Method for delaying skeletal muscle senescence by using small extracellular vesicle miR-146a-5p derived from adipose tissue

Abstract

The invention discloses a method for delaying skeletal muscle senescence by using adipose tissue-derived extracellular vesicles mi R-146a-5p, relates to the technical field of biological information, and comprises application of overexpressed adipose tissue-derived extracellular vesicles mi R-146a-5p in delaying skeletal muscle senescence, and the application is a non-disease treatment purpose. The method for overexpressing the small extracellular vesicles mi R-146a-5p from the adipose tissue comprises the following steps: transgene overexpression or treatment by using small molecules or compounds for promoting transcription and translation of mi R-146a-5p genes; according to the method for delaying skeletal muscle senescence by using the small extracellular vesicle mi R-146a-5p derived from the adipose tissue, related signal channels and molecular mechanisms in skeletal muscles can be regulated in a targeted manner by using the mi R-146a-5p in the small extracellular vesicle derived from the adipose tissue, and the senescence process of the skeletal muscles is effectively delayed.

Description

Technical Field

[0001] The present invention relates to bioinformatics, and in particular to a method for delaying skeletal muscle aging by utilizing small extracellular vesicles miR-146a-5p derived from adipose tissue. Background Art

[0002] As the global population ages, skeletal muscle health is receiving increasing attention. Skeletal muscle atrophy, a typical and widespread condition, can limit patients' mobility, slow their movements, increase their risk of accidents like falls and fractures, and severely impact their quality of life.

[0003] Adipose tissue plays a crucial role in regulating human physiology. Far from being merely an energy storage depot, it is also a crucial endocrine organ, secreting a diverse array of bioactive molecules that participate in systemic metabolic regulation. Small extracellular vesicles (sEVs), crucial mediators of adipose tissue secretion, carry abundant miRNAs and other regulatory molecules, shuttle between adipose tissue and other organs, transmitting key regulatory signals.

[0004] Currently, clinical interventions for skeletal muscle aging are very limited. Traditional interventions, such as exercise therapy, can enhance muscle strength and improve endurance to a certain extent, but their effectiveness in reversing established muscle atrophy and aging-related pathological changes is extremely limited. While nutritional supplements can provide the necessary raw materials for muscle metabolism, they struggle to precisely target key regulatory pathways and effectively slow the progression of skeletal muscle aging. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for delaying skeletal muscle aging by utilizing small extracellular vesicles miR-146a-5p derived from adipose tissue, so as to solve the above-mentioned deficiencies in the prior art.

[0006] In order to achieve the above-mentioned object, the present invention provides the following technical solution: the use of overexpressing adipose tissue-derived small extracellular vesicles miR-146a-5p in delaying skeletal muscle aging, wherein the use is for non-disease treatment purposes, and the method of overexpressing adipose tissue-derived small extracellular vesicles miR-146a-5p includes transgenic overexpression, or treatment with small molecules or compounds that promote the transcription and translation of the miR-146a-5p gene.

[0007] Furthermore, the host for delaying skeletal muscle aging is a cell, a non-genetic individual or a genetic individual.

[0008] A method for delaying skeletal muscle aging by utilizing small extracellular vesicles miR-146a-5p derived from adipose tissue, comprising the following steps: overexpressing small extracellular vesicles miR-146a-5p derived from adipose tissue in a host, or treating with small molecules or compounds that promote transcription, translation or functional activation of the miR-146a-5p gene.

[0009] Furthermore, the method for overexpressing adipose tissue-derived small extracellular vesicles miR-146a-5p is to construct a recombinant expression vector containing the miR-146a-5p gene, and then overexpress it in animal cells, non-genetic individuals or genetic individuals.

[0010] Compared with the prior art, the present invention provides a method for delaying skeletal muscle aging using small extracellular vesicles (EVs) miR-146a-5p derived from adipose tissue. By utilizing miR-146a-5p in small extracellular vesicles derived from adipose tissue, the method can target and regulate related signaling pathways and molecular mechanisms in skeletal muscle, effectively delaying the aging process of skeletal muscle. It is clarified that miR-146a-5p targets Fbx32 and affects the FoxO3 signaling axis, thereby regulating key physiological processes such as autophagy, apoptosis, and differentiation of skeletal muscle cells. This makes the treatment method more specific and targeted, and can more effectively intervene in the pathophysiological changes of skeletal muscle aging, reduce the impact on non-target tissues, and reduce potential side effects. By regulating related processes such as mitochondrial autophagy and apoptosis, miR-146a-5p helps to enhance the repair and regeneration capacity of skeletal muscle, improve the patient's quality of life and exercise ability, and reduce the burden of disease.

[0011] This invention provides a new molecular target for the treatment of diseases related to skeletal muscle atrophy, mitochondrial dysfunction, etc. The treatment strategy based on miR-146a-5p is expected to play an important role in the treatment of various diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0013] Figure 1 Schematic diagram of the expression of miR-146a-5p in pig skeletal muscle tissue and skeletal muscle development characteristics provided in the embodiments of the present invention;

[0014] Figure 2 Schematic diagram of the expression and developmental characteristics of miR-146a-5p in mouse skeletal muscle tissue provided in an embodiment of the present invention;

[0015] Figure 3 Schematic diagram of the effect of miR-146a-5p on the proliferation and differentiation of porcine skeletal muscle satellite cells provided in an embodiment of the present invention;

[0016] Figure 4 Schematic diagram of the effect of adipose-specific miR-146a-5p knockdown on mouse skeletal muscle differentiation, mitochondrial autophagy, and apoptosis provided in an embodiment of the present invention;

[0017] Figure 5 Schematic diagram of the effects of miR-146a-5p on apoptosis, autophagy, ROS, and ATP during C2C12 cell differentiation provided in an embodiment of the present invention;

[0018] Figure 6 Schematic diagram of miR-146a-5p overexpression in small extracellular vesicles of adipose tissue alleviating muscle fiber atrophy, apoptosis and autophagy provided in an embodiment of the present invention;

[0019] Figure 7 Schematic diagram of miR-146a-5p regulating mouse skeletal muscle differentiation, regeneration and repair, mitochondrial autophagy and apoptosis through WAT-sEV provided in the embodiments of the present invention;

[0020] Figure 8 Schematic diagram of miR-146a-5p regulating skeletal muscle cell differentiation, mitochondrial autophagy and apoptosis by targeting Fbx32 provided in an embodiment of the present invention;

