A pharmaceutical composition for treating sarcopenia and use thereof

Abstract

The application belongs to the field of medicine, and discloses a kind of drug composition for treating sarcopenia, the drug composition comprises DNase I and PMI.The application discloses the application of the drug composition in preparing a kind of drug for treating sarcopenia.DNase I of the application is combined with mitochondrial autophagy agonist PMI, simultaneously enhances mitochondrial autophagy in the clearance NET, synergistically reduces cell senescence and oxidative stress, promotes myogenic differentiation, significantly improves muscle mass, strength and pathological histology in D-galactose induced sarcopenia mouse model and SAMP8 rapid aging mouse model, and the curative effect is better than single drug treatment.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to a pharmaceutical composition for treating sarcopenia and its application, specifically the application of DNase I combined with PMI in the preparation of a drug for treating sarcopenia. Background Technology

[0002] Sarcopenia is an age-related geriatric syndrome characterized by progressive and systemic loss of skeletal muscle mass, strength, and physical function, and is one of the most significant challenges to healthy aging. The prevalence of sarcopenia among older adults living in Asian communities ranges from 7.3% to 12%, increasing significantly with age. The clinical consequences of sarcopenia are severe, including falls, fractures, metabolic disorders, increased all-cause mortality, and a heavy burden on the healthcare system. However, there are currently no effective pharmacological treatments, and clinical management is primarily limited to lifestyle interventions such as nutritional support and exercise rehabilitation, with limited and inconsistent effectiveness.

[0003] The regeneration and repair capacity of skeletal muscle primarily relies on muscle satellite cells (MuSCs), a type of adult stem cell located between the basal layer and sarcolemma of muscle fibers. Aging profoundly impacts the muscle satellite cell population, not only reducing their numbers but, more importantly, causing a functional decline in their proliferation and myogenic differentiation capabilities. This is a direct cause of sarcopenia, muscle atrophy, and weakness. Chronic low-grade systemic inflammation is considered a key driver of this process, but the specific molecular mechanisms connecting the inflammatory environment with impaired muscle satellite cell function remain unclear.

[0004] Neutrophil extracellular traps (NETs) are network structures released by activated neutrophils, composed of chromatin and embedded granular proteins such as neutrophil elastase (NE), myeloperoxidase (MPO), and citrullinated histone H3. In recent years, NETs have been shown to drive tissue damage in various chronic inflammatory diseases through mechanisms such as physical injury, enzymatic degradation, and inflammasome activation.

[0005] Mitophagy is a crucial cellular process for clearing damaged mitochondria and maintaining mitochondrial quality control. Myogenic differentiation of muscle satellite cells is a highly energy-intensive process that requires sufficient energy support from functional mitochondria. When mitophagy is impaired, damaged mitochondria accumulate, leading to energy metabolism disorders and thus inhibiting the differentiation capacity of muscle satellite cells.

[0006] Currently, there are no reports of drug compositions targeting NETs clearance in combination with enhanced mitophagy for the treatment of sarcopenia. Summary of the Invention

[0007] The inventors, through clinical samples, naturally aging mouse models, and cell experiments, have for the first time demonstrated that NETs significantly accumulate in the skeletal muscle of sarcopenic muscular tract (MST) patients and naturally aging mice, and are closely related to impaired differentiation of muscle satellite cells. Mechanistically, MSTs directly inhibit mitophagy in muscle satellite cells, leading to a shift in metabolism from oxidative phosphorylation to glycolysis, increased lactate production, and consequently driving histone H3K9 lactation. Epigenetically, this inhibits the transcription of genes related to mitophagy and myogenic differentiation. DNase I-mediated MST clearance can restore myogenic differentiation and improve sarcopenic muscular tract disease. More importantly, the combined use of DNase I and the mitophagy agonist PMI (P62-mediated mitophagy inducer) enhances mitophagy while clearing MSTs, synergistically reducing cellular senescence and oxidative stress, and promoting myogenic differentiation. In two independent mouse models, this significantly improved muscle mass, strength, and histopathological appearance, demonstrating superior efficacy compared to monotherapy.

[0008] The purpose of this invention is to provide a pharmaceutical composition for treating sarcopenia.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A pharmaceutical composition for treating sarcopenia, the pharmaceutical composition comprising DNase I and PMI, wherein the mass ratio of DNase I to PMI is 1:0.075 to 1:1.

[0011] The structure of PMI is shown below:

[0012]

[0013] Preferably, the mass ratio of DNase I to PMI is 1:1.

[0014] Preferably, the pharmaceutical composition uses DNase I and PMI as active ingredients and also includes pharmaceutically acceptable excipients.

[0015] The present invention also provides the use of the pharmaceutical composition in the preparation of a medicament for treating sarcopenia.

[0016] Preferably, the application is the use of the pharmaceutical composition in the preparation of a medicament for treating sarcopenia by performing at least one of the following actions:

[0017] (1) Synergistically clear neutrophil extracellular traps (NETs) accumulated in skeletal muscle and enhance mitochondrial autophagy in muscle satellite cells;

[0018] (2) Restore mitochondrial morphology and membrane potential of muscle satellite cells and promote the clearance of damaged mitochondria;

[0019] (3) Reduces muscle satellite cell senescence and oxidative stress;

[0020] (4) Increase the ratio of skeletal muscle cell oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) and improve mitochondrial energy metabolism;

[0021] (5) Promotes myogenic differentiation of myosatellite cells and upregulates the expression of MyoD, LC3, Parkin and PINK1 proteins.

