Application of compound in preparation of medicine for treating diabetes-induced muscle atrophy

Abstract

The invention discloses an application of a compound in preparation of a medicine for treating diabetes-induced muscle atrophy, the compound is an exosome modified by an aptamer, and the sequence of the aptamer is shown as SEQ ID NO.1. The invention further discloses a preparation method of the compound for treating diabetes-induced muscle atrophy. The invention discloses the mechanism of the exosome for regulating and controlling muscle atrophy from the angle of mitochondrial function for the first time, and verifies that the exosome can improve diabetes-induced muscle atrophy, and the aptamer combination can enhance the effect of the exosome on muscle atrophy. Therefore, the compound (the aptamer-modified exosome) has a good prospect in the application of preparing the medicine for treating diabetes-induced muscle atrophy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine industry, and in particular to application of a composite in preparing a drug for treating diabetic-induced muscle atrophy. Background Art

[0002] Muscle atrophy refers to the reduction in muscle volume due to thinning or even disappearance of muscle fibers. Aging, immobilization, and some chronic diseases such as type 2 diabetes mellitus (T2DM) may all lead to muscle atrophy.

[0003] Skeletal muscle fibers include fast-twitch fibers and slow-twitch fibers. Slow-twitch fibers contain more myoglobin and mitochondria than fast-twitch fibers, and produce abundant ATP through oxidative phosphorylation (OXPHOS), which can improve exercise endurance. Studies have shown that the conversion of muscle fiber type from slow to fast can reduce the level of skeletal muscle oxidative metabolism and exercise capacity. Mitochondrial dysfunction plays an important role in muscle atrophy. Impaired mitochondrial function and abnormal mitochondrial accumulation in aged mice can lead to muscle atrophy. Disorders of glucose and lipid metabolism, insulin resistance, and inflammatory responses in diabetic patients may lead to skeletal muscle mitochondrial dysfunction, accompanied by abnormal muscle fiber type conversion and muscle atrophy. Forkhead box O (FoxO) family members are key transcription factors that regulate muscle atrophy. They are transported to the nucleus after dephosphorylation and / or acetylation to promote the expression of E3 ubiquitin ligases MuRF1 and Atrogin 1. Studies have shown that FoxO1 / 3 / 4 deficiency can enhance the function of muscle mitochondria and the proportion of muscle tissue, while FoxO3 activation reduces muscle content by inducing the expression of MuRF1 and Atrogin 1. Sirtuins, especially SIRT1, are associated with metabolic diseases such as obesity and diabetes. SIRT1 is a nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase plays a crucial role in skeletal muscle remodeling by regulating FoxO transcriptional activity. Activation of SIRT1 in skeletal muscle of diabetic mice promotes mitochondrial biogenesis.

[0004] Stem cells have unique biological characteristics, strong self-renewal ability, multidirectional differentiation potential, and the ability to secrete a variety of cytokines. They are the best seed cells for organ and tissue regeneration. Stem cell therapy can enable some diabetic patients to stop insulin therapy or reduce insulin dosage, and its effectiveness and safety in treating diabetes have also been effectively verified. Exosomes (EXO) are extracellular vesicles secreted by cells that contain bioactive molecules, including proteins, lipids, and nucleic acids. At present, many studies at home and abroad have shown that treatment based on mesenchymal stem cells (MSC) plays an important role in muscle atrophy caused by various diseases, especially mesenchymal stem cells and their exosomes have good application prospects in the treatment of muscle atrophy caused by T2DM.

[0005] However, systemic application of exosomes has low selectivity for diseased tissues, which has implications for their efficacy and safety related to nonspecific biodistribution in vivo. Summary of the invention

[0006] The present disclosure is made in view of the above-mentioned prior art conditions, and its purpose is to provide an application of a complex in the preparation of a drug for treating diabetic-induced muscle atrophy.

[0007] To this end, the first aspect of the present disclosure provides the use of a complex in the preparation of a drug for treating muscle atrophy induced by diabetes, wherein the complex is an exosome modified by an aptamer, and the sequence of the aptamer is shown in SEQ ID NO. 1. In the present disclosure, the mechanism of exosome regulation of muscle atrophy is revealed for the first time from the perspective of mitochondrial function, and it is verified that exosomes can improve muscle atrophy induced by diabetes, and aptamer binding can enhance the effect of exosomes on muscle atrophy. Therefore, the complex (exosome modified by an aptamer) has good prospects in the application of preparing a drug for treating muscle atrophy induced by diabetes.

[0008] In the applications involved in the present disclosure, optionally, the exosomes are derived from human umbilical cord mesenchymal stem cells.

[0009] In the application of the first aspect of the present disclosure, optionally, the aptamer is folded to form a secondary structure.

[0010] In the application of the first aspect of the present disclosure, optionally, the aptamer is resuspended in TE buffer and then folded in folding buffer to form a secondary structure under the following folding conditions: 10 min at 70°C, rapid cooling on ice for 5 min, and slow cooling at 37°C for 30 min.

[0011] In the application of the first aspect of the present disclosure, optionally, the step of modifying the exosomes using the aptamer includes: adding an aldehyde modification to the 5' end of the aptamer, and incubating the aldehyde-modified aptamer with the exosomes to obtain the complex.

[0012] In the application of the first aspect of the present disclosure, optionally, the exosomes improve diabetes-induced muscle atrophy by enhancing SIRT1 / foxo3a-mediated mitochondrial function.

[0013] In the application of the first aspect involved in the present disclosure, optionally, the aptamer promotes the internalization efficiency of the exosomes in muscle cells, and enhances the effect of the exosomes on muscle atrophy and muscle fiber type transformation.

[0014] In the application of the first aspect of the present disclosure, optionally, the aptamer enhances the effect of the exosomes through SIRT1-mediated mitochondrial function.

[0015] The second aspect of the present disclosure provides an application of a complex in the preparation of a drug for treating muscle diseases, characterized in that the complex is an exosome modified by an aptamer, and the sequence of the aptamer is shown in SEQ ID NO. 1. In the present disclosure, it is confirmed that aptamer modification enhances the effect of exosomes on diabetes-induced muscle atrophy and muscle fiber type transformation, providing a new idea for improving the efficacy of exosomes in treating muscle diseases.