[0021] Figure 9 The effects of miR-146a-5p on mouse skeletal muscle differentiation, mitochondrial autophagy and apoptosis provided in the embodiments of the present invention;

[0022] Figure 10 Schematic diagram of the effect of miR-146a-5p knockdown on mouse skeletal muscle differentiation, mitochondrial autophagy and apoptosis provided in an embodiment of the present invention;

[0023] Figure 11 Schematic diagram of the effect of overexpression of miR-146a-5p on muscle fiber atrophy, apoptosis and autophagy provided in an embodiment of the present invention;

[0024] Figure 12 Schematic diagram of mi-R146a-5p regulating skeletal muscle differentiation, regeneration and repair, mitochondrial autophagy and apoptosis through small extracellular vesicles of white adipose tissue (WAT-sEV) provided in an embodiment of the present invention;

[0025] Figure 13 Schematic diagram of miR-146a-5p regulating skeletal muscle cell differentiation, mitochondrial autophagy and apoptosis by targeting Fbx32 provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0027] The experimental description used in this application:

[0028] Animals for this study were obtained from a commercial farm in Xinfeng County, Shaoguan City, Guangdong Province, China. Twenty boars were selected, including five lean-type piglets (LR) and five Chinese fat-type piglets (LT) at one day of age, and five lean-type piglets (CS) at 60 and 90 days of age. Samples of adipose tissue and longissimus dorsi muscle were promptly collected and stored in liquid nitrogen for future analysis.

[0029] Adipose-specific miR-146a-5p knockout (aKO) mice were created using the Cre-loxP system. The breeding process involved miR-146a-5p flox / flox (miR-146a-5-5p flox + / - The resulting F1 generation (miR-146a-5p flox + / - , Cre + / - ) and miR-146a-5flox + / - mice were crossed to generate aKO (miR-146a-5p flox + / - , Cre + / - ) and control flox (miR-146a-5p flox -+ / - ) mice. Mice were housed in individually ventilated cages under pathogen-free conditions at 24°C with a 12-h light / dark cycle and provided with a standard diet from the Animal Experiment Center of Tianjin Hospital. This study primarily used male mice aged 8 to 12 weeks, with additional young (4 weeks) and older (18 months) mice used for specific analyses.

[0030] 2. Immunofluorescence: Muscle tissue samples were snap-frozen in isopentane cooled with liquid nitrogen and embedded in Tissue Tek OCT (Fisher Scientific / Thermo Scientific). 10-μm-thick cryosections were prepared at −25°C and fixed with 4% paraformaldehyde for 10 minutes. Sections were washed three times with PBS for 5 minutes and then blocked 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 and included mouse anti-MyoD (1:100), rabbit anti-laminin (1:1000), mouse anti-MyHC (1:2000), and mouse anti-Pax7 (1:50). After three PBS washes, sections were incubated with secondary antibodies: goat anti-mouse IgM / Alexa Fluor 555 (1:2000) and goat anti-rabbit FITC (1:2000) for 1 hour at room temperature, and sections were treated with 0.05% Sudan Black for 5 minutes. Fluorescence images were acquired using a Nikon Eclipse Ti inverted microscope.

[0031] C2C12 cells were fixed with 4% paraformaldehyde and permeabilized with 0.4% Triton X-100 in PBS for 20 minutes each at room temperature. Cells were initially blocked with 5% goat serum for 1 hour and then incubated overnight at 4°C with primary antibodies: anti-MyoD, anti-MyHC, and anti-Pax7. Cells were then incubated with Alexa Fluor 555-conjugated goat anti-mouse IgM (1:2000) for 1 hour at room temperature in the dark. Cell nuclei were stained with DAPI. Fluorescence images were acquired with a Nikon Eclipse Ti microscope, and myotube diameter was quantified using ImageJ software.

[0032] 3. Extraction and culture of skeletal muscle satellite cells: Muscles of 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 handled under sterile conditions. After disinfecting the mice in 75% ethanol, the skin of the hind limbs was carefully removed, and the blood vessels, connective tissue, fat, and bones were discarded, leaving only the muscle tissue. For piglets, euthanasia was performed by cardiac puncture, and the longissimus dorsi muscle was isolated aseptically. The muscle tissue was then washed with PBS until the rinse solution was clear and minced into pieces of approximately 1 mm. 3The minced muscle was incubated in 0.2% collagenase II at 37°C in a water bath shaker for 1 hour with gentle stirring every 10 minutes. After digestion, the mixture was centrifuged at 1000 rpm for 10 minutes at 4°C and the supernatant was removed. The precipitate was treated with 0.25% trypsin for 30 minutes at 37°C with stirring every 10 minutes. Trypsin activity was inhibited by adding growth medium containing 20% ​​fetal bovine serum and 1% penicillin-streptomycin. The cell suspension was filtered through 100, 200 and 400 mesh filters in sequence and then centrifuged at 1000 rpm for 10 minutes at 4°C. The precipitate was resuspended in fresh growth medium and transferred to a 25cm 2 Cells were incubated at 37°C in a 5% CO2 environment. To purify MuSCs, a differential adhesion technique was used, in which isolated muscle cells were incubated for two hours to allow fibroblasts to attach to the surface while satellite cells remained suspended. Non-adherent cells were moved to new culture flasks, and the medium was changed on day 4 to remove residual blood and dead cells. Cell growth was monitored and cells were passaged when they reached 75%-90% confluency. At each passage, a 30-minute differential adhesion was performed to further eliminate fibroblasts.

[0033] 4. Cell culture: C2C12 myoblasts were maintained in 12-well plates in high-glucose DMEM supplemented with 10% FBS and 1% P / S. After reaching confluency, the medium was replaced with differentiation medium containing DMEM and 2% horse serum for a 6-day induction period. The medium was changed every two days. MuSC or C2C12 cells were plated at 1×10 5 The number of cells per well in a 12-well plate used for transfection experiments. Cells at 60-70% confluency were exposed to 40 nM miR-146a-5p mimic, 80 nM miR-146 / 5p inhibitor, 50 nM si-Fbx32, or 10 μg / mL sEV. Transfections were performed using siRNAs from GenePharma and Tsingke Biological Technology (China) in combination with Lipofectamine 2000.