[0022] The drug is available in the form of an injection, a skeletal muscle-targeting delivery system, or a high-end non-injection formulation.

[0023] The injectable formulations include solution-type injectables, in-situ gels, injectable microspheres, and lyophilized powder injections; the skeletal muscle-targeting delivery systems include skeletal muscle-targeting ligand-modified liposomes or lipid nanoparticles, exosomes, or extracellular vesicles (EVs) engineered carriers; and the high-end dosage forms via non-injection routes include oral nanodelivery systems, pulmonary inhalers, and soluble microneedle patches.

[0024] The in-situ gel is an in-situ thermosensitive hydrogel.

[0025] Another object of the present invention is to provide the use of 2-DG (2-deoxy-D-glucose) in the preparation of a medicament for treating sarcopenia.

[0026] The drug is available in the form of an injection, a skeletal muscle-targeting delivery system, or a high-end non-injection formulation.

[0027] The injectable formulations include solution-type injectables, in-situ gels, injectable microspheres, and lyophilized powder injections; the skeletal muscle-targeting delivery systems include skeletal muscle-targeting ligand-modified liposomes or lipid nanoparticles, exosomes or extracellular vesicle-engineered carriers; and the high-end dosage forms via non-injection routes include oral nanodelivery systems, pulmonary inhalers, and soluble microneedle patches.

[0028] The in-situ gel is an in-situ thermosensitive hydrogel.

[0029] The present invention has the following beneficial effects:

[0030] This invention reveals for the first time the direct pathogenic role of NETs in the pathogenesis of sarcopenia, elucidating the complete molecular mechanism by which NETs inhibit myoblast differentiation by suppressing mitochondrial autophagy in muscle satellite cells, inducing metabolic reprogramming and histone lactation, thus providing new theoretical basis for the study of the pathogenesis of sarcopenia.

[0031] This invention is the first to combine the NETs scavenger DNase I with the mitophagy agonist PMI. By synergistically clearing NETs and enhancing mitophagy, it effectively breaks the vicious cycle of NETs-mediated mitophagy inhibition and impaired muscle regeneration. Experiments have shown that the combined treatment is significantly superior to monotherapy in restoring mitochondrial morphology and membrane potential, promoting the clearance of damaged mitochondria, reducing cellular senescence and reactive oxygen species levels, and promoting myogenic differentiation.

[0032] This invention has verified the therapeutic effect of combined drug administration in both D-galactose (D-gal) induced sarcopenia mouse models and SAMP8 rapid aging mouse models. It can significantly improve muscle mass, grip strength, exercise endurance, restore muscle histological structure, reduce inflammation and oxidative stress levels, and has good safety.

[0033] This invention provides a novel combination therapy regimen for the clinical treatment of sarcopenia, which has significant clinical application value and translational potential. Attached Figure Description

[0034] Figure 1 HE staining results of gastrocnemius muscle tissue from a patient with sarcopenia.

[0035] Figure 2 The results show the serum NETs markers (dsDNA, MPO-DNA, CitH3) levels in patients with sarcopenia.

[0036] Figure 3 The results show the immunofluorescence staining of skeletal muscle tissue from patients with sarcopenia; where A shows the immunofluorescence staining results of CitH3 and neutrophils (NE) in skeletal muscle tissue from patients with sarcopenia, and B shows the immunofluorescence staining results of MyoD in skeletal muscle tissue from patients with sarcopenia.

[0037] Figure 4 HE staining results of gastrocnemius muscle tissue from naturally aged mice; where 6m, 15m, and 24m represent 6-month-old, 15-month-old (pre-sarcopenia), and 24-month-old (sarcopenia) male C57BL / 6 mice, respectively.

[0038] Figure 5 Serum dsDNA and inflammatory factor levels in naturally aging mice at different ages.

[0039] Figure 6 The results of CitH3 and NE immunofluorescence staining in the skeletal muscle of naturally aging mice.

[0040] Figure 7 The results of MyoD immunofluorescence staining of skeletal muscle from naturally aging mice.

[0041] Figure 8The effects of NETs on C2C12 cell differentiation are shown in Figure A, where Giemsa staining is used to detect the effect of NETs on myogenic differentiation of C2C12 cells; Figure B is qPCR detection of the effect of NETs on the expression levels of genes related to C2C12 cell differentiation; and Figure C is Western blot detection of the effect of NETs on the expression of MyoD protein in C2C12 cells and primary myosatellite cells.

[0042] Figure 9 To detect the effect of NETs on the C2C12 cell cycle using flow cytometry.

[0043] Figure 10 To detect the effect of NETs on apoptosis in C2C12 cells by flow cytometry.

[0044] Figure 11 Results of the effects of DNase I on NETs clearance on myosatellite cell differentiation and senescence; where A is the effect of DNase I on myogenic differentiation of C2C12 cells detected by Giemsa staining; and B is the effect of DNase I on cell senescence detected by SA-β-gal staining.

[0045] Figure 12 To detect the effect of DNase I on MyoD expression using immunofluorescence

[0046] Figure 13 The DCFH-DA probe was used to detect the effect of DNase I on ROS levels.