[0016] The third aspect of the present disclosure provides a drug for treating diabetic-induced muscle atrophy, including a complex, wherein the complex is an exosome modified by an aptamer, and the sequence of the aptamer is shown in SEQ ID NO. 1. In the present disclosure, it is confirmed that aptamer modification enhances the effect of exosomes on diabetic-induced muscle atrophy and muscle fiber type transformation, providing a new idea for improving the efficacy of exosomes in treating muscle diseases.

[0017] According to the present disclosure, there can be provided a use of a complex in preparing a drug for treating diabetic-induced muscle atrophy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a test result diagram of the MSC-EXOs involved in the present invention in alleviating diabetes-induced muscle atrophy and muscle fiber type transformation.

[0019] Figure 2 This is a test result diagram of the MSC-EXOs involved in the present invention repairing atrophy-related SIRT1 / FoxO3a signals and mitochondrial function damage.

[0020] Figure 3This is a test result diagram showing that the MSC-EXOs involved in the present invention alleviates PA-induced C2C12 myotube atrophy and mitochondrial dysfunction.

[0021] Figure 4 This is a test result diagram showing that the MSC-EXOs involved in the present invention combat myotube atrophy by enhancing SIRT1-mediated mitochondrial function.

[0022] Figure 5 This is a test result diagram showing that the binding of the aptamer involved in the present invention promotes the internalization of MSC-EXOs in skeletal muscle.

[0023] Figure 6 The figure is a test result showing that the aptamer binding of the present invention enhances the effect of MSC-EXOs on muscle atrophy and muscle fiber type transformation.

[0024] Figure 7 This is a test result diagram of the effect of the aptamer involved in the present invention on enhancing MSC-EXOs through SIRT1-mediated mitochondrial function. DETAILED DESCRIPTION

[0025] Hereinafter, with reference to the accompanying drawings, the preferred embodiments of the present disclosure will be described in detail. In the following description, the same symbols are assigned to the same components, and repeated descriptions are omitted. In addition, the accompanying drawings are only schematic diagrams, and the ratio of the dimensions of the components or the shapes of the components may be different from the actual ones.

[0026] It should be noted that the terms "including" and "having" and any variations thereof in the present invention, such as a process, method, system, product or device that includes or has a series of steps or units, are not necessarily limited to those steps or units clearly listed, but may include or have other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0027] In addition, the subheadings and the like involved in the following description of the present invention are not intended to limit the content or scope of the present invention, but are merely used as a reminder for reading. Such subheadings should neither be understood as being used to divide the content of the article, nor should the content under the subheading be limited to the scope of the subheading.

[0028] Abbreviations and their interpretations of the proper nouns involved in this disclosure:

[0029] T2DM: type 2diabetes mellitus type 2 diabetes;

[0030] MSC / MSCs: Mesenchymal stem cell / cells;

[0031] hucMSCs: human umbilical cord MSCs human umbilical cord mesenchymal stem cells;

[0032] MSC-EXOs / MSC-EXO:hucMSC-derived exosomes human umbilical cord mesenchymal stem cell-derived exosomes;

[0033] HFD: High-fat diet;

[0034] IM: hindlimb immobilization;

[0035] PBS: Phosphate-buffered saline;

[0036] IPGTT: Intraperitoneal glucose tolerance test;

[0037] IPITT: Intraperitoneal insulin tolerance test.

[0038] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below by the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be interpreted as limiting the present invention.

[0039] It should be understood by those skilled in the art that unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.

[0040] It should also be understood that terms, such as those defined in commonly used dictionaries, should be understood to have a meaning consistent with that in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless as defined herein.

[0041] Those skilled in the art will appreciate that, unless otherwise stated, the singular forms "a", "an", "said" and "the" used herein may also include plural forms. It should be further understood that the term "comprising" used in the specification of the present invention refers to the presence of the features, integers, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements and / or groups thereof.

[0042] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

[0043] The present disclosure relates to:

[0044] Application of the complex in the preparation of a drug for treating diabetic-induced muscle atrophy;

[0045] Application of the complex in the preparation of drugs for treating muscle diseases;

[0046] Drugs, including compounds, for treating diabetes-induced muscle atrophy;

[0047] Application of the complex in the preparation of a drug for treating muscle atrophy induced by type 2 diabetes;

[0048] Application of the complex in preparing a drug for treating disuse muscular atrophy.

[0049] In the present disclosure, the complex refers to exosomes modified by aptamers. The complex can be targeted to muscle. Thus, the effect of the complex on muscle diseases can be improved.

[0050] In some examples, aptamers can contribute to the targeted muscle delivery of exosomes. In some examples, aptamer binding can enhance the effect of exosomes on muscle atrophy. In some examples, the sequence of the aptamer can be 5'-CAGGAGCCGAGAACCGGTTGGTGGGTAATCCTGTTAGCGC-3' (as shown in SEQ ID NO.1). In the present disclosure, the mechanism of exosome regulation of muscle atrophy is revealed for the first time from the perspective of mitochondrial function, and it is verified that exosomes can improve diabetes-induced muscle atrophy, and aptamer binding can enhance the effect of exosomes on muscle atrophy. Therefore, the complex (exosomes modified by aptamers) has good prospects in the application of preparing drugs for treating diabetes-induced muscle atrophy.

[0051] In the present disclosure, exosomes modified by aptamers may also be referred to as aptamer-conjugated exosomes, aptamer-coupled exosomes, and the like.

[0052] In some examples, the aptamer can fold to form a secondary structure, thereby helping to improve the stability and functionality of the aptamer.

[0053] In some examples, the aptamer can be resuspended in TE buffer and then folded in folding buffer to form a secondary structure. In some examples, the folding conditions are: 70° C. for 10 min, rapid cooling on ice for 5 min, and slow cooling at 37° C. for 30 min.

[0054] In some examples, the step of modifying the exosomes with the aptamer may include: adding an aldehyde modification to the 5' end of the aptamer, and incubating the aldehyde-modified aptamer with the exosomes to obtain a complex.

[0055] In some examples, exosomes can improve diabetes-induced muscle atrophy by enhancing SIRT1 / foxo3a-mediated mitochondrial function.

