Animals
Animals for the examine had been sourced from a business farm in Xinfeng County, Shaoguan Metropolis, Guangdong Province, China. A complete of 20 male pigs had been chosen, together with 5 lean-type piglets (LR) and 5 Chinese language fat-type piglets (LT) at 1 day of age, in addition to 5 lean-type pigs (CS) at 60 and 90 days of age. Adipose tissue and samples from the longest dorsal muscle had been swiftly harvested and preserved in liquid nitrogen for future evaluation.
Adipose-specific miR-146a-5p knockout (aKO) mice had been created using the Cre-loxP system (Cyagen, Suzhou, China). The breeding course of concerned crossing miR-146a-5p flox/flox (miR-146a-5p flox+/+, Flox) mice with adipose tissue-specific Cre mice. The ensuing F1 era (miR-146a-5p flox+/−, Cre+/−) was then bred with miR-146a-5p flox+/+ mice to generate aKO (miR-146a-5p flox+/+, Cre+/−) and management Flox (miR-146a-5p flox+/+) mice. The mice had been housed in individually ventilated cages underneath pathogen-free situations at 24℃, maintained on a 12-hour gentle/darkish cycle, and supplied with a regular eating regimen from the Tianjin Hospital Animal Experiment Middle (Tianjin, China). The examine predominantly used male mice aged 8 to 12 weeks, with further teams of youthful (4 weeks) and older (18 months) mice for particular analyses. All experimental procedures complied with the Nationwide Institutes of Well being tips for laboratory animal care and use and had been authorised by the Tianjin Hospital Animal Analysis Ethics Committee (Moral approval quantity: 2024YLS208).
Immunofluorescence
Muscle tissue samples had been quickly frozen in isopentane cooled with liquid nitrogen and embedded in Tissue-Tek OCT (Fisher Scientific/Thermo Scientific). Cryosections, 10 μm thick, had been ready at -25℃ and stuck with 4% paraformaldehyde for 10 min. The sections underwent three 5-minute washes with PBS, adopted by blocking with an answer containing PBS, 0.5 mL goat serum, 0.2 g BSA, 0.2 mL 10% Triton X-100, and 0.01 g sodium azide. Major antibodies had been incubated in a single day 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 Programs, MAB4470), and mouse anti-Pax7 (1:50, DSHB, 528428). Following three PBS washes, the sections had been 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 reduce background staining, the sections had been handled with 0.05% Sudan black for five min. Fluorescent pictures had been obtained utilizing a Nikon Eclipse Ti inverted microscope (Tokyo, Japan).
C2C12 cells had been fastened with 4% paraformaldehyde and permeabilized with 0.4% Triton X-100 in PBS, every for 20 min at room temperature. Cells had been initially blocked with 5% goat serum for 1 h after which incubated in a single day at 4℃ with major antibodies: anti-MyoD (sc-377460, Santa Cruz), anti-MyHC (MAB4470, R&D Programs), and anti-Pax7 (528428, DSHB). The cells had been then incubated at nighttime at room temperature for 1 h with goat anti-mouse IgM conjugated to Alexa Fluor 555 (1:2000, bs-0368G-AF555, Bioss). Nuclei had been DAPI-stained. Fluorescent pictures had been acquired with a Nikon Eclipse Ti microscope, and myotube diameters had been quantified utilizing ImageJ software program.
Skeletal muscle satellite tv for pc cell extraction and culturing
Muscle tissues from each mice and piglets had been used as tissue sources for isolating skeletal muscle satellite tv for pc cells (MuSCs). Mice, roughly 10 days previous, had been euthanized by cervical dislocation and processed underneath aseptic situations. After sterilizing the mice in 75% ethanol, the hind limb pores and skin was fastidiously eliminated, and blood vessels, connective tissue, fats, and bone had been discarded, leaving solely the muscle tissue. For piglets, euthanasia was carried out by way of cardiac puncture, and the longissimus dorsi muscle was aseptically remoted. The muscle tissue was then washed with PBS till the rinse resolution was clear, and it was minced into roughly 1 mm³ items. The minced muscle was incubated in 0.2% collagenase II at 37℃ for 1 h in a water bathtub shaker, with light agitation each 10 min. Put up-digestion, the combination was centrifuged at 1000 rpm for 10 min at 4℃, and the supernatant was eliminated. The pellet was handled with 0.25% trypsin at 37 °C for 30 min, with agitation each 10 min. Trypsin exercise was inhibited by including a progress medium containing 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) (Gibco). The cell suspension was filtered via 100, 200, and 400 mesh filters in sequence, then centrifuged at 1000 rpm for 10 min at 4℃. The pellet was resuspended in contemporary progress medium and transferred to 25 cm² tradition flasks after the supernatant was discarded. The cells had been incubated at 37℃ in a 5% CO₂ atmosphere. To purify MuSC, a differential apposition approach was used. The remoted muscle cells had been incubated for 2 hours, enabling fibroblasts to connect to the floor whereas the satellite tv for pc cells remained in suspension. Non-adherent cells had been moved to new tradition flasks, and on day 4, the medium was modified to eradicate residual blood and lifeless cells. Cell progress was monitored, and cells had been passaged upon reaching 75-90% confluence. At every passaging, a 30-minute differential walling was carried out to additional eradicate fibroblasts.