[0034] 5. CCK-8 content assay: The cells were seeded in 96-well plates 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. The absorbance was measured at 450 nm using a microplate reader to determine cell viability. Cell viability was assessed using the CCK-8 assay. C2C12 cells were plated on 96-well plates at a density of 10,000 cells per well, with 6 replicates for each condition. After treatment, each well received a mixture of 10 μL of CCK-8 reagent and 90 μL of culture medium, followed by incubation for 1 hour. The absorbance at 450 nm was measured using a BioTek microplate reader, and blank wells were used as controls to assess cell viability and proliferation according to the instructions.

[0035] 6. EdU incorporation assay: using BeyoClick with Alexa Fluor 488 TM EdU cell proliferation kit was used to evaluate cell proliferation. C2C12 cells were plated at 1×10 4 Cells were counted per well and incubated with 10 μM EdU for 2 hours at 37°C in a CO2 incubator. Following incubation, cells were fixed in 4% paraformaldehyde for 20 minutes and then permeabilized with 0.3% Triton X-100 for 15 minutes. The click reaction mixture was incubated at room temperature in the dark for 30 minutes and then stained with Hoechst 33342 for 10 minutes. EdU-positive cells were quantified using ImageJ software.

[0036] 7. Flow cytometric analysis of apoptosis: Apoptosis was assessed by flow cytometry using the Annexin V-FITC / PI apoptosis detection kit. Cells were first harvested by trypsinization and transferred to a 15 mL centrifuge tube. After centrifugation at 1000 g for 5 minutes, the supernatant was discarded and the cell pellet was washed twice with cold PBS. After 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 were additionally centrifuged, and the supernatant was removed. The pellet was resuspended in 500 μL of Annexin V binding buffer, followed by the addition of 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI). The mixture was gently vortexed and incubated in the dark at room temperature (20-25°C) for 15 minutes. After incubation, the level of apoptosis was assessed by flow cytometry to distinguish between early apoptotic, late apoptotic, and necrotic cells.

[0037] 8. Transmission electron microscopy: Mice were humanely euthanized and perfused with 100 mM sodium phosphate buffer (pH 7.4). The tissue was immersed in a phosphate buffer (PB) fixative containing 2.5% glutaraldehyde and 1% paraformaldehyde. The tibialis anterior (TA) muscle was carefully dissected, cut into small pieces, and stored in the same fixative at 4°C overnight. After rinsing in PB, the samples were incubated in 0.2 M imidazole for 15 minutes and then fixed with 1% osmium tetroxide. After additional rinsing with high-purity water, the samples were incubated in a 4% aqueous solution of lead nitrate at 4°C overnight. Dehydration was performed using a series of graded acetone solutions, and the samples were then immersed in epoxy resin at 60°C for 24 hours. Ultrathin sections were then prepared using an ultramicrotome, placed on copper grids, and examined by transmission electron microscopy under double-blind analytical conditions.

[0038] 9. sEV Isolation, Characterization, and Administration: sEVs were isolated from white adipose tissue of 3-month-old mice. The tissue was rinsed three times with PBS and minced into fragments less than 3 mm in size. The cultures were maintained in 75 cm² flasks containing 25 ml of DMEM supplemented with 1% P / S and 10% sEV-depleted FBS at 37°C for 24 hours. After incubation, the supernatant was centrifuged in stages: first at 300 × g for 10 minutes to remove suspended cells, then at 2000 × g for 10 minutes to eliminate dead cells, and finally at 10,000 × g for 30 minutes to remove cellular debris. The supernatant was concentrated using a 100 kDa protein concentrator and subsequently purified using a 0.22 μm PVDF filter. The concentrated sample was ultracentrifuged at 120,000 × g for 90 minutes using a 38.5 mL Beckman Coulter tube. After ultracentrifugation, sEVs were washed with PBS, resuspended, and aliquoted into 100 μL in PBS and stored at −80° C. sEVs isolated from white adipose tissue (WAT) of Flox and aKO mice were labeled as Flox-sEVs and aKO-sEVs, respectively.

[0039] Western blot analysis was performed to characterize sEVs using the extracellular vesicle markers TSG101, Alix, CD9, and CD63, with calnexin as a negative control for the endoplasmic reticulum. The concentration and size distribution of sEVs were assessed using a Nanosight instrument, and their morphology was analyzed by transmission electron microscopy.

[0040] For in vivo administration, recipient mice were injected with 100 μg of sEVs into the TA muscle three times a week. In vitro, cells were treated with sEVs at a concentration of 10 μg / mL, and sEV uptake was tracked by labeling the vesicles with the fluorescent dye PKH67.

[0041] 10. Dual luciferase reporter assay: To evaluate gene expression, a dual luciferase reporter assay was performed. HEK293T cells were cultured in 96-well plates at 2.5 × 10 4 Cells were cultured at a density of 100 μg / well and allowed to reach 60%-70% confluency. 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 miR-146a-5p mimic or negative control. After 24 hours of incubation, luciferase activity was measured using the Dual-GLO Luciferase Assay System according to the manufacturer's instructions.

[0042] 11. Detection of Reactive Oxygen Species (ROS): Intracellular ROS levels in C2C12 cells were measured using a ROS detection kit, and fluorescence emission was visualized and recorded using a fluorescence microscope.

[0043] 12. Analysis of Cellular ATP Levels: Quantify ATP levels in cell lysates using an ATP assay kit. Mix 20 μL of lysate with 10 μL of ATP assay reagent and measure luminescence using a BioTek microplate reader. Measure protein concentration using a Pierce BCA assay kit. ATP levels are then normalized to protein content for calculation.

[0044] 13. Quantitative Real-Time PCR (RT-qPCR): Total RNA was extracted using TRIzol reagent. To remove genomic DNA contamination, RNA samples were treated with DNase I. 1–2 μg of RNA was reverse transcribed using EZB 4× EZscript Reverse Transcription Mix II to synthesize cDNA. Quantitative real-time PCR (qPCR) was performed on a Bio-Rad C1000 Touch using 2× RealStar Fast SYBR qPCR Mix (GenStar, catalog number A301). GAPDH served as an internal control for mRNA quantification, while U6 RNA was used to normalize miRNA expression levels.