[0056] In some examples, the aptamer can promote the internalization efficiency of exosomes in muscle cells. In some examples, the aptamer can enhance the effect of exosomes on muscle atrophy and muscle fiber type transition. In some examples, the aptamer can enhance the effect of exosomes through SIRT1-mediated mitochondrial function.

[0057] In some examples, exosomes can be derived from human umbilical cord mesenchymal stem cells. In this case, the umbilical cord, as a source of mesenchymal stem cells and their exosomes, has the advantages of reducing harm to the human body and facilitating collection compared to other materials such as bone marrow and liver, and mesenchymal stem cells derived from umbilical cord have stronger proliferation and differentiation capabilities.

[0058] In some examples, human umbilical cord mesenchymal stem cells and their exosomes can be obtained from fresh umbilical cord tissue of newborns. In some examples, human umbilical cord mesenchymal stem cells can be obtained by culturing umbilical cord tissue. In some examples, the cells after dissociation of human umbilical cord tissue can be cultured using α-MEM medium. The culture medium can contain 10% fetal bovine serum, 100U / mL penicillin and 100μg / mL streptomycin. Thus, human umbilical cord mesenchymal stem cells can be cultured.

[0059] In some examples, exosomes derived from human umbilical cord mesenchymal stem cells can be isolated after culturing human umbilical cord mesenchymal stem cells with a culture medium. In some examples, the cultured supernatant can be filtered using a 0.22 μm filter, thereby removing components with larger particle sizes in the supernatant and retaining the exosomes in the supernatant as much as possible. In some examples, the filtered supernatant can be centrifuged, and the precipitate after centrifugation is the exosome. The filtered supernatant can be centrifuged at a speed of 120,000 g for 70 minutes, and the precipitate after centrifugation is the exosome. Thus, exosomes derived from human umbilical cord mesenchymal stem cells can be prepared.

[0060] As mentioned above, the present disclosure also provides a drug for treating diabetic-induced muscle atrophy, including a complex, wherein the complex is an exosome modified by an aptamer, and the sequence of the aptamer is shown in SEQ ID NO. 1. Thus, a drug having a therapeutic effect on diabetic-induced muscle atrophy can be obtained.

[0061] In some examples, the drug may also include an excipient. Among them, the excipient may be a pharmaceutically acceptable excipient. Thus, adding an excipient can help improve the performance of the drug and make the drug more suitable for clinical needs. In some examples, the excipient may be a pharmaceutically acceptable carrier, excipient, diluent, etc.

[0062] In this disclosure, the mechanism of exosome regulation of muscle atrophy is revealed for the first time from the perspective of mitochondrial function, and it is verified that exosomes can improve diabetes-induced muscle atrophy, and aptamer binding can enhance the effect of exosomes on muscle atrophy. Therefore, the complex (exosomes modified by aptamers) has good prospects for application in the preparation of drugs for the treatment of muscle diseases.

[0063] To facilitate understanding of the present invention, the present invention is further explained below with reference to specific embodiments in conjunction with the accompanying drawings, and the specific embodiments do not constitute a limitation on the embodiments of the present invention.

[0064] Experimental methods:

[0065] Human umbilical cord mesenchymal stem cells were obtained from fresh umbilical cords of healthy newborns with informed consent from their parents. This study was approved by the Ethics Committee of Qilu Hospital of Shandong University. Human umbilical cord mesenchymal stem cells (hucMSCs) were cultured in α-MEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco), penicillin 100 U / mL, and streptomycin 100 μg / mL. The third to fifth passages of cells were used for flow cytometry, differentiation induction, and preparation of conditioned medium.

[0066] Isolation and characterization of exosomes

[0067] After 48 h of culture in exosome-free medium, hucmsc-conditioned medium and helf-conditioned medium were centrifuged and filtered through 0.22 μm filters, respectively. The medium was ultracentrifuged at 120,000 g for 70 min at 4 °C to collect exosome particles. The morphology of exosomes was observed by transmission electron microscopy; G2 Spiriti FEI, Tecnai, USA). The size of exosomes was quantified using ZetaVIEW S / N17-310 (PARTICLE METRIX, Germany). The expression of CD9, TSG101, HSP70, and Calnexin was detected by Western blotting.

[0068] Cell culture and RNAi

[0069] Human embryonic lung fibroblasts (HELFs) were obtained from the Chinese Center for Cell Culture (Shanghai, China).

[0070] Mouse podocyte cell line MPC5 and mouse monocyte / macrophage cell line RAW264.7 were purchased from American Type Culture Collection (ATCC, USA) and sold to Mingjing Biotechnology (Shanghai, China).

[0071] HELFs, MPC5 and RAW264.7 were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Alpha mouse liver 12 (AML12) cell line was obtained from the Shanghai Cell Bank and cultured in DMEM / F12 medium (Gibco, USA) containing 10% bovine serum, 100 U / mL penicillin, 100 μmol / mL streptomycin, 1% insulin-transferrin-selenium and 0.1 μmol / L dexamethasone.

[0072] Mouse C2C12 myoblasts were purchased from China Infrastructure of Cell Line Resource (Beijing, China) and cultured in high-glucose DMEM containing 10% fetal bovine serum and antibiotics. Differentiation medium consisting of heat-inactivated horse serum and antibiotics was introduced 4 days after reaching 80-90% confluency. The medium was changed every 2 days. Fully differentiated myotubes were stimulated with palmitic acid (PA, 0.6 mM) and exosomes derived from human umbilical cord mesenchymal cells (MSC-EXOs) (25 μg / ml) or exosomes derived from human embryonic lung fibroblasts (HELF-EXOs) (25 μg / ml) for 24 h.

[0073] To further investigate its mechanism, small interfering RNA (siRNA) was transfected into myotubes using Lipofectamine 2000 transfection reagent (Invitrogen, USA) before treatment with MSC-EXOs. Briefly, C2C12 myoblasts were placed in 6-well plates for complete differentiation and then switched to Opti-MEM I reduced serum medium (Gibco) containing SIRT1 siRNA (125nM) for 6 hours. Subsequently, the medium was replaced with differentiation medium and myotubes were treated with MSC-EXOs for 24 hours. siRNA oligonucleotides were synthesized by GenePharma Co., Ltd. (Shanghai, China).