Cell tradition
C2C12 myoblasts had been maintained in 12-well plates (Corning) with high-glucose DMEM (Gibco), supplemented with 10% FBS and 1% P/S. Upon reaching confluence, the tradition medium was changed with a differentiation medium containing DMEM and a couple of% horse serum (HS, Gibco) for a 6-day induction interval. Exchange the medium each two days. MuSCs or C2C12 cells had been seeded at 1 × 10⁵ cells per effectively in 12-well plates for transfection experiments. Cells at 60–70% confluence had been uncovered 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 Organic Know-how, China, along side Lipofectamine 2000 (Thermo Fisher), adhering to the producer’s tips.
CCK-8 assay
Cells had been seeded in a 96-well plate and handled in accordance with the experimental design. After incubation, Cell Counting Package-8 (CCK-8) resolution was added to every effectively, and the plate was additional incubated. Absorbance was measured at 450 nm utilizing a microplate reader to find out cell viability. Cell viability was evaluated with the CCK-8 assay. C2C12 cells had been plated in 96-well plates at a density of 10,000 cells per effectively, with six replicates per situation. Following remedy, every effectively obtained 10 µL of CCK-8 reagent and 90 µL of the medium combine, then incubated for 1 h. Absorbance at 450 nm was measured utilizing a BioTek microplate reader, with clean wells as controls. Cell viability and proliferation had been assessed following the producer’s tips (Beyotime, Haimen, China).
EdU incorporation assay
Cell proliferation was evaluated utilizing the BeyoClick™ EdU Cell Proliferation Package with Alexa Fluor 488 (Beyotime, C0071) in accordance with the producer’s protocol. C2C12 cells had been plated in 96-well plates at a density of 1.0 × 10⁴ cells per effectively and incubated with 10 µM EdU for two h at 37℃ in a CO₂ incubator. Following incubation, the cells had been fastened in 4% paraformaldehyde for 20 min, then permeabilized with 0.3% Triton X-100 for 15 min. The Click on Response Combination was incubated at room temperature at nighttime for 30 min, then stained with Hoechst 33,342 for 10 min. EdU-positive cells had been quantified utilizing ImageJ software program [15].
Move cytometric evaluation of cell apoptosis
Apoptosis was assessed by way of movement cytometry (BD FACSCalibur) utilizing the Annexin V-FITC/PI Apoptosis Detection Package (BD Biosciences). Cells had been first harvested by trypsinization and transferred into 15 mL centrifuge tubes. After centrifuging at 1,000 g for five min, the supernatant was discarded, and the cell pellet was washed twice with chilly PBS. Following every wash, the cells had been re-centrifuged, resuspended in PBS, and counted to make sure the proper focus. For apoptosis evaluation, 50,000 to 100,000 cells underwent further centrifugation, adopted by supernatant elimination. 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) had been added. The combination was gently vortexed and left to incubate at nighttime at room temperature (20–25℃) for 15 min. Apoptosis ranges had been assessed post-incubation by way of movement cytometry, permitting for the excellence of early apoptotic, late apoptotic, and necrotic cells.
Transmission Electron microscopy
Mice had been humanely euthanized and perfused with 100 mM sodium phosphate buffer (pH 7.4). The tissues had been immersed in a phosphate buffer (PB) fixative containing 2.5% glutaraldehyde and 1% paraformaldehyde. The tibialis anterior (TA) muscle was fastidiously dissected, minimize into smaller items, and saved in a single day in the identical fixative at 4℃. After a PB rinse, samples had been incubated in 0.2 M imidazole for 15 min and subsequently fastened with 1% osmium tetroxide. Following an extra rinse with high-purity water, the samples had been incubated in a single day at 4℃ in a 1% aqueous lead nitrate resolution. A sequence of graded acetone options had been employed for dehydration, and the samples had been subsequently embedded in epoxy resin at a temperature of 60℃ for twenty-four h. Extremely-thin sections had been then ready utilizing an ultramicrotome, positioned on copper grids, and examined with a transmission electron microscope underneath situations of double-blind evaluation [16].
sEV isolation, characterization, and administration
sEV had been remoted from the white adipose tissue of 3-month-old mice. The tissue was rinsed thrice with PBS and minimize into items underneath 3 mm. The tradition was stored in 75 cm² flasks containing 25 mL of DMEM, with the addition of 1% P/S and 10% sEV-depleted FBS, for twenty-four h at 37℃. After incubation, the supernatant was centrifuged in phases: first at 300×g for 10 min to take away suspended cells, then at 2,000×g for 10 min to eradicate lifeless cells, and at last at 10,000×g for 30 min to clear mobile particles. The supernatant was concentrated utilizing a 100 kDa protein concentrator (Macrosep) and subsequently purified with a 0.22 μm PVDF filter (Millipore). The concentrated pattern underwent ultracentrifugation at 120,000×g for 90 min utilizing a 38.5 mL Beckman Coulter tube. After ultracentrifugation, the sEV had been washed with PBS, resuspended, and aliquoted into 100 µL parts in PBS for storage at -80℃. The sEV remoted from the white adipose tissue (WAT) of Flox and aKO mice had been labeled as Flox-sEV and aKO-sEV, respectively.