[0045] 14. Western Blot: Western blotting was used to assess the expression of various functional proteins. Protein samples were lysed with RIPA buffer and quantified using the Thermo Fisher Rapid Gold BCA Protein Assay Kit. 15 μg of protein per sample was subjected to SDS-PAGE and transferred to a PVDF membrane (Millipore) for analysis. The primary antibodies included anti-TSG101 (rabbit, ZEN BIO, 381538), anti-Alix (rabbit, Sangon Biotech, D262028), anti-Calnexin mouse, ZEN BIO, 200947), anti-MyHC (mouse, R&D Systems, MAB4470), anti-MyoD (rabbit, ZEN BIO, 252249), anti-myosin (rabbit, ZEN BIO, 382257), anti-Fbx32 (rabbit, Abclonal, A3193), anti-MuRFabcam, ab243506), anti-P62 (rabbit, Abclonal, A19700) and anti-tubulin (rabbit, Bioworld, AP0064). The membrane was incubated with the primary antibody overnight and then exposed to the secondary antibody (Bioworld) for one hour at room temperature. Protein bands were visualized by gel imaging system, and Tubulin was used as a normalization control. Data analysis was performed using ImageJ software.

[0046] 15. Statistical Analysis: Statistical analyses, including one-way analysis of variance, independent t-test, and data visualization, were performed using SPSS (v27) and GraphPad Prism (v9.0). Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using unpaired Student's t-test or one-way analysis of variance. Statistical significance was defined as *P < 0.05 and **P < 0.01. The letters a, b, and c indicate different levels of significance between groups; different letters indicate significant differences, while the same letter indicates no significant difference.

[0047] Example 1:

[0048] The application of overexpressing adipose tissue-derived small extracellular vesicles miR-146a-5p in delaying skeletal muscle aging is for non-disease treatment purposes. The methods for overexpressing adipose tissue-derived small extracellular vesicles miR-146a-5p include transgenic overexpression, or treatment with small molecules or compounds that promote the transcription and translation of the miR-146a-5p gene; the host for delaying skeletal muscle aging is a cell, a non-genetic individual, or a genetic individual.

[0049] miR-146a-5p is a microRNA molecule that plays an important regulatory role in various physiological and pathological processes in the body. It can regulate gene expression by binding to specific sites on target mRNAs, leading to mRNA degradation or inhibiting their translation.

[0050] Adipose tissue-derived small extracellular vesicles (sEVs) are membrane-bound vesicles secreted by adipocytes and other cells, typically ranging in diameter from 30 to 150 nm. These sEVs play an important role in intercellular communication and transport, carrying a variety of bioactive molecules such as proteins, lipids, and nucleic acids, including miR-146a-5p. They can transmit signals between cells, influencing the physiological functions and gene expression of recipient cells.

[0051] Methods for overexpressing miR-146a-5p:

[0052] Transgenic overexpression: Through genetic engineering technology, the gene sequence of miR-146a-5p is introduced into adipose tissue cells, enabling the cells to continuously and stably express miR-146a-5p at a high level, and load it into small extracellular vesicles for secretion. This method can increase the production of miR-146a-5p from the source.

[0053] Treatment with small molecules or compounds that promote the transcription and translation of the miR-146a-5p gene: Some chemical small molecules or natural compounds can regulate gene transcription and translation. For example, certain histone deacetylase inhibitors can alter chromatin structure and enhance the transcriptional activity of the miR-146a-5p gene; while some protein synthesis enhancers can accelerate the processing and maturation of the miR-146a-5p precursor, thereby increasing its intracellular content and promoting its loading into small extracellular vesicles.

[0054] Host types that delay skeletal muscle aging:

[0055] At the cellular level: In in vitro cell experiments, co-culture of small extracellular vesicles derived from adipose tissue that overexpress miR-146a-5p with skeletal muscle cells revealed decreased expression of skeletal muscle cell aging markers, enhanced cell viability and proliferation, and alleviated cell cycle arrest. This provides cellular-level evidence for studying the mechanism by which miR-146a-5p delays skeletal muscle aging.

[0056] Non-genetic factors: For skeletal muscle aging caused by non-genetic factors, such as decreased muscle strength and mass due to aging, lack of exercise, and malnutrition, small extracellular vesicles overexpressing miR-146a-5p are expected to be an effective means of delaying the onset of aging. Through local injection or systemic administration, miR-146a-5p can act on skeletal muscle tissue, improve its physiological function, and delay the aging process.

[0057] Genetic individuals: For some hereditary skeletal muscle diseases, such as Duchenne muscular dystrophy, although the main cause is gene mutation, miR-146a-5p may also play an ameliorative role to a certain extent.

[0058] Overexpression of adipose tissue-derived small extracellular vesicles (miR-146a-5p) has broad application prospects in delaying skeletal muscle aging. Small extracellular vesicles can serve as natural carriers to specifically deliver miR-146a-5p to skeletal muscle cells, thereby increasing the concentration of the drug in the target tissue and reducing side effects on non-target tissues. Small extracellular vesicles (EVs) derived from adipose tissue are components produced by the organism itself and have good biocompatibility, low immunogenicity, and high safety. Adipose tissue is an abundant source and relatively easy to obtain. In addition, the extraction and preparation technology of small extracellular vesicles is also constantly developing and improving, which is conducive to large-scale production and application.

[0059] Example 2:

[0060] This embodiment provides a technical solution based on the first embodiment: a method for delaying skeletal muscle aging by using small extracellular vesicles miR-146a-5p derived from adipose tissue, comprising the following steps: overexpressing small extracellular vesicles miR-146a-5p derived from adipose tissue in a host body, or treating with small molecules or compounds that promote transcription, translation or functional activation of the miR-146a-5p gene; the method for overexpressing small extracellular vesicles miR-146a-5p derived from adipose tissue is to construct a recombinant expression vector containing the miR-146a-5p gene, and then overexpress it in animal cells, non-genetic individuals or genetic individuals.

[0061] Animal cells can be cell lines used in experimental research, such as mouse embryonic fibroblasts. Non-genetically afflicted individuals refer to normal humans or animals, while genetically afflicted individuals refer to humans or animals with a certain genetic background. By increasing the content of small extracellular vesicle miR-146a-5p in adipose tissue, the miR-146a-5p protein can function more effectively, thereby delaying the aging process of skeletal muscle.

[0062] Construct a recombinant expression vector: Obtain the miR-146a-5p gene sequence from a gene library, amplify the gene fragment using PCR, and clone it into the selected recombinant expression vector. Verify the constructed recombinant expression vector using enzyme digestion and sequencing to ensure that the miR-146a-5p gene is correctly inserted into the appropriate position in the vector and that the sequence is correct.