[0074] The siRNA sequences for the negative control (NC) are sense 5'-UUCUCCGAACGUGUCACGUTT-3' and antisense 5'-ACGUGACACGUUCGGAGAATT-3'.

[0075] The sequences of SIRT1 siRNA are sense 5'-GGGAUCAAGAGGUUGUUAATT-3' and antisense 5'-UUAACAACCUCUUGAUCCCTT-3'.

[0076] Formation of aptamer

[0077] The aptamer oligonucleotide was synthesized by Shanghai Sangon Biotech Co., Ltd. The sequence is: 5'-CAGGAGCCGAGAACCGGTTGGTGGGTAATCCTGTTAGCGC-3' (SEQ ID NO.1). A random sequence with the same number of bases was used as a control. The oligonucleotide was resuspended in Tris-EDTA (TE) buffer (pH 8.0; Sangon Biotech, Shanghai, China) at a final concentration of 100 μM. Then it was folded in folding buffer (DPBS [pH 7.5] containing 1 mM MgCl 2 ) to form a secondary structure. The folding conditions were: 10 min at 70 °C, rapid cooling on ice for 5 min, and slow cooling at 37 °C for 30 min.

[0078] The secondary structure of the aptamer was predicted using the RNA Structure website (https: / / rna.urmc.rochester.edu / RNAstructureWeb / index.html).

[0079] Confirmation of aptamer affinity

[0080] To confirm the affinity of the aptamer for C2C12, the suspended C2C12, α mouse liver 12 (AML12) cell line, mouse podocyte line MPC5, and mouse monocyte / macrophage RAW264.7 were randomly reacted with 200 nM cy3-labeled aptamer or cy3-labeled random sequence. After centrifugation to remove the excess aptamer, the percentage of cy3-positive cells was measured by flow cytometry. To directly observe the binding of the aptamer to all the above cells, 200 nM cy3-labeled aptamer or random sequence was added to the cultured cells, incubated at 37 °C for 3 h, washed 3 times with PBS, stained with DAPI, and the signal of the conjugated aptamer was captured by a fluorescence microscope (Olympus BX53, Japan).

[0081] To confirm the specificity of the aptamer for skeletal muscle, mice were injected with 2.5 μM Cy3-labeled aptamers or Cy3-labeled random sequences dissolved in 300 μl folding buffer via the tail vein. 30 minutes after injection, the mice were killed, and the hearts, livers, spleens, lungs, kidneys, and skeletal muscles of the mice were dissected and scanned using an in vivo imaging system (IVIS) (Tanon ABL X5, China).

[0082] Binding of aptamers to exosomes

[0083] Aldehyde modification was added to the 5' end of the aptamer, and the aldehyde group can react with the amino group on the MSC-EXOs membrane through the Schiff base reaction. Specifically, 200nM aldehyde-modified aptamer and 1.0mg / mL MSC-EXOs were added to PBS, incubated overnight in a rotating mixer at 4°C, and unbound aptamer was removed by centrifugation in a 100kDa ultrafiltration tube. Apt-EXOs (exosomes modified by aptamers, i.e., complexes) were prepared for animal experiments.

[0084] Animal experiments

[0085] All animal experiments were approved by the Animal Ethics Committee of Qilu Hospital of Shandong University. Four-week-old male db / db mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. (Suzhou) and fed with ordinary mouse chow. The mice were housed at 22-25°C and 55±5% humidity with a light / dark cycle of 12 h. T2DM was defined as two consecutive fasting blood glucose levels ≥16.7 mmol / l. Then 200 μg of MSC-EXOs or Apt-EXOs were suspended in PBS and injected into db / db mice through the tail vein every 3 days for 6 weeks.

[0086] Exosomal Tracer

[0087] For in vivo tracking, MSC-EXOs or Apt-EXOs were labeled with DIR (Invitrogen, USA) and injected into mice via the tail vein. Mice were scanned with IVIS 6 hours after injection. For in vitro tracking, MSC-EXOs or Apt-EXOs were labeled with PKH67 green fluorescent cell attachment kit (PKH67, Sigma-Aldrich) and incubated with fully differentiated myotubes for 3 hours. Fluorescence signals were captured using a fluorescence microscope (Olympus BX53, Japan).

[0088] Metabolic testing and in vivo muscle performance analysis

[0089] Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed. Forelimb grip strength was measured using an electronic dynamometer (Handpi HP-5N, Beijing, China). Mice were trained to grasp a horizontal bar connected to the dynamometer with their forelimbs and gently pull backward in a horizontal direction. Each force applied to the bar was recorded before the mouse lost control. Each mouse was tested three times, and the measured values ​​were averaged.

[0090] Histology and immunohistochemical staining

[0091] After mice were anesthetized, bilateral tibialis anterior (TA), soleus (SO), and gastrocnemius muscles were dissected and weighed. Muscle weights were corrected for body weight. One part of the muscle was treated with liquid nitrogen and frozen at -80°C, and the other part was fixed with 4% paraformaldehyde for hematoxylin-eosin (H&E) staining and immunohistochemical staining. The fixed TA muscle was embedded in paraffin and sectioned at a thickness of 5 μm at the maximum cross-section. After dewaxing, hematoxylin and eosin (H&E) staining was performed according to standard procedures. For immunohistochemical staining, briefly, after slides were dewaxed, antigen retrieval was performed using antigen retrieval solution, endogenous peroxidase was inactivated with hydrogen peroxide (3%) for 15 minutes, and then blocked with protein blocking solution (10% normal goat serum) for 30 minutes at room temperature. The slides were then incubated with anti-fast myosin skeletal heavy chain antibody (Abcam, USA, Cat. No. ab91506, 1:1,000) and anti-slow myosin skeletal heavy chain antibody (Abcam, Cat. No. ab234431, 1:1,000) at 4°C overnight.

[0092] After incubation with the secondary antibody at room temperature for 60 min, 3,3′-diaminobenzidine (DAB) solution was used for color development. Images were acquired using a microscope (Olympus BX53, Japan). The cross-sectional area (CSA) of muscle fibers was calculated using Image-Pro Plus software.

