Animals
Animals for the study were sourced from a commercial farm in Xinfeng County, Shaoguan City, Guangdong Province, China. A total of 20 male pigs were selected, including 5 lean-type piglets (LR) and 5 Chinese fat-type piglets (LT) at 1 day of age, as well as 5 lean-type pigs (CS) at 60 and 90 days of age. Adipose tissue and samples from the longest dorsal muscle were swiftly harvested and preserved in liquid nitrogen for future analysis.
Adipose-specific miR-146a-5p knockout (aKO) mice were created utilizing the Cre-loxP system (Cyagen, Suzhou, China). The breeding process involved crossing miR-146a-5p flox/flox (miR-146a-5p flox+/+, Flox) mice with adipose tissue-specific Cre mice. The resulting F1 generation (miR-146a-5p flox+/−, Cre+/−) was then bred with miR-146a-5p flox+/+ mice to generate aKO (miR-146a-5p flox+/+, Cre+/−) and control Flox (miR-146a-5p flox+/+) mice. The mice were housed in individually ventilated cages under pathogen-free conditions at 24℃, maintained on a 12-hour light/dark cycle, and provided with a standard diet from the Tianjin Hospital Animal Experiment Center (Tianjin, China). The study predominantly used male mice aged 8 to 12 weeks, with additional groups of younger (4 weeks) and older (18 months) mice for specific analyses. All experimental procedures complied with the National Institutes of Health guidelines for laboratory animal care and use and were approved by the Tianjin Hospital Animal Research Ethics Committee (Ethical approval number: 2024YLS208).
Immunofluorescence
Muscle tissue samples were rapidly frozen in isopentane cooled with liquid nitrogen and embedded in Tissue-Tek OCT (Fisher Scientific/Thermo Scientific). Cryosections, 10 μm thick, were prepared at -25℃ and fixed with 4% paraformaldehyde for 10 min. The sections underwent three 5-minute washes with PBS, followed by blocking with a solution containing PBS, 0.5 mL goat serum, 0.2 g BSA, 0.2 mL 10% Triton X-100, and 0.01 g sodium azide. Primary antibodies were incubated overnight at 4 °C, comprising mouse anti-MyoD (1:100, Santa Cruz, sc-377460), rabbit anti-laminin (1:1000, Thermo Fisher, PA1-16730), mouse anti-MyHC (1:2000, R&D Systems, MAB4470), and mouse anti-Pax7 (1:50, DSHB, 528428). Following three PBS washes, the sections were incubated at room temperature for 1 h with secondary antibodies: goat anti-mouse IgM/Alexa Fluor 555 (1:2000, bs-0368G-AF555, Bioss) and goat anti-rabbit FITC (1:2000, bs-0295G-FITC, Bioss). To minimize background staining, the sections were treated with 0.05% Sudan black for 5 min. Fluorescent images were obtained using a Nikon Eclipse Ti inverted microscope (Tokyo, Japan).
C2C12 cells were fixed with 4% paraformaldehyde and permeabilized with 0.4% Triton X-100 in PBS, each for 20 min at room temperature. Cells were initially blocked with 5% goat serum for 1 h and then incubated overnight at 4℃ with primary antibodies: anti-MyoD (sc-377460, Santa Cruz), anti-MyHC (MAB4470, R&D Systems), and anti-Pax7 (528428, DSHB). The cells were then incubated in the dark at room temperature for 1 h with goat anti-mouse IgM conjugated to Alexa Fluor 555 (1:2000, bs-0368G-AF555, Bioss). Nuclei were DAPI-stained. Fluorescent images were acquired with a Nikon Eclipse Ti microscope, and myotube diameters were quantified using ImageJ software.