[0063] For animal cells, the constructed recombinant expression vector is introduced into animal cells through transfection technology (liposome transfection, electroporation, etc.), and cell lines that stably express miR-146a-5p are screened. Then, the small extracellular vesicles secreted by these cells are collected for subsequent research; for non-genetic individuals, the recombinant vector can be introduced into adipose tissue by local injection, or through viral vector-mediated gene delivery, the recombinant expression vector can be introduced into adipose tissue cells to overexpress miR-146a-5p in vivo and exert its effect through the secretion of small extracellular vesicles; in genetic individuals, due to their specific genetic background, the recombinant expression vector may need to be optimized and adjusted according to the individual's genetic characteristics to ensure the effective overexpression of miR-146a-5p.

[0064] Treatment with small molecules or compounds: Treatment can be performed in vivo or in vitro. In vitro, adipocytes can be incubated with small molecules or compounds, and the small extracellular vesicles secreted by the cells can be collected. In vivo, small molecules or compounds can be introduced into the animal body through injection, oral administration, or other routes to act on adipose tissue.

[0065] Example 3:

[0066] See also Figure 1 and Figure 2 This embodiment provides a technical solution based on the first embodiment: the expression of miR-146a-5p in skeletal muscle tissues of different animals and the characteristics of skeletal muscle development.

[0067] 1. Expression of miR-146a-5p in pig skeletal muscle tissue and skeletal muscle development characteristics:

[0068] The formation and development of porcine skeletal muscle is a highly complex process, with significant differences in growth rate and meat quality between different pig breeds. Skeletal muscle fiber growth and development are influenced by various factors, with breed and age being key factors. We examined the expression levels of miR-146a-5p in adipose and skeletal muscle tissue and investigated the differential expression of genes associated with skeletal muscle development in one-day-old Chinese obese Langtang (LT) and lean Landrace (LR) piglets.

[0069] RT-qPCR and Western Blot were used to evaluate the mRNA and protein expression of genes related to skeletal muscle differentiation. Figure 1 a, The expression levels of miR-146a-5p in fat and skeletal muscle of LR piglets were significantly higher than those in LT piglets; see Figure 1 b to Figure 1 c, Western blot analysis showed that the expression of cell proliferation-related proteins (Cyclin A2, Cyclin D1, Cyclin E1 and PCNA) in skeletal muscle of LR piglets was significantly decreased compared with that of LT piglets. Figure 1 f, The mRNA and protein expressions of MyoG and MyoD were significantly increased, the MyHC protein level was significantly elevated, and the expressions of Fbx32 and MuRF were significantly decreased in the skeletal muscles of LR piglets.

[0070] Subsequently, we analyzed the expression of miR-146a-5p and changes in skeletal muscle development-related genes in 1-day-old lean LR piglets, 60-day-old lean three-cross (CS) pigs, and 90-day-old CS pigs. Figure 1 g, Compared with other pig groups, LR piglets showed the highest expression of miR-146a-5p, while 90-day-old CS pigs showed the lowest expression; see Figure 1 h, RT-qPCR analysis showed that MyoD and MyoG mRNA levels were significantly downregulated, while Fbx32 and MuRF levels were significantly upregulated in 90-day-old CS pigs; see Figure 1 i to Figure 1 j, Western blot analysis confirmed that the protein levels of MyoG, MyoD, and MyHC were significantly decreased in 90-day-old CS pigs. In contrast, the protein levels of Fbx32 and MuRF were significantly increased.

[0071] In summary, the expression of miR-146a-5p is significantly correlated with the skeletal muscle development of pigs, and there are significant differences between different breeds and ages.

[0072] 2. Expression and developmental characteristics of miR-146a-5p in mouse skeletal muscle tissue: Effects of miR-146a-5ps on skeletal muscle growth in young and old wild-type mice.

[0073] See also Figure 2 a to Figure 2 b, Examination of TA muscle sections revealed smaller skeletal muscle fiber area (0–200 μm) in aged mice compared with young mice. 2 ) increased, with larger fiber area (1000-3000μm 3 ) is significantly reduced; see Figure 2c, RT-qPCR analysis showed that the expression of miR-146a-5p in skeletal muscle of aged mice was significantly decreased compared with that of young mice; see Figure 2 d to Figure 2 e, Western blot analysis showed that proliferation-related proteins (PCNA, Cycl in D1, Cycl in A2, and Cycl in E1) were significantly increased in skeletal muscle of aged mice compared with young mice; see Figure 2 f to Figure 2 h, RT-qPCR and Western Blot analysis showed that the MyHC protein level in the skeletal muscle of aged mice decreased significantly, the mRNA expression and protein abundance of the differentiation markers MyoD and MyoG decreased, and the mRNA expression and protein levels of the skeletal muscle atrophy genes Fbx32 and MuRF increased simultaneously.

[0074] In summary, decreased miR-146a-5p levels in aged mice hindered skeletal muscle differentiation and exacerbated muscle atrophy, highlighting its key role in the regulation of muscle development and aging.

[0075] Example 4:

[0076] See also Figure 3 This embodiment provides a technical solution based on the first embodiment: the effect of miR-146a-5p on the proliferation and differentiation of pig MuSCs.

[0077] By isolating porcine MuSCs, we investigated the biological role of miR-146a-5p, particularly its effects on cell proliferation and differentiation. Figure 3 a, The extracted MuSCs were first characterized. In growth medium (GM), the early proliferating myofibroblast marker Pax7 was positive in up to 85% of cells, indicating that these cells were myogenic precursors. When switched to differentiation medium (DM), an increase in the number of myotubes and more obvious fusion were observed on day 5. The differentiation marker MyHC was successfully enriched in MuSCs-DM; see Figure 3 b, Western blot analysis showed that the expression levels of Cyclin A2, Cyclin D1, Cyclin E1, and Pax7 were significantly increased in GM culture compared with DM; see Figure 3 c, The expression of MyoG and MyoD was significantly increased in DM compared with GM. These results confirmed the successful isolation of porcine MuSCs.