[0047] Figure 14 To detect the effect of DNase I on the expression of MyoD and P16 proteins using Western blot.

[0048] Figure 15 The therapeutic effect of DNase I on D-galactose-induced sarcopenia mice (mouse grip strength, treadmill exhaustion and rotarod test).

[0049] Figure 16 Results of high-resolution ultrasound detection of quadriceps muscle thickness in D-galactose-induced sarcopenic mice treated with DNase I.

[0050] Figure 17 HE staining and quantitative results of muscle fiber cross-sectional area of ​​gastrocnemius muscle in D-galactose-induced sarcopenic mice treated with DNase I.

[0051] Figure 18 Western blot was used to detect the expression of MyoD and P16 proteins in the gastrocnemius muscle of D-galactose-induced sarcopenic mice treated with DNase I.

[0052] Figure 19The results show the serum SOD, GSH, MDA, and ATP levels in D-galactose-induced sarcopenic mice treated with DNase I.

[0053] Figure 20 To detect the effect of DNase I on MyoD expression in gastrocnemius muscle using immunofluorescence.

[0054] Figure 21 Results of proteomics and KEGG enrichment analysis after NETs treatment of C2C12 cells.

[0055] Figure 22 To detect mitochondrial morphology in NETs-treated C2C12 cells using transmission electron microscopy (TEM) and label-free live-cell imaging (IDT).

[0056] Figure 23 To investigate the effects of NETs on oxygen consumption rate and extracellular acidification rate for Seahorse.

[0057] Figure 24 The effect of NETs on mitochondrial membrane potential was detected using JC-1 assay.

[0058] Figure 25 To detect lactate levels in C2C12 cells using a lactate assay kit.

[0059] Figure 26 The lactate level in the serum of SAMP8 mice was detected using a lactate assay kit.

[0060] Figure 27 A lactate assay kit was used to detect serum lactate levels in patients with sarcopenia.

[0061] Figure 28 To detect the effects of NETs on pan-lactic acidification and H3K9la levels in C2C12 cells using Western blot.

[0062] Figure 29 To investigate the effects of pan-lactic acidification and H3K9la levels in the SAMP8 sarcopenia mouse model using Western blot.

[0063] Figure 30 To detect the regulation of H3K9la on the transcription of Pink1 and Prkn promoters by ChIP-qPCR.

[0064] Figure 31 To detect mitochondrial morphology in C2C12 cells after 2-DG treatment using label-free live-cell imaging.

[0065] Figure 32 Giemsa staining was used to detect the effect of 2-DG on myogenic differentiation of C2C12 cells.

[0066] Figure 33 The effect of 2-DG on mitochondrial membrane potential was detected using JC-1 assay.

[0067] Figure 34 To detect LC3 expression in C2C12 cells using immunofluorescence.

[0068] Figure 35 To detect the effect of 2-DG on MyoD expression using immunofluorescence.

[0069] Figure 36 To investigate the effects of 2-DG on oxygen consumption rate and extracellular acidification rate for Seahorse.

[0070] Figure 37 The results of high-resolution ultrasound detection of quadriceps muscle thickness.

[0071] Figure 38 This is a comparative diagram of mouse muscle anatomy; in the diagram, QA represents the quadriceps femoris, GA represents the gastrocnemius, TA represents the tibialis anterior, SOL represents the soleus, and EDL represents the extensor digitorum longus.

[0072] Figure 39 Results of H&E staining and quantitative analysis of cross-sectional area of ​​gastrocnemius muscle fibers.

[0073] Figure 40 Western blot was used to detect gastrocnemius muscle differentiation and autophagy protein expression.

[0074] Figure 41 To detect the effect of combined DNase I and PMI on mitochondrial morphology using label-free live-cell imaging.

[0075] Figure 42 The effect of combined use of DNase I and PMI on cell senescence was detected by SA-β-gal staining.

[0076] Figure 43 To detect the effect of combined DNase I and PMI on myoblast differentiation using Giemsa staining.

[0077] Figure 44 To detect the effect of combined administration of DNase I and PMI on mitophagy using MitoTracker and LysoTracker co-localization analysis.

[0078] Figure 45 To detect the effect of combined DNase I and PMI on MyoD expression in primary myosatellite cells using immunofluorescence.

[0079] Figure 46 The DCFH-DA probe was used to detect the effect of combined DNase I and PMI on ROS levels.

[0080] Figure 47 To investigate the effect of combined use of DNase I and PMI on OCR / ECAR for Seahorse.

[0081] Figure 48 The effect of combined DNase I and PMI on the expression of LC3, Parkin, PINK1 and MyoD proteins was detected by Western blot.

[0082] Figure 49 The results show the levels of SOD, GSH, MDA, and ATP in C2C12 cells.

[0083] Figure 50 Changes in thigh circumference in D-galactose-induced and SAMP8 sarcopenia mouse models.

[0084] Figure 51 Changes in grip strength in D-galactose-induced and SAMP8 sarcopenic mouse models.

[0085] Figure 52 To detect the thickness of the quadriceps femoris muscle in a D-galactose-induced mouse model using high-resolution ultrasound.

[0086] Figure 53 To detect the thickness of the quadriceps femoris muscle in the SAMP8 sarcopenia mouse model using high-resolution ultrasound.