[0093] Western blotting

[0094] TA muscle and C2C12 myotubes were lysed with RIPA lysis buffer (P0013B, Beyotime, Shanghai, China), and proteins were isolated and transferred to polyvinylidene difluoride (PVDF) membranes (IPVH00010 0.45 μm, Millipore, USA). After blocking with 5% skim milk for 1 hour at room temperature, the membranes were incubated with specific primary antibodies at 4°C overnight. After incubation with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature, protein expression was detected using enhanced chemiluminescence.The primary antibodies used were as follows: Atrogin1 (Proteintech, China, Cat. No.67172-1-Ig, 1:5,000), MuRF1 (Proteintech, Cat. No.55456-1-AP, 1:1,000), GAPDH (Abways, China, Cat. No.AB0037, 1:5,000), sirtuin 1 (SIRT1; Proteintech, Cat. No.13161-1-AP, 1:1,000), forkhead box O3a (FoxO3a, CST, Cat. No.2497S, 1:1,000), phosphorylated-(p-)FoxO3a(Ser253) (Bioss, Cat. No.bs-3140R, 1:500); NADH dehydrogenase(ubiquinone)1beta subcomplex8 (NDUFB8; Proteintech, Cat. No. 14794-1-AP, 1:1,000), succinate dehydrogenase complexsubunit B (SDHB; Proteintech, Cat. No. 10620-1-AP, 1:5,000), ubiquinol-cytochrome creductase core protein II (UQCRC2; Proteintech, Cat. No. 14742-1-AP, 1:1,000), cytochrome coxidase II (MTCO2, Proteintech, Cat. No. 55070-1-AP, 1:1,000), ATPsynthase, H+transport, mitochondrial F1 complex, alpha subunit 1(ATP5A1,Proteintech,Cat.No.14676-1-AP,1:2,000),heat shock protein90 (HSP90; ABclonal, China; Cat. No. A5027, 1:1,000).

[0095] Transmission Electron Microscopy (TEM)

[0096] TA muscles were dissected and rapidly fixed with 2.5% glutaraldehyde and 1% phosphate-buffered osmium tetroxide. After embedding, sectioning, and double staining with uranyl acetate and lead citrate, electron micrographs of the ultrastructure of TA muscles were taken using TEM (JEM-1200EX II, JEOL; Tokyo, Japan).

[0097] Succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) activity assay

[0098] SDH activity detection kit (Solarbio, China, order number: BC0955) and LDH activity detection kit (Solarbio, China, order number: BC0685) were used to detect the succinate dehydrogenase (SDH) activity and lactate dehydrogenase (LDH) activity of gastrocnemius muscle and C2C12 myotubes. The SDH and LDH activities of muscles were corrected by muscle weight, while the activities of myotubes were corrected by protein concentration.

[0099] Seahorse Analysis

[0100] Oxygen consumption rate (OCR) was measured using the Mitochondrial Stress Test Kit (Agilent, Order No. 103015-100) according to the manufacturer's instructions. 4 Cells were seeded at a density of 10 cells / well into XF96 cell culture microwells and allowed to fully differentiate. Cells were then cultured in XF assay medium (Seahorse XF DMEM (pH 7.4) supplemented with 10-mM glucose, 2-mM pyruvate-glutamine, and 1-mM pyruvate) and treated as indicated. Oligomycin, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and antimycin A / rotenone were used at concentrations of 1.5, 1.5, and 0.5 μM, respectively. OCR was measured and analyzed using a Seahorse XF96 analyzer (Agilent Technologies).

[0101] Statistical analysis

[0102] All data are presented as mean ± SEM. Unpaired Student's t test or one-way analysis of variance (ANOVA) combined with Tukey's test were used to analyze the differences among the groups using GraphPad Prism 8 software. P < 0.05 was considered statistically significant.

[0103] Results Analysis

[0104] 1. MSC-EXOs alleviate diabetes-induced muscle atrophy and muscle fiber type transformation

[0105] Figure 1 This is a test result diagram of the MSC-EXOs involved in the present invention in alleviating diabetes-induced muscle atrophy and muscle fiber type transformation.

[0106] Figure 1 Part A is a schematic diagram of grip strength (n=5-6 mice); Figure 1Part B is a diagram showing the percentage of tibialis anterior muscle weight to body weight (n=5-6 mice); Figure 1 Part C is a diagram showing the percentage of soleus muscle weight to body weight (n=5-6 mice); Figure 1 Part D in the middle shows hematoxylin-eosin (H&E) staining and immunohistochemical staining of fast and slow myosin heavy chains of TA muscle (scale bar: 50 μm), as well as a schematic diagram of myofiber cross-sectional area (CSA), fast and slow muscle fibers, and the percentage of slow to fast muscle fibers (n=5 mice); Figure 1 Part E in the middle is a schematic diagram of Western blot analysis of atrogin1 and MuRF1 in TA muscle (n=3 mice). In each figure of the present disclosure, ImageJ software was used for band quantitative analysis. Data are mean ± SEM. (*p<0.05, **p<0.01, ***p<0.001).

[0107] We investigated the effects of hucMSC-derived exosomes (MSC-EXOs) on muscle atrophy and myofiber type transition in db / db mice. Flow cytometry analysis showed that hucMSCs were positive for CD105 and CD73 (>95%) and negative for CD34 and HLA-DR (<2%). HucMSCs had the potential for adipogenic, osteogenic, and chondrogenic differentiation as indicated by Oil Red O, Alizarin Red S, and Alizarin Blue staining, respectively. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) showed that MSC-EXOs isolated from hucMSC-conditioned medium were cup-shaped vesicles with a diameter of approximately 130 nm. Western blotting results showed that MSC-EXOs were positive for protein markers CD9, TSG101, and HSP70, and negative for the endoplasmic reticulum marker calnexin.

