DR.

Multi-lineage differentiation potency of ESC–MSCs was evaluated by inducing osteogenic, adipogenic, and chondrogenic differentiation. All differentiation media and dyes are purchased from Cyagen (China). For osteogenic differentiation, ESC–MSCs (2 × 104 cells/cm2) were cultured in 6-well plates coated with 1% gelatin and grown until reaching 60–70% confluency. The medium for promoting the formation of osteogenic differentiation was changed every 3 d. After a 3-w induction period, calcium nodules were identified by staining with alizarin red. For chondrogenic differentiation, 5 × 105 was formed through centrifugation at 1000 rpm for 5 min. The pellet was then cultured in 15-mL centrifuge tubes containing chondrogenic differentiation medium for a duration of 3 w. After 4% paraformaldehyde fixation and frozen sections, alcian blue is used to dye frozen sections. Adipogenic induction commenced upon the full confluence of the cell culture. After being isolated, the cells were cultured in adipogenic differential medium A for 3 d. Following this initial phase, the cells were then transitioned to adipogenic differential medium B for a single day of maintenance. This dynamic process was repeated for a total of 6 cycles to promote the synthesis and accumulation of lipid droplets within the cells. Subsequently, the cultures underwent fixation utilizing the 4% paraformaldehyde solution, followed by staining using oil red O.

Extracellular vesicles (EVs) isolation and identification

ESC–MSCs were cultured using a serum-free MSC proliferation medium (ExCell Bio, China). EVs were extracted using ultracentrifugation previously described [21]. Briefly, the supernatant of the P4 generation ESC–MSCs cell culture was collected and subjected to centrifugation at 4 °C and 300 g for 5 min. To eliminate any remaining cells and cell fragments, the centrifugation was extended for 15 min at 3000g (4 °C). After filtration, an ultra-high-speed centrifuge (Beckman, USA) was used to centrifuge at 4 °C and 10,000 g for 30 min to remove cellular organelles, followed by centrifugation at 4 °C and 120,000 g for 70 min to obtain EVs. A suitable amount of PBS solution was then added to resuspend.

The procedural method for observing samples using transmission electron microscopy (TEM) is as follows: A volume of 10 μL of the sample was dispensed onto a copper mesh, subsequently permitting sedimentation for 1 min, followed by absorption of the supernatant using filter paper. Subsequently, a volume of 10 μL of a 4% uranyl acetate solution was added to the copper mesh, allowing it to settle for 10 min, after which the supernatant was once more absorbed using filter paper. After several min of air-drying at room temperature, the imaging was inspected using an electron microscope (JEOL, Japan) at 120 kV.

The particle size analysis procedure is as follows: 30 μL of the sample was taken, and the analysis of particle size was conducted employing NanoSight NS500 (Malvern Instruments, UK).

Western blotting was used to further examine the surface characteristic proteins Calnexin, HSP70, TSG101, CD63, and CD9.

Pathological analysis

Paraffin sections with a thickness of 4 μm were prepared and subsequently subjected to staining using either H and E or Masson trichrome methods. The stained sections were analyzed using a light microscope (Leica, Germany) at 200× magnification. The determination of the proportion of injured myofibers was conducted following the protocol outlined by McCormack et al. [22]. Injured myofibers are defined as ragged cellular edges, vacuolation, lymphocyte infiltration, or rhabdomyolysis. Myofibers that contain central nuclei were identified as regenerative [23]. Regenerated myofibers were calculated as described in our previous study [17]. Briefly, for each sample, five random fields were chosen and observed using a light microscope (Leica) at 200× magnification. The aim was to determine the total count of regenerative myofibers. Additionally, the minor axis diameters of these fibers in each TA muscle were measured with the assistance of ImageJ software (NIH, USA). In order to evaluate the fibrotic region in sections of skeletal muscle, five random fields were chosen from each sample using a light microscope (Leica) at 200× magnification. The percentage of the fibrotic area was determined utilizing ImageJ software.

TUNEL and dystrophin staining analysis

Skeletal muscle apoptosis was measured by performing TUNEL and dystrophin immunofluorescence staining on muscle tissue frozen sections (10 μm). The TUNEL staining was carried out following the protocol of the manufacturer (Roche Inc., Switzerland). Subsequently, the sections were subjected to an incubation step with anti-dystrophin (1:200, 12715-1-AP, Proteintech) and succeeded by an anti-rabbit IgG cyanin 3 (Cy3) secondary antibody (1:200, SA00009-2, Proteintech). DAPI (Meilunbio, China) was used for counterstaining the nuclei. The sections were analyzed using an upright fluorescence microscope (Olympus, Japan) at 200× magnification. The photomicrographs were combined using the Image-Pro Plus software (Olympus). The quantity of TUNEL-positive and DAPI-positive nuclei was enumerated, taking into account solely those labeled nuclei which exhibited co-localization with dystrophin staining. The findings were articulated in the form of the TUNEL index, acquired by dividing the tally of TUNEL-positive nuclei by the aggregate number of nuclei. The calculation of the TUNEL index was performed for each section by scrutinizing five random and non-overlapping fields.