[0087] Figure 54 The serum NE level in D-galactose-induced and SAMP8 sarcopenia mouse models.

[0088] Figure 55 The serum MPO-DNA level in D-galactose-induced and SAMP8 sarcopenia mouse models.

[0089] Figure 56 The results of H&E staining of gastrocnemius muscle in D-galactose-induced and SAMP8 sarcopenia mouse models.

[0090] Figure 57 To detect the expression of LC3 and MyoD in the gastrocnemius muscle of a D-galactose-induced and SAMP8 sarcopenia mouse model using immunofluorescence.

[0091] Figure 58 Western blot was used to detect the expression of MyoD protein in the gastrocnemius muscle of a D-galactose-induced and SAMP8 sarcopenia mouse model.

[0092] Figure 59 Serum ATP levels in D-galactose-induced and SAMP8 sarcopenia mouse models. Detailed Implementation

[0093] The technical solution of the present invention will be specifically described below through embodiments. It should be noted that the listed embodiments are only for illustrative purposes and are used to explain the present invention, and do not constitute a limitation on the scope of protection of the present invention in any way.

[0094] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The use of these terms in this specification is merely for describing particular embodiments and is not intended to limit the scope of the invention.

[0095] Unless otherwise specified, any method that does not specify the operating conditions or steps is a standard method in the field; all reagents and materials used are commercially available.

[0096] The comparison between two groups in this invention was performed using a two-tailed unpaired Student's t test (i.e., independent samples t test), and the comparison between multiple groups was performed using analysis of variance (ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0097] Experimental materials:

[0098] 1. Drugs: DNase I (purchased from MedChemExpress, USA, HY-P72974); PMI (purchased from MedChemExpress, USA, HY-115576).

[0099] 2. Cell lines: C2C12 mouse myoblast cell line (purchased from Suzhou Haixing Biotechnology Co., Ltd.); primary myoblast satellite cells were isolated from 4-week-old male C57BL / 6 mice (purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.).

[0100] 3. Animals: 8-week-old male C57BL / 6 mice (purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.); 6-month-old, 15-month-old and 24-month-old male C57BL / 6 mice (purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.); 8-month-old male SAMP8 rapidly aging mice (purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.).

[0101] Example 1

[0102] Correlation between NET accumulation and impaired differentiation of muscle satellite cells in sarcopenia

[0103] 1. Clinical Sample Analysis

[0104] A total of 192 patients, aged 65–105 years (mean 89.63 ± 8.79 years), were included in the outpatient follow-up study at the Affiliated Geriatric Hospital of Nanjing Medical University. 73 patients (38.02%) were female and 119 were male. They were divided into a young group (non-sarcopenia controls, age 36.54 ± 8.16 years, 50% female) and an elderly group (sarcopenia patients, age 78.23 ± 5.12 years, 33.33% female). Serum samples were collected from both sarcopenia patients (according to the AWGS 2019 diagnostic criteria) and non-sarcopenia controls. Additionally, 12 patients who underwent knee replacement surgery for tibial fractures, aged 23–82 years, were included. They were divided into a young group (<60 years, 6 cases, non-sarcopenia controls) and an elderly group (≥60 years, 6 cases, sarcopenia patients). Lower limb skeletal muscle tissue samples were collected from both sarcopenia patients (according to the AWGS 2019 diagnostic criteria) and non-sarcopenia controls. Serum levels of NETs markers dsDNA, CitH3, and MPO (mouse myeloperoxidase)-DNA complex were detected using quantitative real-time immunofluorescence assay and ELISA. Immunofluorescence staining was used to detect the expression of NETs markers CitH3 (red fluorescence) and Ly6G (green fluorescence), as well as the expression of muscle satellite cell markers PAX7 (green fluorescence) and differentiation marker MyoD (red fluorescence) in skeletal muscle tissue.

[0105] HE staining results are as follows Figure 1 The results showed that, compared to the non-sarcopenia (Non-Sp) group, patients in the sarcopenia (Sp) group had decreased muscle fiber density, widened interfibrillary spaces, and increased inflammatory cell infiltration, with vacuolar formation observed in some muscle tissue. Serum NETs marker levels were significantly elevated in sarcopenia patients compared to non-sarcopenia controls. Figure 2 NET accumulation in skeletal muscle tissue was significantly increased, while MyoD expression was significantly decreased. Figure 3 ).

[0106] 2. Natural aging mouse model

[0107] Five male C57BL / 6 mice were collected at 6 months (healthy control), 15 months (pre-sarcopenia), and 24 months (sarcopenia). Serum and skeletal muscle tissue were collected. The levels of NETs markers and differentiation markers were detected, and skeletal muscle was subjected to H&E staining and immunofluorescence staining.

[0108] HE staining results of skeletal muscle tissue are as follows Figure 4 As shown, aged (24-month-old) mice exhibited decreased muscle fiber density, widened interfibrillary spaces, and increased inflammatory cell infiltration, with vacuolation observed in some muscle tissues. Serum NETs marker levels significantly increased with age. Figure 5 NETs accumulate in skeletal muscle ( Figure 6 ), decreased expression of myosatellite cell differentiation markers ( Figure 7 ).