Skeletal muscle satellite cell extraction and culturing
Muscles from both mice and piglets were used as tissue sources for isolating skeletal muscle satellite cells (MuSCs). Mice, approximately 10 days old, were euthanized by cervical dislocation and processed under aseptic conditions. After sterilizing the mice in 75% ethanol, the hind limb skin was carefully removed, and blood vessels, connective tissue, fat, and bone were discarded, leaving only the muscle tissue. For piglets, euthanasia was performed via cardiac puncture, and the longissimus dorsi muscle was aseptically isolated. The muscle tissue was then washed with PBS until the rinse solution was clear, and it was minced into approximately 1 mm³ pieces. The minced muscle was incubated in 0.2% collagenase II at 37℃ for 1 h in a water bath shaker, with gentle agitation every 10 min. Post-digestion, the mixture was centrifuged at 1000 rpm for 10 min at 4℃, and the supernatant was removed. The pellet was treated with 0.25% trypsin at 37 °C for 30 min, with agitation every 10 min. Trypsin activity was inhibited by adding a growth medium containing 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) (Gibco). The cell suspension was filtered through 100, 200, and 400 mesh filters in sequence, then centrifuged at 1000 rpm for 10 min at 4℃. The pellet was resuspended in fresh growth medium and transferred to 25 cm² culture flasks after the supernatant was discarded. The cells were incubated at 37℃ in a 5% CO₂ environment. To purify MuSC, a differential apposition technique was used. The isolated muscle cells were incubated for two hours, enabling fibroblasts to attach to the surface while the satellite cells remained in suspension. Non-adherent cells were moved to new culture flasks, and on day 4, the medium was changed to eliminate residual blood and dead cells. Cell growth was monitored, and cells were passaged upon reaching 75-90% confluence. At each passaging, a 30-minute differential walling was performed to further eliminate fibroblasts.
Cell culture
C2C12 myoblasts were maintained in 12-well plates (Corning) with high-glucose DMEM (Gibco), supplemented with 10% FBS and 1% P/S. Upon reaching confluence, the culture medium was replaced with a differentiation medium containing DMEM and 2% horse serum (HS, Gibco) for a 6-day induction period. Replace the medium every two days. MuSCs or C2C12 cells were seeded at 1 × 10⁵ cells per well in 12-well plates for transfection experiments. Cells at 60–70% confluence were exposed to 40 nM miR-146a-5p mimics, 80 nM miR-146a-5p inhibitor, 50 nM si-Fbx32 or 10 µg/mL sEV. Transfection utilized siRNAs from GenePharma and Tsingke Biological Technology, China, in conjunction with Lipofectamine 2000 (Thermo Fisher), adhering to the manufacturer’s guidelines.
CCK-8 assay
Cells were seeded in a 96-well plate and treated according to the experimental design. After incubation, Cell Counting Kit-8 (CCK-8) solution was added to each well, and the plate was further incubated. Absorbance was measured at 450 nm using a microplate reader to determine cell viability. Cell viability was evaluated with the CCK-8 assay. C2C12 cells were plated in 96-well plates at a density of 10,000 cells per well, with six replicates per condition. Following treatment, each well received 10 µL of CCK-8 reagent and 90 µL of the medium mix, then incubated for 1 h. Absorbance at 450 nm was measured using a BioTek microplate reader, with blank wells as controls. Cell viability and proliferation were assessed following the manufacturer’s guidelines (Beyotime, Haimen, China).
EdU incorporation assay
Cell proliferation was evaluated using the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, C0071) according to the manufacturer’s protocol. C2C12 cells were plated in 96-well plates at a density of 1.0 × 10⁴ cells per well and incubated with 10 µM EdU for 2 h at 37℃ in a CO₂ incubator. Following incubation, the cells were fixed in 4% paraformaldehyde for 20 min, then permeabilized with 0.3% Triton X-100 for 15 min. The Click Reaction Mixture was incubated at room temperature in the dark for 30 min, then stained with Hoechst 33,342 for 10 min. EdU-positive cells were quantified using ImageJ software [15].