Western blotting

Protein extraction was performed from EVs, cells, or tissues using a protein lysis buffer containing PMSF (Biosharp, China). For the detection of phosphorylated proteins, an additional phosphatase inhibitor (Biosharp) was added to the lysis buffer. The quantification of proteins was accomplished through the utilization of a BCA kit (Biosharp), while their separation was performed via sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by their transfer onto PVDF membranes. Subsequently, the PVDF membranes underwent a blocking process utilizing a solution composed of 5% skimmed milk in Tris-buffered saline. In the subsequent step, the membranes were subjected to overnight incubation at 4 °C with primary antibodies. Primary antibodies that we used include anti-Calnexin (abcam, ab133615), anti-HSP70 (abcam, ab181606), anti-TSG101 (abcam, ab125011), anti-CD63 (abcam, ab134045), anti-CD9 (abcam, ab263019), anti-β-actin (CST, #4970S), anti-iNOS (Invitrogen, #PA3-030A), anti-Bcl-2 (CST, #3498S), anti-Bax (CST, #14796S), anti-Cleaved caspase 3 (CST, #9664S), anti-CD206 (CST, #24595), anti-CD68 (abcam, ab283654), anti-CD86 (abcam, ab220188), anti-Arg1 (CST, #93668S), anti-P65 (CST, #8242S), anti-phospho-P65 (CST, #3033S), anti-IKBα (CST, #4812S), anti-phospho-IKBα (CST, #2859S), anti-JAK1 (CST, #3344T), anti-phospho-JAK1 (CST, #74129S), anti-STAT6 (CST, #5397S), anti-phospho-STAT6 (CST, #56554S), anti-STAT3 (CST, #12640S), anti-phospho-STAT3 (CST, #9145S), anti-PI3K (CST, #4249T), anti-phospho-PI3K (CST, #13857S), anti-AKT (CST, #9272S), anti-phospho-AKT (CST, #4060T), anti-PTEN (Biolegend, USA, 4C11A11), anti-NLRP3 (CST, #15101S), anti-mTOR (CST, #2983T), and anti-TLR3 (CST, #6961S). Next, the PVDF membranes were exposed to the appropriate HRP-linked antibodies and incubated at room temperature for a duration of 2 h. The bands were made visible through the use of an ECL kit (Millipore, USA) and measured by ImageJ software.

Serum and cell culture supernatants analysis

In this study, serum was obtained by collecting blood samples from the abdominal aorta. Creatine kinase was measured in samples using an LW C400 Clinical Chemistry Analyzer (Landwind Medical, China). Additionally, ELISA kits were employed to measure the concentrations of TNF-α, IL-6, and IL-10 (Elabscience, China) in both the serum and cell culture supernatant. The samples were evaluated for OD at a wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific, USA).

Rat ethology

In this study, skeletal muscle function was assessed using the hanging grid test and grip strength test. The hanging grid test involved placing rats individually at the center of a wire mesh screen suspended 50 cm above a plastic cage filled with sawdust bedding. The hanging duration was recorded in three independent trials, and the data from all three trials were averaged to obtain a final value. This test is commonly used to evaluate muscle strength and endurance in rodents [17, 24].

The grip strength test was utilized in this study to assess muscle strength in rats. The test was conducted using a grip strength meter (Handpi HP-5N, China). During the test, the rats were held by the tail and approached the grid slowly until their hind claws grasped the grid. They were then gently pulled by the tail until they released their grip, and the forces of three trials were recorded and averaged to obtain the final value. This test is commonly used to evaluate muscle strength and has been previously described [17, 25].

Immunofluorescence staining

The paraffin-embedded muscle tissue sections (4 μm) were utilized. These sections were subjected to antigen retrieval, blocking, and labeling with primary antibodies against CD86 (Santa Cruz, sc-20060) and CD206 (Santa Cruz, sc-58986), followed by staining with FITC- or Cy3-labeled secondary antibodies. Negative controls were included in the experiment to ensure the specificity of the staining. After the staining process, the sections were observed using an upright fluorescence microscope (Olympus) at 200× magnification. Immunofluorescent staining of CD31 (R and D Systems, USA, AF3628) was performed on muscle tissue frozen sections (10 μm) using the method described above.

Quantitative real‑time PCR

The extraction of RNA was carried out by utilizing TRIzol reagent (Invitrogen, USA) according to the instructions provided by the manufacturer. To determine the concentration and purity of the RNA sample, the Nanodrop spectrophotometer (Thermo Fisher) was used for quantifying the extracted RNA. After extracting RNA, the PrimeScript RT reagent kit (Yeason, China) was utilized to perform reverse transcription, converting RNA into cDNA. The Mx3000P real-time PCR system (Agilent Technologies, USA) was used to perform quantitative real-time PCR with SYBR Mixture (Yeason), specific primers (as indicated in Additional file 1: Table S1), and the cDNA samples. For normalization purposes, the internal reference gene β-actin was employed in housekeeping.