[0078] See also Figure 3 d and Figure 3k, The effects of miR-146a-5ps on MuSC proliferation and differentiation were explored by transfecting cells with mimics and inhibitors of miR-146a-5ps. Figure 3 For example, treatment with miR-146a-5p mimics significantly reduced the number of EdU-positive cells and cell viability, as shown by EdU staining and CCK-8 assays, and these effects were abolished by miR-146a-5p inhibitors. Figure 3 h to Figure 3 j, miR-146a-5p mimics significantly reduced PCNA protein levels and the mRNA and protein expressions of Cyclin D1, Cyclin A2, and Cyclin E1, whereas miR-146a-5p inhibition produced the opposite effects. Figure 3 l, miR-146a-5p mimics enhanced MyoG and MyoD mRNA expression and decreased Fbx32 and MuRF mRNA levels in MuSCs, whereas miR-146a-5p inhibitors produce the opposite effects. Figure 3 m to Figure 3 n, Western blot analysis showed that miR-146a-5p mimics increased MyHC protein levels in MuSCs, whereas miR-146a-5p inhibitor significantly reduced MyHC expression.

[0079] Overall, miR-146a-5p is crucial in controlling the growth and differentiation of MuSCs and may affect their biological activities by regulating cell cycle proteins and skeletal muscle differentiation markers.

[0080] Embodiment 5:

[0081] See also Figure 4 、 Figure 9 and Figure 10 This embodiment provides a technical solution based on the first embodiment: the effect of adipose-specific miR-146a-5p knockdown on skeletal muscle differentiation, mitochondrial autophagy and apoptosis in mice.

[0082] A fat-specific miR-146a-5p knockout mouse model (aKO mouse, Cre) was created using CRISPR-Cas9. + / - Loxp + / - ), Flox mice (Loxp + / -+ ) as a control, and the effect of miR-146a-5p deletion in adipose tissue on skeletal muscle development was studied ( Figure 4 a). Please refer to Figure 4 a and Figure 4 b, Genotype identification ( Figure 4a) and real-time fluorescence quantitative PCR ( Figure 4 b) The analysis confirmed that the expression of miR-146a-5p in WAT of aKO mice was significantly reduced compared with that of Flox mice, verifying the successful establishment of the aKO mouse model.

[0083] See also Figure 9 a to Figure 9 b, In aKO mice, miR-146a-5p expression in TA muscle was significantly reduced. The effect of deleting adipose-specific miR-146a-5p on muscle development was evaluated by comparing skeletal muscle growth in young and old 146aKO mice. Histological analysis of TA muscle showed more pronounced muscle atrophy and smaller muscle fibers (0-200 μm) in old 146aKO mice compared with young mice. 2 ) increased, larger fibers (1000-5000μm 3 )reduce.

[0084] See also Figure 9 c, RT-qPCR analysis showed that miR-146a-5p levels were significantly decreased in TA muscles of aged 146aKO mice compared with those of young mice. Figure 9 de, RT-qPCR and Western Blot analysis confirmed that the levels of proliferation-related proteins were increased in the skeletal muscle of aged 146aKO mice. Figure 9 f to Figure 9 h, In aged 146aKO mice, the mRNA and protein levels of atrophy-related genes Fbx32 and MuRF were significantly increased, while the expression of differentiation-related genes was significantly decreased compared with young mice.

[0085] These findings are similar to the characteristics of skeletal muscle development in wild-type mice at different ages, further confirming that adipose-specific miR-146a-5p deletion has an age-dependent effect on skeletal muscle development.

[0086] Pluripotent stem cells are essential for the maintenance and regeneration of skeletal muscle. MuSCs were isolated from muscle tissue of Flox and aKO mice to evaluate the effects of adipose-specific loss of miR-146a-5p on their proliferation and differentiation. Figure 4 c, The expression of miR-146a-5p was significantly reduced in MuSCs from aKO mice (aKO-MuSCs). Figure 4 e to Figure 4 f, Adipocyte-specific deletion of miR-146a-5p resulted in a significant increase in cell survival and EdU positivity in aKO-MuSCs. Figure 4 g. Figure 4 h and Figure 10a, The mRNA levels of PCNA, Cyclin D1, Cyclin A2, and Cyclin E1 were significantly increased in aKO-MuSCs, and Western Blot confirmed a similar trend at the protein level ( Figure 4 h and Figure 10 a). Please refer to Figure 4 i to Figure 4 k, Myotube diameter and nuclear fusion index of aKO-MuSCs were significantly reduced. Figure 4 l to Figure 4 m and Figure 10 b, The expression levels of MyoD and MyoG at the mRNA and protein levels, as well as MyHC protein, were significantly decreased, while the mRNA and protein levels of atrophy-related genes Fbx32 and MuRF were significantly increased. In conclusion, adipose-specific miR-146a-5p knockout exacerbates MuSC atrophy in mice.

[0087] See also Figure 4 n, RT-qPCR analysis showed that the expression of pro-apoptotic genes Bax, Caspase-3, and Caspase-9 was significantly increased in the TA muscle of aKO mice; see Figure 4 o to Figure 4 p, Immunoblotting showed that the Bcl-2 protein level was significantly decreased in the TA muscle of aKO mice, while the levels of Bax and Caspase-3 were increased; see Figure 4 q, Electron microscopy analysis shows mitochondrial swelling and autophagic vacuoles in TA muscle of aKO mice; see Figure 4 r, RT-qPCR analysis showed that the mRNA levels of autophagy-related genes ULK1, ULK2, ATG16L1, P62, LC3a, and LC3b were significantly increased in TA muscles of aKO mice compared with those in the control group; see Figure 4 s to Figure 4 u, Western blot analysis further confirmed the increased expression of autophagy-related proteins LC3 and P62 in TA muscles of aKO mice; see Figure 10 c to Figure 10 e, Bcl-2 mRNA and protein levels in aKO-MuSCs were significantly decreased, while Bax and Caspase-3 levels were significantly increased, and Caspase-9 mRNA was upregulated. Figure 10 f to Figure 10 i, RT-qPCR and Western blot analyses also showed that autophagy-related genes and proteins were significantly upregulated in aKO MuSCs, mirroring the changes observed in skeletal muscle tissue of aKO mice.

[0088] In conclusion, adipose-specific loss of miR-146a-5p in a mouse model enhances MuSC proliferation while impeding skeletal muscle differentiation and exacerbating muscle atrophy. These changes underscore the critical role of miR-146a-5ps in muscle development through apoptotic and autophagic pathways.