[0108] We injected MSC-EXOs (200 μg dissolved in 200 μl PBS) into db / db mice via the tail vein every 3 days for 6 weeks; 200 μl / mouse PBS injection was used as a control. IPGTT and IPITT 1 week after the last injection showed that MSC-EXOs improved glucose and insulin tolerance. Grip strength test showed that MSC-EXOs increased muscle strength in db / db mice ( Figure 1 Injection of MSC-EXOs did not affect body weight but increased muscle mass in the tibialis anterior (TA) and soleus (SO) muscles ( Figure 1 H&E and immunohistochemical staining showed that MSC-EXO treatment not only alleviated the diabetes-induced decrease in myofiber CSA (including fast and slow muscle fibers), but also increased the ratio of slow to fast muscle fibers ( Figure 1In addition, MSC-EXOs also inhibited the upregulation of diabetes-related E3-ubiquitin ligases Atrogin1 and MuRF1 ( Figure 1 (Part E).

[0109] These results suggest that MSC-EXOs injection can alleviate diabetes-related muscle atrophy and myofiber type transition in db / db mice.

[0110] 2. MSC-EXOs repair atrophy-related SIRT1 / FoxO3a signaling and mitochondrial function damage

[0111] Figure 2 This is a test result diagram of the MSC-EXOs involved in the present invention repairing atrophy-related SIRT1 / FoxO3a signals and mitochondrial function damage.

[0112] Figure 2 Part A in the middle is a schematic diagram of Western blot analysis of SIRT1, p-FoxO3a (S253), and FoxO3a in TA muscle of db / db mice (n=3 mice); Figure 2 Part B in the middle is a transmission electron microscopy (TEM) image of TA muscle mitochondria (scale bar, 0.6 μm); Figure 2 Part C in the middle is a schematic diagram of Western blot analysis of mitochondrial complexes NDUFB8, SDHB, UQCRC2, MTCO2, and ATP5A1 in TA muscle of db / db mice (n = 3 mice); Figure 2 Part D in the middle is a schematic diagram of SDH activity in gastrocnemius muscle of db / db mice (n=5 mice); Figure 2 Part E in the middle is a schematic diagram of LDH activity in the gastrocnemius muscle of db / db mice (n=5 mice).

[0113] Mitochondrial dysfunction plays an important role in muscle atrophy and myofiber type transition. To explore the mechanism of MSC-EXOs-mediated alleviation of muscle atrophy, we focused on the regulation of mitochondrial function and the related SIRT1 / FoxO3a pathway. Western blotting results showed that the expression of SIRT1 and the phosphorylation level of FoxO3a in db / db mice were lower than those in db / m mice, and the expression of SIRT1 and p-FoxO3a was upregulated by injection of MSC-EXOs ( Figure 2 Transmission electron microscopy showed that MSC-EXO treatment rescued the mitochondrial structural damage caused by diabetes ( Figure 2 The expression of mitochondrial complexes, including SDHB, UQCRC2, MTCO2, and ATP5A1, was increased by MSC-EXOs ( Figure 2MSC-EXOs can also enhance SDH activity and reduce LDH activity ( Figure 2 These results suggest that MSC-EXOs activate SIRT1 / FoxO3a signaling and alleviate mitochondrial dysfunction in skeletal muscle.

[0114] 3. MSC-EXOs alleviate PA-induced C2C12 myotube atrophy and mitochondrial dysfunction

[0115] To further investigate the direct autonomous effects of MSC-EXOs on muscle cells, we used an in vitro C2C12 myocyte model. HELF-derived exosomes (HELF-EXOs) were used as a control.

[0116] Figure 3 This is a test result diagram showing that the MSC-EXOs involved in the present invention alleviates PA-induced C2C12 myotube atrophy and mitochondrial dysfunction.

[0117] Figure 3 Part A in the middle is a schematic diagram of the effects of PA and MSC-EXOs on atrogin1 and MuRF1 in C2C12 myotubes analyzed by Western blot (n=4); Figure 3 Part B in the middle is a schematic diagram of the diameter of C2C12 myotubes treated with PA and MSC-EXOs; Figure 3 Part C in the middle is a schematic diagram of Western blot analysis of the expression of SIRT1, p-FoxO3a (S253) and FoxO3a in C2C12 myotubes (n=4). Figure 3 Part D in the middle is a schematic diagram of Western blot analysis of mitochondrial complexes NDUFB8, SDHB, UQCRC2, MTCO2, and ATP5A1 (n=3-4); Figure 3 Part E in the middle is a schematic diagram of Seahorse analysis of OXPHOS in C2C12 myocyte tubes treated with PA and MSC-EXO (n=3), including basal respiration, maximal respiration, reserve respiratory capacity, and ATP production; Figure 3 Part F is a schematic diagram of SDH and LDH activities of C2C12 myotubes treated with PA and MSC-EXOs (n=3).

[0118] PA treatment increased the expression of Atrogin 1 and MuRF1 ( Figure 3 Middle A), reduced myotube diameter ( Figure 3 B), while MSC-EXOs treatment abolished the PA-induced increase in Atrogin 1 and MuRF1 expression and increased myotube diameter ( Figure 3MSC-EXOs increased the expression of SIRT1 and promoted the phosphorylation of FoxO3a ( Figure 3 C), upregulating the expression of mitochondrial complexes, including NDUFB8, SDHB, and MTCO2 ( Figure 3 Seahorse analysis showed that MSC-EXOs treatment enhanced mitochondrial oxidative phosphorylation (OXPHOS), as evidenced by increased basal respiration, maximal respiration, reserve respiratory capacity, and ATP production ( Figure 3 In addition, MSC-EXOs enhanced SDH activity and decreased LDH activity in myotubes ( Figure 3 These results support a direct, myocyte-autonomous effect of MSC-EXOs and suggest that MSC-EXOs modulate SIRT1 / FoxO3a signaling and mitochondrial function in vitro.

[0119] 4. MSC-EXOs counteract myotube atrophy by enhancing SIRT1-mediated mitochondrial function

[0120] To determine whether the effect of MSC-EXOs on muscle atrophy requires SIRT1, we pretreated C2C12 myotubes with siRNA targeting SIRT1.

[0121] Figure 4 This is a test result diagram showing that the MSC-EXOs involved in the present invention combat myotube atrophy by enhancing SIRT1-mediated mitochondrial function.