Western blot evaluation was carried out to characterize the sEV utilizing extracellular vesicle markers TSG101, Alix, CD9, and CD63, with Calnexin serving as a damaging management for the endoplasmic reticulum. The focus and measurement distribution of sEV had been evaluated utilizing a Nanosight instrument, and their morphology was analyzed via transmission electron microscopy (TEM) [17].
For in vivo administration, recipient mice obtained three weekly injections of 100 µg sEV into the TA muscle. In vitro, cells had been handled with sEV at a focus of 10 µg/mL. The uptake of sEV was tracked by labeling the vesicles with PKH67 fluorescent dye, as per the producer’s tips (Sigma-Aldrich).
Twin-Luciferase reporter assay
To judge gene expression, a dual-luciferase reporter assay was carried out. HEK293T cells had been cultured in 96-well plates (Corning) at a density of two.5 × 10⁴ cells per effectively and allowed to succeed in 60-70% confluence. Cells had been co-transfected with 100 ng of a dual-luciferase reporter plasmid (containing wild-type, mutant, or deleted goal sequences) and three pmol of both the miR-146a-5p mimic or a damaging management. After 24 h of incubation, luciferase actions had been measured utilizing the Twin-GLO luciferase assay system (Promega), following the producer’s directions. Firefly luciferase exercise was normalized to Renilla luciferase exercise to right for transfection effectivity [18].
Detection of reactive oxygen species (ROS)
Intracellular ROS ranges in C2C12 cells had been measured utilizing the ROS assay equipment (S0033, Beyotime, Shanghai, China) in accordance with the producer’s directions. Fluorescent emissions had been visualized and documented utilizing a fluorescence microscope.
Mobile ATP stage evaluation
ATP ranges in cell lysates had been quantified utilizing the ATP assay equipment (Beyotime, Cat# S0026). A 20 µL aliquot of lysate was combined with 10 µL of ATP detection reagent, and luminescence was measured utilizing a BioTek microplate reader. Protein concentrations had been measured utilizing the Pierce BCA assay equipment. ATP ranges had been then normalized to the protein content material for calculation.
Quantitative Actual-Time PCR
Whole RNA was extracted following the producer’s protocol utilizing TRIzol reagent (Thermo Fisher). To take away genomic DNA contamination, RNA samples underwent remedy with DNase I (EZB, Shanghai, China). cDNA was synthesized by reverse transcribing 1–2 µg of RNA utilizing the EZB 4×EZscript Reverse Transcription Combine II (EZB, Shanghai, China). Quantitative real-time PCR (qPCR) was carried out on the Bio-Rad C1000 Contact (QuantStudio Actual-Time PCR System) with the two×RealStar Quick SYBR qPCR Combine (GenStar, Cat No. A301), as per the producer’s tips. GAPDH served as the interior management for mRNA quantification, whereas U6 RNA was used to normalize miRNA expression ranges. The two-ΔΔCt technique was used to quantify relative mRNA ranges [19].
Western blotting
The expression of assorted useful proteins was evaluated utilizing Western blotting. Protein samples had been lysed utilizing RIPA buffer and quantified with Thermo Fisher’s Fast Gold BCA Protein Assay Package. Every pattern’s 15 µg protein underwent SDS-PAGE and was transferred to PVDF membranes (Millipore) for evaluation. Major 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 Programs, 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 first antibody in a single day, adopted by a one-hour publicity to the secondary antibody (Bioworld) at room temperature. Protein bands had been visualized by a gel imaging system and normalized utilizing Tubulin as an inner management. Quantitative outcomes of protein bands had been expressed in arbitrary models (AU) relative to the management. Information evaluation was carried out utilizing ImageJ software program [20].
Statistical evaluation
Statistical analyses, comprising one-way ANOVA, impartial t-tests, and information visualization, had been carried out utilizing SPSS (v27) and GraphPad Prism (v9.0). Information are offered as means ± customary error of the imply (SEM). Teams had been in contrast utilizing both an unpaired Pupil’s t-test or one-way ANOVA for statistical evaluation. Statistical significance was outlined as *P < 0.05 and **P < 0.01. The letters a, b, and c denote various significance ranges between teams; distinct letters point out a major distinction, whereas equivalent letters suggest no important distinction [21].