[0109] 3. In vitro validation of NETs on myosatellite cell differentiation

[0110] Primary myosatellite cell (MuSC) extraction: Male 4-week-old C57BL / 6 mice (15–16 g) were sterilized with 75% ethanol, fixed supine on a dissecting board, and the skin at the ankle joint was aseptically incised to expose the quadriceps femoris muscle, which was collected in ice-cold PBS. The mice were then turned over and fixed prone, the lower body skin was removed, and the intact hind limb skeletal muscles were separated and placed in ice-cold PBS. The muscles were rinsed with PBS containing 1% penicillin-streptomycin, minced, and rinsed again. The tissue blocks were transferred to 15 mL tubes and digested at 37 °C with DMEM medium containing 0.2% type I collagenase and 0.1% dispersin for 1 h. Digestion was terminated by adding 2–3 volumes of DMEM medium containing 2% heat-inactivated FBS. The cells were centrifuged at 500 g for 3 min, the supernatant was discarded, and the erythrocyte lysis buffer was filtered through 100 μm and 70 μm cell filters, followed by centrifugation at 500 g for 3 min. Cell pellets were resuspended in DMEM complete medium (10% FBS, 2.5 ng / mL bFGF, 1% penicillin-streptomycin) and inoculated. After 1 h, the supernatant of non-adherent cells was collected, transferred to a new culture flask, and cultured in a 37 ℃, 5% CO2 incubator.

[0111] Bone marrow neutrophils were isolated from 8-week-old male C57BL / 6 mice and NETs were released by stimulation with PMA (phorbol ester). The supernatant containing NETs was carefully collected and centrifuged at 2000 rpm for 10 min at room temperature to remove residual cells and large debris. The supernatant was then centrifuged at 14000 rpm for 15 min at 4°C. The precipitate was washed with 2 mL of cold PBS and resuspended in a small amount of PBS. The DNA concentration was measured using a microplate reader to represent the NET quantity.

[0112] Differentiation medium: DMEM high glucose medium + 1% penicillin / streptomycin solution + 2% horse serum (HS); Differentiation medium containing 5 ng / mL NETs: NETs are first prepared into a high-concentration stock solution with PBS, and then diluted with differentiation medium to a final concentration of 5 ng / mL; DMEM high glucose complete medium: DMEM high glucose medium + 1% penicillin / streptomycin solution + 10% fetal bovine serum; DMEM high glucose complete medium containing 5 ng / mL NETs: NETs are first prepared into a high-concentration stock solution with PBS, and then diluted with DMEM high glucose complete medium to a final concentration of 5 ng / mL.

[0113] C2C12 cells were divided into four groups: NETs treatment (C2C12+2%HS+NETs) group: C2C12 cells were induced to differentiate for 7 days in differentiation medium containing 5 ng / mL NETs at 37℃ and 5% CO2; C2C12+2%HS group (differentiation induction group): C2C12 cells were induced to differentiate for 7 days in differentiation medium at 37℃ and 5% CO2; C2C12+NETs group: C2C12 cells were co-cultured with NETs in DMEM high-glucose complete medium containing 5 ng / mL NETs at 37℃ and 5% CO2 for 24 h; Control group: C2C12 cells were cultured in DMEM high-glucose complete medium at 37℃ and 5% CO2 for 24 h.

[0114] MuSCs cells were divided into several groups: NETs treatment group (MuSCs + 2% HS + NETs): MuSCs cells were induced to differentiate for 7 days in differentiation medium containing 5 ng / mL NETs at 37°C and 5% CO2; MuSCs + 2% HS group (differentiation induction group): MuSCs cells were induced to differentiate for 7 days in differentiation medium at 37°C and 5% CO2; MuSCs + NETs group: MuSCs cells were co-cultured with NETs (5 ng / mL) in DMEM high-glucose complete medium containing 5 ng / mL NETs at 37°C and 5% CO2 for 24 h; Control group: MuSCs cells were cultured in DMEM high-glucose complete medium at 37°C and 5% CO2 for 24 h.

[0115] The expression of differentiation-related genes and proteins was detected by qPCR, Giemsa staining and Western blot, and the cell cycle and apoptosis of C2C12 cells were detected by flow cytometry.

[0116] like Figures 8-10 As shown, NETs treatment significantly inhibited the differentiation ability of C2C12 myoblasts: Giemsa staining revealed a significant reduction in myotube formation and poor fusion ( Figure 8 A); qPCR detection showed that the mRNA levels of a series of myogenic differentiation marker genes, including MyHC, Myogenin, Desmin, α-actin, CK, TnT, MyoD, and MEF2, were significantly downregulated. Figure 8 B). At the protein level, NETs not only inhibited MyoD expression in C2C12 cells, but also reduced the MyoD protein content in primary myosatellite cells (MuSCs). Figure 8 (C) This suggests that NETs have a direct inhibitory effect on key regulatory factors in the early stages of myoblastic differentiation. Furthermore, NETs can also inhibit cell proliferation (C). Figure 9), and promotes apoptosis ( Figure 10 These results indicate that NETs can simultaneously impair the differentiation, proliferation, and survival of myoblasts, leading to impaired myotube formation and weakened muscle regeneration. Their cellular effects are highly consistent with the pathological characteristics of sarcopenia, such as skeletal muscle regeneration and repair defects, reduced muscle fiber numbers, and muscle mass loss, suggesting that abnormal accumulation of NETs may be one of the important mechanisms that exacerbate the development of sarcopenia.