[0089] Example 6:

[0090] See also Figure 5 This embodiment provides a technical solution based on the first embodiment: the effect of miR-146a-5p on apoptosis, autophagy, ROS and ATP during the differentiation of C2C12 cells.

[0091] See also Figure 5 a, The effects of miR-146a-5p on apoptosis, autophagy, ROS, and ATP levels in C2C12 cells were investigated by transfecting C2C12 cells with miR-146a-5p mimics or inhibitors and performing various experiments; see Figure 5 b to Figure 5 c, Flow cytometric analysis showed that miR-146a-5p mimics significantly reduced cell apoptosis, whereas inhibitors enhanced cell apoptosis; see Figure 5 d, RT-qPCR analysis showed that the mimic increased Bcl-2 expression while decreasing the levels of Bax, Caspase-3, and Caspase-9; these effects were reversed by the inhibitor; see Figure 5 e to Figure 5 f, Western blot analysis confirmed the regulatory effect of miR-146a-5p on apoptosis-related proteins.

[0092] For autophagy, see Figure 5 g, RT-qPCR showed that miR-146a-5p mimics significantly reduced the mRNA levels of autophagy-related genes (ULK1, ULK2, ATG16L1, P62, LC3a, and LC3b), while the inhibitor significantly increased their expression; see Figure 5 h to Figure 5 j, Immunoblotting showed that miR-146a-5p mimics significantly reduced LC3 and P62 protein levels, while the inhibitor significantly increased their expression. In addition, ROS plays a crucial role in skeletal muscle atrophy and aging and helps maintain mitochondrial homeostasis by promoting mitophagy. Figure 5 k to Figure 5m, Elevated miR-146a-5p expression significantly reduced ROS production and increased ATP levels in C2C12 cells. Conversely, miR-146a-5p inhibitor enhanced ROS production and decreased ATP levels. miR-146a-5p significantly affected the differentiation of C2C12 cells by inhibiting apoptosis, autophagy, and ROS production.

[0093] Embodiment seven:

[0094] See also Figure 6 and Figure 11 , this embodiment provides a technical solution based on the first embodiment: overexpression of miR-146a-5p in adipose tissue sEV alleviates muscle fiber atrophy, apoptosis and autophagy.

[0095] sEVs are crucial for intercellular communication, transporting biomolecules such as lipids, proteins, and miRNAs to target cells. Figure 6 a and Figure 6 b, WAT-derived sEVs (WAT-sEVs) were extracted from Flox mice and aKO mice, and their morphology was observed using transmission electron microscopy, and their diameters were determined to be mainly distributed between 20 and 170 nm using a nanoparticle size analyzer. Figure 6 c, Western blot analysis confirmed the presence of typical sEV markers Alix, TSG101, CD63, and CD9 in WAT sEVs, while the endoplasmic reticulum marker calnexin was not detected, verifying the successful isolation of WAT sEVs; see Figure 6 d, qPCR analysis showed that the level of miR-146a-5p in adipose tissue-derived sEVs of aKO mice was significantly decreased compared with that of Flox mice.

[0096] To further verify the biological function of WAT-sEVs, C2C12 cells were treated with PKH67 fluorescent labeling and their intracellular uptake was observed after 12 hours. Figure 6 e, Immunofluorescence staining results showed that sEVs successfully entered the cytoplasm. In subsequent cell culture experiments, C2C12 cells were co-transfected with sEVs from Flox mice (Flox-sEV) or aKO mice (aKO-sEV) and miR-146a-5p mimics (aKO-sEV+M), resulting in a significant increase in myofiber diameter and nuclear fusion percentage. Figure 6 f to Figure 6 g and Figure 11 C2C12 cells co-transfected with aKO-sEV or Flox-sEV and a miR-146a-5p inhibitor (Flox-sEV+I) showed a significant decrease in myofiber diameter and nuclear fusion rate; see Figure 6 g and Figure 6 I, Western Blot analysis showed that Flox-sEV and aKO-sEV+M treatment significantly increased the expression of muscle-specific markers MyHC and MyoD, while decreasing the levels of atrophy-related proteins Fbx32 and MuRF. In contrast, aKO-sEV and Flox-sEV+I treatment showed the opposite effect, significantly decreasing the levels of MyHC and MyoD, while increasing the levels of Fbx32 and MuRF; see Figure 6 j and Figure 6 k, Western blot analysis showed that Flox-sEV and aKO-sEV+M increased the expression of Bcl-2 and decreased the levels of Caspase-3 and Bax. In contrast, aKO-sEV and Flox-sEV+I counteracted these effects by decreasing the level of Bcl-2 and increasing the levels of Bax and Caspase-3.

[0097] See also Figure 6 l and Figure 6 M, Autophagy analysis showed that Flox-sEV and aKO-sEV+M significantly reduced the expression of LC3 and P62 autophagy markers, while aKO-sEV and Flox-sEV+I enhanced their expression. In conclusion, overexpression of miR-146a-5p can counteract the inhibition of myofiber differentiation, atrophy, apoptosis, and autophagy induced by aKO-sEV, while enhancing the differentiation of C2C12 cells.

[0098] Embodiment 8:

[0099] See also Figure 7 and Figure 12 This embodiment provides a technical solution based on the first embodiment: MiR-146a-5p regulates mouse skeletal muscle differentiation, regeneration and repair, mitochondrial autophagy and apoptosis through WAT-sEV.

[0100] Wild-type aged mice were selected as experimental subjects, and PKH67 was used to fluorescently label WAT-sEVs. Labeled WAT-sEVs were injected into the TA muscles of the mice. Figure 7 a and Figure 7 b, The results show that these particles are primarily located in the gastrocnemius (GAS) and TA muscles and, once in the systemic circulation, they accumulate in epididymal WAT and inguinal WAT as well as intestinal fat. Figure 7 c, PBS, Flox-sEVs, and aKO-sEVs were serially injected into TA muscles over a 17-day period, and muscle samples were collected for analysis on day 19. Figure 7d, miR-146a-5p levels were significantly increased in TA muscles of aged mice treated with Flox-sEVs compared with those in the aKO-sEV group. Figure 7 e to Figure 7 f and Figure 12 a to Figure 12 d, The expression of differentiation-related genes was significantly upregulated, while the expression of proliferation- and atrophy-related genes was significantly downregulated. Figure 7 i to Figure 7 l, Compared with the aKO-sEV group, the myofiber area (>5000 μm) in the TA muscle of aged mice after Flox-sEV injection decreased. 2 ) increased significantly, and smaller muscle fibers (<200 μm 2 ). See Figure 7 m to Figure 7 n, The fluorescence intensity of MyoD and Pax7 in TA muscles of mice injected with Flox-sEVs was significantly increased, indicating enhanced skeletal muscle repair and regeneration, and apoptosis markers were significantly reduced in TA muscles of aged mice treated with Flox-sEVs. Figure 7 o to Figure 7 q and Figure 12 e to Figure 12 f, Autophagy was significantly improved in the TA muscles of aged mice receiving Flox-sEVs, whereas mitophagy was observed in the aKO-sEV group.

