Animals
Animals for the examine have been sourced from a business farm in Xinfeng County, Shaoguan Metropolis, Guangdong Province, China. A complete of 20 male pigs have 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 have been swiftly harvested and preserved in liquid nitrogen for future evaluation.
Adipose-specific miR-146a-5p knockout (aKO) mice have 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 have been housed in individually ventilated cages below pathogen-free circumstances at 24℃, maintained on a 12-hour mild/darkish cycle, and supplied with a typical weight loss plan 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 pointers for laboratory animal care and use and have been authorised by the Tianjin Hospital Animal Analysis Ethics Committee (Moral approval quantity: 2024YLS208).
Immunofluorescence
Muscle tissue samples have been quickly frozen in isopentane cooled with liquid nitrogen and embedded in Tissue-Tek OCT (Fisher Scientific/Thermo Scientific). Cryosections, 10 μm thick, have been ready at -25℃ and glued 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. Main antibodies have 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 have 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 attenuate background staining, the sections have been handled with 0.05% Sudan black for five min. Fluorescent photos have been obtained utilizing a Nikon Eclipse Ti inverted microscope (Tokyo, Japan).
C2C12 cells have been fastened with 4% paraformaldehyde and permeabilized with 0.4% Triton X-100 in PBS, every for 20 min at room temperature. Cells have 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 have been then incubated in the dead of night at room temperature for 1 h with goat anti-mouse IgM conjugated to Alexa Fluor 555 (1:2000, bs-0368G-AF555, Bioss). Nuclei have been DAPI-stained. Fluorescent photos have been acquired with a Nikon Eclipse Ti microscope, and myotube diameters have been quantified utilizing ImageJ software program.
Skeletal muscle satellite tv for pc cell extraction and culturing
Muscle groups from each mice and piglets have been used as tissue sources for isolating skeletal muscle satellite tv for pc cells (MuSCs). Mice, roughly 10 days outdated, have been euthanized by cervical dislocation and processed below aseptic circumstances. After sterilizing the mice in 75% ethanol, the hind limb pores and skin was fastidiously eliminated, and blood vessels, connective tissue, fats, and bone have been discarded, leaving solely the muscle tissue. For piglets, euthanasia was carried out through cardiac puncture, and the longissimus dorsi muscle was aseptically remoted. The muscle tissue was then washed with PBS till the rinse answer 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 mild agitation each 10 min. Publish-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 development medium containing 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) (Gibco). The cell suspension was filtered by 100, 200, and 400 mesh filters in sequence, then centrifuged at 1000 rpm for 10 min at 4℃. The pellet was resuspended in recent development medium and transferred to 25 cm² tradition flasks after the supernatant was discarded. The cells have been incubated at 37℃ in a 5% CO₂ atmosphere. To purify MuSC, a differential apposition method was used. The remoted muscle cells have 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 have been moved to new tradition flasks, and on day 4, the medium was modified to remove residual blood and useless cells. Cell development was monitored, and cells have been passaged upon reaching 75-90% confluence. At every passaging, a 30-minute differential walling was carried out to additional remove fibroblasts.
Cell tradition
C2C12 myoblasts have 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 pair of% horse serum (HS, Gibco) for a 6-day induction interval. Substitute the medium each two days. MuSCs or C2C12 cells have been seeded at 1 × 10⁵ cells per properly in 12-well plates for transfection experiments. Cells at 60–70% confluence have 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, at the side of Lipofectamine 2000 (Thermo Fisher), adhering to the producer’s pointers.
CCK-8 assay
Cells have been seeded in a 96-well plate and handled in keeping with the experimental design. After incubation, Cell Counting Equipment-8 (CCK-8) answer was added to every properly, 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 have been plated in 96-well plates at a density of 10,000 cells per properly, with six replicates per situation. Following remedy, every properly 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 have been assessed following the producer’s pointers (Beyotime, Haimen, China).
EdU incorporation assay
Cell proliferation was evaluated utilizing the BeyoClick™ EdU Cell Proliferation Equipment with Alexa Fluor 488 (Beyotime, C0071) in keeping with the producer’s protocol. C2C12 cells have been plated in 96-well plates at a density of 1.0 × 10⁴ cells per properly and incubated with 10 µM EdU for two h at 37℃ in a CO₂ incubator. Following incubation, the cells have 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 in the dead of night for 30 min, then stained with Hoechst 33,342 for 10 min. EdU-positive cells have been quantified utilizing ImageJ software program [15].