[0122] Figure 4 Part A in the middle is a schematic diagram of Western blot analysis of SIRT1, p-FoxO3a (S253), and FoxO3a in C2C12 myocytes transfected with SIRT1 siRNA and treated with MSC-EXO (n=4); Figure 4 Part B in the middle is a schematic diagram of Western blot analysis of mitochondrial complexes NDUFB8, SDHB, UQCRC2, MTCO2, and ATP5A1 (n=3-4); Figure 4 Part C in the middle is a schematic diagram of Seahorse's analysis of OXPHOS in C2C12 myotubes transfected with SIRT1 siRNA and treated with MSC-EXOs (n=3), including basal respiration, maximal respiration, reserve respiratory capacity, and ATP production; Figure 4 Part D in the middle is a schematic diagram of SDH and LDH activities of C2C12 myocytes transfected with SIRT1 siRNA and treated with MSC-EXOs (n=3); Figure 4 Part E in the middle is a schematic diagram of Western blot analysis of Atrogin1 and MuRF1 (n=4); Figure 4Part F in the middle is a schematic diagram of the diameter of C2C12 myocytes.

[0123] Western blotting showed that MSC-EXOs-mediated upregulation of SIRT1 / FoxO3a signaling and mitochondrial complexes was partially abolished by siR-SIRT1 ( Figure 4 AB part).

[0124] At the same time, SIRT1 silencing reduced the effects of MSC-EXOs on OXPHOS in myotubes. Figure 4 C) and SDH and LDH activities ( Figure 4 Finally, siR-SIRT1 attenuated the MSC-EXOs-dependent decrease in Atrogin 1 / MuRF1 levels ( Figure 4 Middle E) and the increase in myotube diameter ( Figure 4 These results suggest that MSC-EXOs alleviate muscle atrophy by promoting SIRT1-mediated mitochondrial function in C2C12 myotubes.

[0125] 5. Aptamer binding promotes the internalization of MSC-EXOs in skeletal muscle

[0126] We hypothesized that modification of MSC-EXOs with specific recognizable ligands would achieve muscle-targeted delivery and enhance the effects of MSC-EXOs on muscle atrophy.

[0127] Figure 5 This is a test result diagram showing that the binding of the aptamer involved in the present invention promotes the internalization of MSC-EXOs in skeletal muscle.

[0128] Figure 5 Part A in the middle is a schematic diagram of the predicted secondary structure of the aptamer; Figure 5 Part B in the middle is a schematic diagram of flow cytometry analysis of the affinity of aptamers to myocytes (n=3); Figure 5 Part C in the middle is a schematic diagram of the affinity of the aptamer for myocytes captured by fluorescence microscopy; Figure 5 Part D in the middle is a schematic diagram of the distribution of cy3-labeled aptamers in mice detected by in vivo imaging system (IVIS) 30 minutes after administration; Figure 5 Part E in the middle is a schematic diagram of the distribution of DIR-labeled aptamer-bound MSC-EXOs in mice after 6 hours of infusion using IVIS. Data are mean ± SEM. (***p<0.001)

[0129] We used the RNA Structure website to predict the secondary structure of the aptamer ( Figure 5To confirm its specificity, we labeled the aptamers with Cy3 and incubated them with different cell types, including myoblasts C2C12, hepatocytes AML12, capsular cells MPC5, and macrophages / monocytes RAW264.7 and macrophages / monocytes. Random sequences were used as controls. Flow cytometric analysis showed that the aptamers had a greater affinity for myocytes than for other cell types, with statistical significance ( Figure 5 Figure 2. Part B of Figure 2. Images obtained using a fluorescence microscope showed that the aptamer effectively and specifically entered the muscle cells ( Figure 5 Meanwhile, in vivo tracing showed that the aptamer was specifically recruited in skeletal muscle ( Figure 5 (section D).

[0130] In order to make more exosomes reach skeletal muscle, the above aptamer was coupled to MSC-EXOs. The 5' end of the aptamer was modified with an aldehyde group, which can react with the amino group on the MSC-EXOs membrane through the Schiff base reaction. TEM, NTA and western blot analysis showed that aptamer modification did not affect the basic properties of MSC-EXOs, but promoted the internalization efficiency of MSC-EXOs in muscle cells. It is worth noting that the in vivo biodistribution of systemically delivered aptamer-modified MSC-EXOs was detected. MSC-EXOs labeled with DIR were combined with the aptamer and injected into mice through the tail vein. IVIS analysis showed that aptamer binding promoted the internalization of MSC-EXOs into skeletal muscle ( Figure 5 (Part E).

[0131] 6. Aptamer binding enhances the effects of MSC-EXOs on muscle atrophy and myofiber type transition

[0132] To explore whether Aptamer conjugation can enhance the effect of MSC-EXOs on muscle atrophy, we injected Apt-EXOs or MSC-EXOs (200 μg dissolved in 200 μl PBS) into db / db mice through the tail vein once every 3 days for 6 weeks. IPGTT and IPITT results showed that there was no significant difference between the Apt-EXOs group and the MSC-EXOs group.

[0133] Figure 6 The figure is a test result showing that the aptamer binding of the present invention enhances the effect of MSC-EXOs on muscle atrophy and muscle fiber type transformation.

[0134] Figure 6 Part A is a schematic diagram of grip strength (n=5-6 mice); Figure 6 Part B is a diagram showing the percentage of tibialis anterior muscle weight to body weight (n=5-6 mice); Figure 6Part C is a diagram showing the percentage of soleus muscle weight to body weight (n=5-6 mice); Figure 6 Part D in the middle shows hematoxylin-eosin (H&E) staining and immunohistochemical staining of fast and slow myosin heavy chains of TA muscle (scale bar: 50 μm), as well as a schematic diagram of the myofiber cross-sectional area (CSA), fast and slow muscle fibers, and the percentage of slow to fast muscle fibers (n=5-6 mice); Figure 6 Part E in the middle is a schematic diagram of Western blot analysis of atrogin1 and MuRF1 in TA muscle (n=3 mice).

[0135] However, compared with MSC-EXOs, Apt-EXOs treatment further enhanced the grip strength of db / db mice ( Figure 6 Part A) and increased TA and SO muscle mass without affecting body weight ( Figure 6 H&E and immunohistochemical staining showed that Apt-EXOs further increased the CSA of muscle fibers (including fast and slow muscle fibers) and increased the percentage of slow muscle fibers ( Figure 6 D in the middle), and inhibited the expression of Atrogin 1 / MuRF1 ( Figure 6 These results suggest that Aptamer conjugation potentiates the effects of MSC-EXOs on muscle atrophy and myofiber turnover.