[0117] Example 2

[0118] Effects of DNase I on NETs clearance on myosatellite cell differentiation

[0119] 1. In vitro experiments

[0120] C2C12 cells were divided into a control group, a NETs group (NETs 5 ng / mL), a DNase I + NETs group (NETs 5 ng / mL, DNase I 0.1 mg / mL), and a Proteinase K + NETs group (NETs 5 ng / mL, Proteinase K 50 μg / mL). Control group C2C12 cells were cultured for 7 days in differentiation medium (same as in Example 1) at 37°C and 5% CO2. NETs group C2C12 cells were cultured for 7 days in differentiation medium containing 5 ng / mL NETs (same as in Example 1) at 37°C and 5% CO2. DNase I + NETs group C2C12 cells were cultured for 7 days in differentiation medium containing both 5 ng / mL NETs and 0.1 mg / mL DNase I at 37°C and 5% CO2. Proteinase K + NETs group C2C12 cells were cultured for 7 days in differentiation medium containing both 5 ng / mL NETs and 50 μg / mL Proteinase K at 37°C and 5% CO2. Giemsa staining was used to detect myotube formation, SA-β-gal staining was used to detect cell senescence, immunofluorescence was used to detect MyoD expression and ROS levels, and Western blot was used to detect MyoD and P16 protein expression.

[0121] The results are as follows Figures 11-14 As shown. Giemsa staining results showed that myotube formation was significantly increased after NET removal ( Figure 11 A). β-galactosidase staining showed that the proportion of senescent cells was increased in the NETs group; DNase I scavenging of NETs significantly reduced the proportion of senescent cells, and Proteinase K treatment also reduced the proportion of senescent cells, but its reversal effect was weaker than that of DNase I ( Figure 11B). Immunofluorescence assays further showed that DNase I upregulated MyoD expression in C2C12 cells after NETs clearance; Proteinase K treatment also partially restored MyoD expression, but the upregulation was not as significant as that of DNase I. Figure 12 Immunofluorescence results showed that intracellular ROS levels decreased after NETs removal. Figure 13 Western blot results showed that DNase I scavenging NETs increased MyoD protein expression and decreased the expression of the aging marker P16 in C2C12 cells; Proteinase K treatment also increased MyoD protein and inhibited P16 expression, but the effect was not as good as DNase I. Figure 14 ).

[0122] 2. D-galactose-induced sarcopenia mouse model

[0123] Eight-week-old C57BL / 6 mice were randomly divided into a control group, a model group, and a DNase I treatment group, with six mice in each group. The model group and the DNase I treatment group were induced to develop sarcopenia by intraperitoneal injection of D-galactose (200 mg / kg, every other day for 8 weeks; D-galactose was dissolved in physiological saline before injection). The control group was injected with an equal volume of physiological saline intraperitoneally. Four weeks after D-galactose injection, the DNase I treatment group mice were injected intraperitoneally with DNase I (10 mg / kg, every other day for 4 weeks; DNase I was prepared with physiological saline before injection). After the last dose in week 8, the following tests were performed: grip strength, treadmill time-to-exhaustion, rotarod test, high-resolution ultrasound to detect quadriceps muscle thickness, hematoxylin and eosin (HE) staining of gastrocnemius muscle, serum biochemical indicators (ATP, SOD, GSH, MDA), Western blot and immunofluorescence to detect the expression of related proteins.

[0124] The results are as follows Figures 15-20 As shown: The grip strength, treadmill exhaustion time, and rotarod duration of mice in the DNase I treatment group were all better than those in the model group. Figure 15 The results of ultrasound imaging in small animals showed that the thickness of the quadriceps femoris (QA) muscle in the DNase I treatment group was significantly greater than that in the model group. Figure 16 HE staining of the gastrocnemius muscle showed that the model group mice had decreased fiber density, widened interfiber spaces, and increased inflammatory cell infiltration. These pathological changes were restored after DNase I treatment. Figure 17Western blot results showed that, compared with the model group, mice treated with DNase I showed increased expression of MyoD protein and decreased expression of the aging marker P16. Figure 18 Serum assay results showed that the levels of SOD, GSH, and ATP in the serum of mice treated with DNase I were significantly increased, while the level of MDA was significantly decreased. Figure 19 Immunofluorescence assays further revealed that the expression of the differentiation marker MyoD was reduced in the gastrocnemius muscle tissue of the DNase I treatment group. Figure 20 ).

[0125] Example 3

[0126] Molecular mechanisms by which NETs inhibit mitophagy

[0127] 1. Take C2C12 cells from the NETs-treated (C2C12+2%HS+NETs) group and the C2C12+2%HS (Control) group in Example 1, “3. In vitro verification of NETs on myosatellite cell differentiation”, and perform proteomics analysis to identify differentially expressed proteins and perform KEGG pathway enrichment analysis; use transmission electron microscopy and label-free live cell imaging to observe the mitochondrial morphology of C2C12 cells after NETs treatment.

[0128] Sequencing results showed that compared with the C2C12+2%HS group, 314 proteins were upregulated and 84 proteins were downregulated in the C2C12+2%HS+NETs group. Furthermore, the upregulated proteins were mainly enriched in the glycolysis pathway, while the downregulated proteins were mainly enriched in the oxidative phosphorylation pathway, suggesting that NETs may promote the progression of sarcopenia by affecting the mitochondrial function of muscle satellite cells, thereby causing metabolic reprogramming. Figure 21 Transmission electron microscopy and label-free live-cell imaging results showed that mitochondria in the NETs-treated group were swollen and fragmented, and mitochondrial cristae were damaged, indicating that NETs inhibited mitophagy in C2C12 cells. Figure 22 ).