Stream cytometric evaluation of cell apoptosis
Apoptosis was assessed through move cytometry (BD FACSCalibur) utilizing the Annexin V-FITC/PI Apoptosis Detection Equipment (BD Biosciences). Cells have 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 have 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 removing. 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) have been added. The combination was gently vortexed and left to incubate in the dead of night at room temperature (20–25℃) for 15 min. Apoptosis ranges have been assessed post-incubation through move cytometry, permitting for the excellence of early apoptotic, late apoptotic, and necrotic cells.
Transmission Electron microscopy
Mice have been humanely euthanized and perfused with 100 mM sodium phosphate buffer (pH 7.4). The tissues have been immersed in a phosphate buffer (PB) fixative containing 2.5% glutaraldehyde and 1% paraformaldehyde. The tibialis anterior (TA) muscle was fastidiously dissected, reduce into smaller items, and saved in a single day in the identical fixative at 4℃. After a PB rinse, samples have been incubated in 0.2 M imidazole for 15 min and subsequently fastened with 1% osmium tetroxide. Following a further rinse with high-purity water, the samples have been incubated in a single day at 4℃ in a 1% aqueous lead nitrate answer. A collection of graded acetone options have been employed for dehydration, and the samples have been subsequently embedded in epoxy resin at a temperature of 60℃ for twenty-four h. Extremely-thin sections have been then ready utilizing an ultramicrotome, positioned on copper grids, and examined with a transmission electron microscope below circumstances of double-blind evaluation [16].
sEV isolation, characterization, and administration
sEV have been remoted from the white adipose tissue of 3-month-old mice. The tissue was rinsed thrice with PBS and reduce into items below 3 mm. The tradition was saved 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 remove useless 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 have 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 have been labeled as Flox-sEV and aKO-sEV, respectively.
Western blot evaluation was performed to characterize the sEV utilizing extracellular vesicle markers TSG101, Alix, CD9, and CD63, with Calnexin serving as a destructive management for the endoplasmic reticulum. The focus and dimension distribution of sEV have been evaluated utilizing a Nanosight instrument, and their morphology was analyzed by 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 have 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 pointers (Sigma-Aldrich).
Twin-Luciferase reporter assay
To guage gene expression, a dual-luciferase reporter assay was carried out. HEK293T cells have been cultured in 96-well plates (Corning) at a density of two.5 × 10⁴ cells per properly and allowed to achieve 60-70% confluence. Cells have 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 destructive management. After 24 h of incubation, luciferase actions have 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 appropriate for transfection effectivity [18].
Detection of reactive oxygen species (ROS)
Intracellular ROS ranges in C2C12 cells have been measured utilizing the ROS assay package (S0033, Beyotime, Shanghai, China) in keeping with the producer’s directions. Fluorescent emissions have been visualized and documented utilizing a fluorescence microscope.
Mobile ATP degree evaluation
ATP ranges in cell lysates have been quantified utilizing the ATP assay package (Beyotime, Cat# S0026). A 20 µL aliquot of lysate was blended with 10 µL of ATP detection reagent, and luminescence was measured utilizing a BioTek microplate reader. Protein concentrations have been measured utilizing the Pierce BCA assay package. ATP ranges have 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 pointers. GAPDH served as the interior management for mRNA quantification, whereas U6 RNA was used to normalize miRNA expression ranges. The two-ΔΔCt methodology was used to quantify relative mRNA ranges [19].
Western blotting
The expression of varied useful proteins was evaluated utilizing Western blotting. Protein samples have been lysed utilizing RIPA buffer and quantified with Thermo Fisher’s Speedy Gold BCA Protein Assay Equipment. Every pattern’s 15 µg protein underwent SDS-PAGE and was transferred to PVDF membranes (Millipore) for evaluation. Main 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 have been visualized by a gel imaging system and normalized utilizing Tubulin as an inner management. Quantitative outcomes of protein bands have been expressed in arbitrary items (AU) relative to the management. Knowledge evaluation was performed utilizing ImageJ software program [20].
Statistical evaluation
Statistical analyses, comprising one-way ANOVA, unbiased t-tests, and knowledge visualization, have been performed utilizing SPSS (v27) and GraphPad Prism (v9.0). Knowledge are introduced as means ± normal error of the imply (SEM). Teams have been in contrast utilizing both an unpaired Scholar’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 big distinction, whereas an identical letters suggest no vital distinction [21].