[0136] 7. Aptamer binding enhances the effect of MSC-EXOs through SIRT1-mediated mitochondrial function

[0137] To further verify the regulatory mechanism of Apt-EXOs in muscular atrophy, we detected SIRT1 signaling and mitochondrial function.

[0138] Figure 7 This is a test result diagram of the effect of the aptamer involved in the present invention on enhancing MSC-EXOs through SIRT1-mediated mitochondrial function.

[0139] Figure 7 Part A in the middle is a schematic diagram of Western blot analysis of SIRT1, p-FoxO3a (S253), and FoxO3a in TA muscle of db / db mice (n=3 mice); Figure 7 Part B in the middle is a transmission electron microscopy (TEM) image of TA muscle mitochondria (scale bar, 0.6 μm); Figure 7 Part C in the middle is a schematic diagram of Western blot analysis of TA muscle mitochondrial complexes NDUFB8, SDHB, UQCRC2, MTCO2, and ATP5A1 (n=3 mice); Figure 7Part D in the middle is a schematic diagram of SDH activity in gastrocnemius muscle (n=5-6 mice); Figure 7 Part E in the middle is a schematic diagram of LDH activity in the gastrocnemius muscle of db / db mice (n=5-6 mice).

[0140] Western blotting showed that the expression of SIRT1 and the phosphorylation level of FoxO3a were higher in the Apt-EXOs group compared with the MSC-EXOs group ( Figure 7 TEM showed that the mitochondrial structure of the Apt-EXOs group was superior to that of the MSC-EXOs group ( Figure 7 (B). The expression of mitochondrial complexes (including SDHB and MTCO2) was further increased by Apt-EXOs Figure 7 In addition, Apt-EXOs enhanced SDH activity and decreased LDH activity ( Figure 7 These results suggest that aptamer modification may enhance the effects of MSC-EXOs through SIRT1-mediated mitochondrial function.

[0141] Summarize

[0142] We injected Apt-EXOs or MSC-EXOs into db / db mice via the tail vein and confirmed that aptamer modification enhanced the effects of MSC-EXOs on diabetes-induced muscle atrophy and myofiber type transition, providing a new idea for improving the efficacy of exosomes in treating muscle diseases.

[0143] In db / db mice, skeletal muscle mitochondrial structure was significantly impaired, while MSC-EXOs treatment restored the abnormal mitochondrial structure. MSC-EXOs upregulated the expression of mitochondrial complexes, accompanied by enhanced SDH activity and decreased LDH activity. Seahorse analysis directly demonstrated that MSC-EXOs promoted mitochondrial oxidative phosphorylation (OXPHOS) in C2C12 myotubes, as evidenced by increased basal respiration, maximal respiration, reserve respiration, and ATP production. In addition, aptamer-conjugated MSC-EXOs restored mitochondrial structure and function in db / db mice. This study revealed for the first time the mechanism by which MSC-EXOs regulate muscle atrophy from the perspective of mitochondrial function.

[0144] In our study, MSC-EXOs promoted SIRT1 / FoxO3a signaling in skeletal muscle of db / db mice and PA-treated C2C12 myotubes. SIRT1 knockdown reduced the effects of MSC-EXOs on OXPHOS, SDH and LDH activities, and myotube diameter. These results indicate that SIRT1 is involved in the improvement of mitochondrial dysfunction by MSC-EXOs, providing a new scientific basis and intervention target for exosomes in the treatment of muscle atrophy.

[0145] In conclusion, our results indicate that exosomes secreted by hucMSCs enhance SIRT1 / FoxO3a-mediated mitochondrial function and improve diabetes-induced muscle atrophy. Aptamer binding enhances the ameliorative effect of MSC-EXOs on muscle atrophy. These findings demonstrate the therapeutic potential of muscle-targeted MSC-EXOs for the treatment of muscle atrophy.

[0146] Therefore, according to the present disclosure, there can be provided a use of a complex (exosomes modified by aptamers) in the preparation of a drug for treating diabetes-induced muscle atrophy.

[0147] Although the present disclosure is specifically described above in conjunction with the accompanying drawings and embodiments, it is to be understood that the above description does not limit the present disclosure in any form. Those skilled in the art may modify and change the present disclosure as needed without departing from the essential spirit and scope of the present disclosure, and these modifications and changes all fall within the scope of the present disclosure.

Claims

1. Use of a complex in the preparation of a drug for treating diabetic-induced muscle atrophy, characterized in that: The complex is an exosome modified by an aptamer, and the sequence of the aptamer is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The exosomes are derived from human umbilical cord mesenchymal stem cells.

3. The use according to claim 1, characterized in that: The aptamer folds to form a secondary structure.

4. The use according to claim 3, characterized in that: The aptamer was resuspended in TE buffer and then folded in folding buffer to form a secondary structure. The folding conditions were: 70° C. for 10 min, rapid cooling on ice for 5 min, and slow cooling at 37° C. for 30 min.

5. The use according to claim 1, characterized in that: The step of modifying the exosomes with the aptamer comprises: adding an aldehyde group to the 5' end of the aptamer for modification, and incubating the aldehyde-modified aptamer and the exosomes together to obtain the complex.

6. The use according to claim 1, characterized in that: The exosomes improve diabetes-induced muscle atrophy by enhancing SIRT1 / foxo3a-mediated mitochondrial function.

7. The use according to claim 1, characterized in that: The aptamer promotes the internalization efficiency of the exosomes in muscle cells and enhances the effects of the exosomes on muscle atrophy and muscle fiber type transformation.

8. The use according to claim 1 or 7, characterized in that: The aptamer enhances the effect of the exosomes through SIRT1-mediated mitochondrial function.

9. Use of a complex in preparing a drug for treating muscle diseases, characterized in that: The complex is an exosome modified by an aptamer, and the sequence of the aptamer is shown in SEQ ID NO.

1.

10. A drug for treating diabetic-induced muscle atrophy, characterized in that: The invention comprises a complex, wherein the complex is an exosome modified by an aptamer, and the sequence of the aptamer is shown in SEQ ID NO.1.

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