Flow cytometric analysis of cell apoptosis
Apoptosis was assessed via flow cytometry (BD FACSCalibur) using the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences). Cells were first harvested by trypsinization and transferred into 15 mL centrifuge tubes. After centrifuging at 1,000 g for 5 min, the supernatant was discarded, and the cell pellet was washed twice with cold PBS. Following each wash, the cells were re-centrifuged, resuspended in PBS, and counted to ensure the correct concentration. For apoptosis analysis, 50,000 to 100,000 cells underwent additional centrifugation, followed by supernatant removal. The pellet was resuspended in 500 µL of Annexin V binding buffer, then 5 µL of Annexin V-FITC and 5 µL of propidium iodide (PI) were added. The mixture was gently vortexed and left to incubate in the dark at room temperature (20–25℃) for 15 min. Apoptosis levels were assessed post-incubation via flow cytometry, allowing for the distinction of early apoptotic, late apoptotic, and necrotic cells.
Transmission Electron microscopy
Mice were humanely euthanized and perfused with 100 mM sodium phosphate buffer (pH 7.4). The tissues were immersed in a phosphate buffer (PB) fixative containing 2.5% glutaraldehyde and 1% paraformaldehyde. The tibialis anterior (TA) muscle was carefully dissected, cut into smaller pieces, and stored overnight in the same fixative at 4℃. After a PB rinse, samples were incubated in 0.2 M imidazole for 15 min and subsequently fixed with 1% osmium tetroxide. Following an additional rinse with high-purity water, the samples were incubated overnight at 4℃ in a 1% aqueous lead nitrate solution. A series of graded acetone solutions were employed for dehydration, and the samples were subsequently embedded in epoxy resin at a temperature of 60℃ for 24 h. Ultra-thin sections were then prepared using an ultramicrotome, placed on copper grids, and examined with a transmission electron microscope under conditions of double-blind analysis [16].
sEV isolation, characterization, and administration
sEV were isolated from the white adipose tissue of 3-month-old mice. The tissue was rinsed thrice with PBS and cut into pieces under 3 mm. The culture was kept in 75 cm² flasks containing 25 mL of DMEM, with the addition of 1% P/S and 10% sEV-depleted FBS, for 24 h at 37℃. After incubation, the supernatant was centrifuged in stages: first at 300×g for 10 min to remove suspended cells, then at 2,000×g for 10 min to eliminate dead cells, and finally at 10,000×g for 30 min to clear cellular debris. The supernatant was concentrated using a 100 kDa protein concentrator (Macrosep) and subsequently purified with a 0.22 μm PVDF filter (Millipore). The concentrated sample underwent ultracentrifugation at 120,000×g for 90 min using a 38.5 mL Beckman Coulter tube. After ultracentrifugation, the sEV were washed with PBS, resuspended, and aliquoted into 100 µL portions in PBS for storage at -80℃. The sEV isolated from the white adipose tissue (WAT) of Flox and aKO mice were labeled as Flox-sEV and aKO-sEV, respectively.
Western blot analysis was conducted to characterize the sEV using extracellular vesicle markers TSG101, Alix, CD9, and CD63, with Calnexin serving as a negative control for the endoplasmic reticulum. The concentration and size distribution of sEV were evaluated using a Nanosight instrument, and their morphology was analyzed through transmission electron microscopy (TEM) [17].
For in vivo administration, recipient mice received three weekly injections of 100 µg sEV into the TA muscle. In vitro, cells were treated with sEV at a concentration of 10 µg/mL. The uptake of sEV was tracked by labeling the vesicles with PKH67 fluorescent dye, as per the manufacturer’s guidelines (Sigma-Aldrich).
Dual-Luciferase reporter assay
To evaluate gene expression, a dual-luciferase reporter assay was performed. HEK293T cells were cultured in 96-well plates (Corning) at a density of 2.5 × 10⁴ cells per well and allowed to reach 60-70% confluence. Cells were co-transfected with 100 ng of a dual-luciferase reporter plasmid (containing wild-type, mutant, or deleted target sequences) and 3 pmol of either the miR-146a-5p mimic or a negative control. After 24 h of incubation, luciferase activities were measured using the Dual-GLO luciferase assay system (Promega), following the manufacturer’s instructions. Firefly luciferase activity was normalized to Renilla luciferase activity to correct for transfection efficiency [18].