[0101] In summary, miR-146a-5p promotes skeletal muscle recovery by promoting differentiation, repair and regeneration, as well as inhibiting muscle atrophy and regulating apoptosis and mitochondrial autophagy.

[0102] Embodiment 9:

[0103] See also Figure 8 and Figure 13 This embodiment provides a technical solution based on the first embodiment: MiR-146a-5p regulates skeletal muscle cell differentiation, mitochondrial autophagy and apoptosis by targeting Fbx32.

[0104] See also Figure 8 a, Transcriptomic and bioinformatics analyses suggest that miR-146a-5p may affect mitophagy in skeletal muscle through its interaction with Fbx32. In 293T cell lines, Fbx32 wild-type (pmirGLO WT), mutant (pmirGLO MUT), and deletion (pmirGLO DEL) were co-transfected with negative control (NC) or miR-146a-5p mimics. Figure 8 Co-transfection of bmiR-146a-5p mimic with pmirGLO WT significantly reduced the dual-luciferase activity, confirming its interaction with Fbx32.

[0105] See also Figure 8 c to Figure 8 d, Three siRNAs targeting Fbx32 were constructed and verified by protein knockdown efficiency, and siFbx32-1 was subsequently selected for subsequent experiments. This experiment included three groups: NC, si-Fbx32, and si-Fbx12 combined with a miR-146a-5p inhibitor (si-Fbx2+miR-146a-5p I). Figure 8 e, In C2C12 cells, si-Fbx32 transfection significantly increased the level of miR-146a-5p, while si-Fbx32+miR-146a-5p I significantly reduced its expression. Figure 8 f to Figure 8 g, Western blot analysis showed that si-Fbx32 transfection significantly reduced the protein levels of Cyclin A2, Cycl in D1, Cyclin E1, and PCNA, whereas co-transfection with miR-146a-5p inhibitor counteracted this reduction, leading to increased protein expression. Figure 8 h to Figure 8 I, EdU detection showed that si-Fbx32 transfection significantly reduced the EdU positive rate in C2C12 cells, while si-Fbx12+miR-146a-5p I significantly increased the positive rate. Figure 8 j to Figure 8 l. si-Fbx32 transfection significantly increased myofiber diameter and myotube nuclear fusion rate, but co-transfection with miR-146a-5p inhibitor significantly reduced these improvements. Figure 8 m to Figure 8 p, In C2C12 cells, si-Fbx32 transfection significantly enhanced the fluorescence intensity of MyoD and Pax7, while co-transfection with miR-146a-5p inhibitor significantly reduced their intensities. Figure 8 q, RT-qPCR analysis showed that si-Fbx32 transfection significantly reduced the mRNA levels of skeletal muscle atrophy markers Fbx32 and MuRF, and enhanced the mRNA expression of skeletal muscle differentiation markers MyoD and MyoG. In contrast, the opposite results were observed in the si-Fbx32+miR-146a-5p I group. Figure 8 r and Figure 13 b, Western blot analysis confirmed the results of RT-qPCR, showing that si-Fbx32 transfection significantly increased skeletal muscle differentiation markers and decreased the levels of atrophy-related proteins, and the combined use of si-Fbx32 and miR-146a-5p inhibitor reversed these changes. Figure 13 a, KEGG pathway analysis showed that miR-146a-5p may be involved in regulating the FoxO3 signaling pathway. Figure 8 r and Figure 13 b, Western blot analysis showed that FoxO3 phosphorylation was reduced after si-Fbx32 transfection, while co-transfection with miR-146a-5p inhibitor led to increased FoxO3 phosphorylation. Figure 8 s to Figure 8 t and Figure 13 c, si-Fbx32 transfection significantly reduced the mRNA and protein expression of apoptosis- and autophagy-related genes in C2C12 cells. In contrast, co-transfection with a miR-146a-5p inhibitor increased gene expression levels. Figure 8 u to Figure 8 w, In terms of ROS, si-Fbx32 transfection significantly reduced ROS levels and increased ATP levels, while si-Fbx12+miR-146a-5p I group promoted ROS production and reduced ATP levels.

[0106] In summary, miR-146a-5p regulates key biological processes such as skeletal muscle cell differentiation, mitochondrial autophagy, ROS, and apoptosis by targeting the Fbx32 / FoxO3 signaling pathway, paving the way for its application in delaying skeletal muscle aging.

[0107] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. Application of overexpression of adipose tissue-derived small extracellular vesicle miR-146a-5p in delaying skeletal muscle aging, characterized in that: The application is for non-disease treatment purposes, and the method of overexpressing adipose tissue-derived small extracellular vesicle miR-146a-5p includes transgenic overexpression, or treatment with small molecules or compounds that promote miR-146a-5p gene transcription and translation.

2. The use of overexpressing adipose tissue-derived small extracellular vesicles miR-146a-5p in delaying skeletal muscle aging according to claim 1, characterized in that: The host for delaying skeletal muscle aging is a cell, a non-genetic individual or a genetic individual.

3. A method for delaying skeletal muscle aging using small extracellular vesicles miR-146a-5p derived from adipose tissue, characterized in that: The method comprises the following steps: overexpressing adipose tissue-derived small extracellular vesicle miR-146a-5p in a host, or treating the host with small molecules or compounds that promote transcription, translation or functional activation of the miR-146a-5p gene.

4. The method of claim 3, wherein the method comprises: The method for overexpressing adipose tissue-derived small extracellular vesicle miR-146a-5p is to construct a recombinant expression vector containing the miR-146a-5p gene, and then overexpress it in animal cells, non-hereditary individuals or hereditary individuals.

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