Macrophages depletion

One ml of liposomal clodronate (LC, 5 mg/ml, Vrije Universiteit, The Netherlands) was injected into the rats via the intravenous route. LC administration was conducted 1 d prior and 1 d following the induction of ACS injury, as previously explained [17, 26]. This method has been previously described in detail. Rats were injected with liposomal vehicle (LV, Vrije Universiteit) as the control.

Immunohistochemistry

The paraffin-embedded muscle tissue sections (4 μm) were utilized. These sections were subjected to antigen retrieval, blocked, and labeled overnight at 4 °C using anti-CD68 (1:200, Santa Cruz, sc-20060). HRP-labeled secondary antibodies (Boster, China) were used, followed by the addition of an avidin–biotin–peroxidase conjugate (Boster). The resulting response was observed using a diaminobenzidine (DAB) chromogen solution for substrate (VectorLabs, USA). In the end, the parts were stained with hematoxylin and examined using light microscopy (Leica) at 200× magnification.

Macrophage differentiation and stimulation

The THP-1 cells were grown in a specialized solution (Procell, China) with a cell density ranging from 3 × 105 to  6 × 105 cells/ml. THP-1 cells were subjected to a treatment of phorbol 12-myristate 13-acetate (PMA, 160 ng/ml, Sigma, USA) for 24 h to induce a macrophage-like phenotype. Subsequently, the adherent cells were cultivated in a fresh medium supplemented with LPS (100 ng/ml, Sigma) and IFN-γ (50 ng/ml, Novoprotein, China). Following this, the conditioned medium was substituted with fresh medium alone or in combination with ESC–MSC-EVs (10 μg/ml) for an additional 48 h.

EVs labeling and cellular uptake assay

EVs were labeled with PKH67 (Sigma) for 2 min at room temperature. Addition of PKH67 to PBS solution without EVs as the control. The labeled EVs suspension was filtered with a Spin Column (Invitrogen, USA) to remove unbound dye according to the previous study [27]. THP-1 cells (1 × 106) were grown in a 6-well dish and then exposed to PKH67-labeled EVs (10 ug/ml) for a duration of 6 h. The cells were subsequently treated with DAPI (Meilunbio) and TRITC-conjugated phalloidin (Yeason) and examined using an inverted fluorescence microscope (Olympus) at 400× magnification.

Flow cytometry analysis

TrypLE™ Express trypsin (Biosharp, China) was used to detach the cells from the plate. In order to prevent non-specific binding, 100 μL of a 10% solution of Gamunex (a solution of human immune globulin) was introduced and left to incubate on ice for a duration of 10 min. This was followed by incubation with anti-CD206 (#321103, Biolegend, USA) and anti-CD86 (#374205, Biolegend) antibodies for 30 min in the dark. After centrifugation, cells were washed twice and immediately measured by the flow cytometer (Beckman), and data evaluation was performed using CytExpert 2.4 software (Beckman).

Transcriptome sequencing and bioinformatic analysis

THP-1 cells were differentiated and stimulated, then cultured in either a normal medium or medium containing ESC–MSC-EVs for 48 h. The sequencing was performed by LC-BIO (China), and the methodology was as previously described [28]. Briefly, Illumina Novaseq 6000 (USA) was utilized for paired-end sequencing in PE150 sequencing mode, following standard procedures. During the analysis phase, CleanData was aligned to the Homo sapiens genome using HISAT2 software. mRNA expression levels were subsequently analyzed by both StringTie and Ballgown software packages. The R package was employed to analyze differences in mRNA expression levels between the two groups. Genes with a different fold change greater than 2, or less than 0.5, and a p value less than 0.05 were selected as differential genes. The mRNA exhibiting differential expression was subsequently subjected to enrichment analysis based on GO and KEGG.

Small RNA sequencing

LC-BIO conducted the analysis of small RNA sequencing using the previously described methodology [29]. Briefly, ESC–MSC-EVs were used to extract total RNA with the mirVana miRNA isolation kit (Ambion). Small RNA libraries were constructed using TruSeq Small RNA Sample Prep Kits (Illumina) with 1 μg of total RNA from each sample. The libraries were sequenced on an Illumina HiSeq X Ten platform after PCR amplification and size selection. Differentially expressed miRNAs were identified with a p value threshold of < 0.05.

MicroRNA transfection

The miRNA transfection was performed as previously described [30]. Chemically synthesized and modified miRNA mimics (Additional file 1: Table S1) or mimics NC from Sangon Company (China) were transfected into THP-1 cells using the FuGENE HD transfection reagent (Promega, USA) following the instructions provided by the manufacturer.

Statistical analysis

GraphPad Prism 8.0 (USA) was utilized for both statistical and graphical analyses in this study. The data were expressed in terms of mean and standard deviation (SD). Two-group comparisons were analyzed using Student’s t-tests, whereas multiple group comparisons were performed using one-way ANOVA with Tukey’s post hoc test and two-factor ANOVAs with Bonferroni pairwise comparisons. Statistical significance was considered at a p value < 0.05.