Detection of reactive oxygen species (ROS)
Intracellular ROS levels in C2C12 cells were measured using the ROS assay kit (S0033, Beyotime, Shanghai, China) according to the manufacturer’s instructions. Fluorescent emissions were visualized and documented using a fluorescence microscope.
Cellular ATP level analysis
ATP levels in cell lysates were quantified using the ATP assay kit (Beyotime, Cat# S0026). A 20 µL aliquot of lysate was mixed with 10 µL of ATP detection reagent, and luminescence was measured using a BioTek microplate reader. Protein concentrations were measured using the Pierce BCA assay kit. ATP levels were then normalized to the protein content for calculation.
Quantitative Real-Time PCR
Total RNA was extracted following the manufacturer’s protocol using TRIzol reagent (Thermo Fisher). To remove genomic DNA contamination, RNA samples underwent treatment with DNase I (EZB, Shanghai, China). cDNA was synthesized by reverse transcribing 1–2 µg of RNA using the EZB 4×EZscript Reverse Transcription Mix II (EZB, Shanghai, China). Quantitative real-time PCR (qPCR) was performed on the Bio-Rad C1000 Touch (QuantStudio Real-Time PCR System) with the 2×RealStar Fast SYBR qPCR Mix (GenStar, Cat No. A301), as per the manufacturer’s guidelines. GAPDH served as the internal control for mRNA quantification, while U6 RNA was used to normalize miRNA expression levels. The 2-ΔΔCt method was used to quantify relative mRNA levels [19].
Western blotting
The expression of various functional proteins was evaluated using Western blotting. Protein samples were lysed using RIPA buffer and quantified with Thermo Fisher’s Rapid Gold BCA Protein Assay Kit. Each sample’s 15 µg protein underwent SDS-PAGE and was transferred to PVDF membranes (Millipore) for analysis. Primary antibodies included anti-TSG101 (rabbit, ZEN BIO, 381538), anti-Alix (rabbit, Sangon Biotech, D262028), anti-Calnexin (rabbit, Sangon Biotech, D262986), anti-CD63 (rabbit, Sangon Biotech, D160973), anti-Wnt 10b (rabbit, Bioss, bs-3662R), anti-CD9 (rabbit, Abcepta, AP68-965), anti-Cyclin A2 (rabbit, GeneTex, GTX103042), anti-Cyclin D1 (rabbit, CST, 2978 S), anti-Cyclin E1 (rabbit, ZEN BIO, 340298), anti-PCNA (mouse, ZEN BIO, 200947), anti-MyHC (mouse, R&D Systems, MAB4470), anti-MyoD (rabbit, ZEN BIO, 252249), anti-MyoG (rabbit, ZEN BIO, 382257), anti-Fbx32 (rabbit, Abclonal, A3193), anti-MuRF (rabbit, Abclonal, A3101), anti-Pax7 (rabbit, Abclonal, A7335), anti-Bcl-2 (rabbit, ZEN BIO, 380709), anti-Bax (rabbit, Sangon Biotech, D290151), anti-Caspase 3 (rabbit, ZEN BIO, 300968), anti-LC3 (mouse, abcam, ab243506), anti-P62 (rabbit, Abclonal, A19700), and anti-Tubulin (rabbit, Bioworld, AP0064). The membrane was incubated with the primary antibody overnight, followed by a one-hour exposure to the secondary antibody (Bioworld) at room temperature. Protein bands were visualized by a gel imaging system and normalized using Tubulin as an internal control. Quantitative results of protein bands were expressed in arbitrary units (AU) relative to the control. Data analysis was conducted using ImageJ software [20].
Statistical analysis
Statistical analyses, comprising one-way ANOVA, independent t-tests, and data visualization, were conducted using SPSS (v27) and GraphPad Prism (v9.0). Data are presented as means ± standard error of the mean (SEM). Groups were compared using either an unpaired Student’s t-test or one-way ANOVA for statistical analysis. Statistical significance was defined as *P < 0.05 and **P < 0.01. The letters a, b, and c denote varying significance levels between groups; distinct letters indicate a significant difference, while identical letters imply no significant difference [21].