[0129] II. C2C12 cells from the control group, NETs group, and DNase I + NETs group in Example 2, "Effect of DNase I on NETs Removal on Myosatellite Cell Differentiation 1. In Vitro Experiment," were used. Glycolytic stress and mitochondrial stress were measured using Seahorse assay. The results showed that NETs treatment decreased the OCR and increased the ECAR; DNase I treatment increased the OCR / ECAR ratio. Figure 23 ).

[0130] III. In Example 1, “3. In vitro verification of NETs on myosatellite cell differentiation”, C2C12 cells from the NETs-treated (C2C12+2%HS+NETs) group and the C2C12+2%HS (Control) group were subjected to JC-1 staining to detect mitochondrial membrane potential. It was found that the mitochondrial membrane potential in the NETs-treated group was decreased. Figure 24 ).

[0131] IV. C2C12 cells from the control group, NETs group, and DNase I + NETs group in Example 2, "Effect of DNase I Clearing NETs on Differentiation of Myosatellite Cells 1. In Vitro Experiment," were collected. Lactate levels in C2C12 cells were detected using a lactate assay kit.

[0132] C2C12 cells were divided into: Control group, NETs group, NETs+PMI group (PMI 20 μM), NETs+DNase I+PMI group (DNase I 0.1 mg / mL + PMI 20 μM), and L-LA group (L-lactic acid 20 mM). The control group C2C12 cells were cultured for 7 days in differentiation medium (same as in Example 1) at 37°C and 5% CO2. The NETs group C2C12 cells were cultured for 7 days in differentiation medium containing 5 ng / mL NETs (same as in Example 1) at 37°C and 5% CO2. The NETs + PMI group C2C12 cells were cultured for 7 days in differentiation medium containing 5 ng / mL NETs and 20 μM PMI at 37°C and 5% CO2. The NETs + DNase I + PMI group C2C12 cells were cultured for 7 days in differentiation medium containing 5 ng / mL NETs, ​​0.1 mg / mL DNase I and 20 μM PMI at 37°C and 5% CO2. The L-LA group C2C12 cells were cultured for 7 days in differentiation medium containing 20 mM L-LA (L-lactic acid) at 37°C and 5% CO2. After differentiation ended on day 7, the levels of pan-lactation and H3K9la in C2C12 cells were detected by Western blot.

[0133] In Example 1, “3. In vitro verification of NETs on myosatellite cell differentiation”, MuSCs cells in the NETs-treated (MuSCs + 2%HS + NETs) group, the control group, and C2C12 cells in the NETs-treated (C2C12 + 2%HS + NETs) group and the C2C12 + 2%HS (Control) group were used to detect the regulation of H3K9la on the Pink1 and Prkn promoter regions by ChIP-qPCR.

[0134] SAMP8 mice were randomly divided into a control group, a model group, a PMI group (10 mg / kg), a DNase I + PMI group (DNase I and PMI, 10 mg / kg each), and an L-LA group (250 mg / kg). DNase I was prepared with physiological saline, PMI was prepared with 5% DMSO + 40% PEG300 + 5% Tween-80 + 50% physiological saline, and L-LA was prepared with physiological saline. The control and model groups received the same amount of physiological saline. Administration was by intraperitoneal injection every other day for 4 weeks. Blood was collected from the orbital sinus of SAMP8 mice and centrifuged at 5000 rpm for 10 min at 4°C to obtain serum. Lactate levels in the serum of SAMP8 mice and in the serum of patients with sarcopenia in Example 1, "1. Clinical Sample Analysis," were detected using a lactate assay kit. Pan-lactation and H3K9la levels in the gastrocnemius muscle tissue of SAMP8 model mice were detected by Western blot.

[0135] The results showed that lactate levels were increased in C2C12 cells in the NETs group, and decreased after treatment with DNase I. Lactate levels were increased in SAMP8 mice in the Model group and in patients with sarcopenia. Figures 26-27 The overall lactation level of C2C12 cells in the NETs group was increased, and the H3K9la level was significantly increased. The lactation modification level was reduced after the combined administration of DNase I and PMI. Figure 28 The overall lactation level of SAMP8 mice in the model group was increased, and the H3K9la level was significantly increased. The lactation modification level was decreased in the DNase I and PMI combined administration group. Figure 29 The effect of H3K9la on autophagy gene expression levels in C2C12 cells and primary myosatellite cells (MuSCs) was detected by ChIP-qPCR. The results showed that H3K9la inhibited the transcription levels of Pink and Prkn. Figure 30 ).

[0136] Example 4

[0137] 2-DG inhibits lactate production and reduces the effect of H3K9la levels on mitophagy.

[0138] 1. In vitro experiments

[0139] C2C12 cells were divided into a control group, an L-LA group, and a 2-DG group, and cultured for 7 days at 37°C and 5% CO2 in differentiation medium, differentiation medium containing 20 mM L-LA, and differentiation medium containing 10 mM 2-DG, respectively.

[0140] Label-free live-cell imaging was used to observe mitochondrial status. The results showed that after adding 2-DG to reduce lactate levels, mitochondrial morphology recovered. Figure 31 Giemsa staining was used to detect myotube formation. The staining results showed that L-LA could inhibit C2C12 cell differentiation, and the effect was reversed after the addition of 2-DG. Figure 32 The JC-1 assay was used to detect changes in mitochondrial membrane potential in C2C12 cells. The results showed that mitochondrial membrane potential recovered after lactate levels decreased. Figure 33 Immunofluorescence was used to detect the expression levels of LC3 and MyoD in C2C12 cells. The immunofluorescence results showed that the level of the mitophagy marker LC3 increased after 2-DG treatment. Figure 34 The 2-DG group showed increased cell differentiation and upregulated expression of the differentiation marker MyoD, suggesting that 2-DG restored the differentiation function of C2C12 cells. Figure 35 The oxygen consumption rate and extracellular acidification rate of C21C12 cells were detected using a Seahorse analyzer. The results showed that the OCR / ECAR ratio was increased in the 2-DG group, indicating that mitochondrial function was restored. Figure 36 ).

[0141] 2. D-galactose-induced sarcopenia mouse model

[0142] Eight-week-old C57BL / 6 mice were randomly divided into four groups: Control, Model, 2-DG, and L-LA, with six mice in each group. The model, 2-DG, and L-LA groups were induced to develop sarcopenia by intraperitoneal injection of D-galactose (200 mg / kg, every other day for 8 weeks; D-galactose was dissolved in physiological saline before injection). The control group was injected with an equal volume of physiological saline. Four weeks after D-galactose injection, the 2-DG group was injected intraperitoneally with 2-DG (400 mg / kg, every other day for 4 weeks; 2-DG was dissolved in physiological saline before injection), and the L-LA group was injected intraperitoneally with L-LA (250 mg / kg, every other day for 4 weeks; L-LA was dissolved in physiological saline before injection). The model and control groups were injected with an equal volume of physiological saline. After the last dose in week 8, the following tests were performed: high-resolution ultrasound to measure quadriceps muscle thickness, H&E staining of gastrocnemius muscle, and Western blot to detect the expression of autophagy and differentiation-related proteins.

[0143] Ultrasound examination of small animals showed that, compared with the L-LA group, the quadriceps femoris muscle thickness of the 2-DG group mice was significantly increased. Figure 37The quadriceps femoris (QA), gastrocnemius (GA), tibialis anterior (TA), soleus (SOL), and extensor digitorum longus (EDL) muscles were isolated and weighed for comparison. The results showed that the wet weight of the above muscles in the 2-DG group was significantly increased compared with that in the L-LA group. Figure 38 HE staining of the gastrocnemius muscle showed that exogenous lactic acid treatment resulted in decreased muscle fiber density, widened inter-fiber spaces, and increased inflammatory cell infiltration. These pathological changes were restored after 2-DG intervention. Figure 39 Western blot results showed that 2-DG treatment increased the expression of the differentiation marker MyoD and increased the expression of markers related to mitophagy. Figure 40 ).

[0144] Example 5

[0145] In vitro synergistic effect of DNase I combined with PMI

[0146] 1. Experimental Grouping

[0147] C2C12 cells were treated with a combination of the NETs inhibitor DNase I and the mitophagy agonist PMI, with the mitophagy inhibitor Mdivi-1 serving as a control. C2C12 cells were divided into the following groups: control group, NETs group, NETs+DNase I group (DNase I 0.1 mg / mL), NETs+PMI group (PMI 20 μM), combined treatment group (NETs+DNase I+PMI group, DNase I 0.1 mg / mL + PMI 20 μM), NETs+Mdivi-1 group (Mdivi-1 10 μM, mitophagy inhibitor control), and NETs+DNase I+Mdivi-1 group (DNase I 0.1 mg / mL + Mdivi-1 10 μM). All C2C12 cells were cultured in differentiation medium at 37°C and 5% CO2 for 7 days to induce differentiation. After differentiation, the control group cells were cultured in fresh differentiation medium, while the cells in the NETs group, NETs+DNase I group, NETs+PMI group, combined drug administration group, NETs+Mdivi-1 group, and NETs+DNase I+Mdivi-1 group were cultured in differentiation medium containing the corresponding drugs for 24 hours. Specifically, NETs and DNase I were first prepared into high-concentration stock solutions with PBS and then diluted with differentiation medium, while Mdivi-1 and PMI were prepared into high-concentration stock solutions with 1% DMSO+PBS and then diluted with differentiation medium.

[0148] 2. Detection indicators

[0149] Label-free live-cell imaging was used to observe mitochondrial morphology; SA-β-gal staining was used to detect cellular senescence; Giemsa staining was used to detect myoblastic differentiation; MitoTracker and LysoTracker colocalization analysis was used to detect mitophagy; immunofluorescence was used to detect MyoD expression in primary myoblast satellite cells; DCFH-DA probe was used to detect ROS levels; Seahorse analysis was used to detect OCR / ECAR; Western blot was used to detect the expression of LC3, Parkin, PINK1 and MyoD proteins; and cellular ATP, SOD, MDA and GSH levels were detected.

[0150] The results are as follows Figures 41-49 As shown. Label-free live-cell imaging observations showed that the combination of DNase I and PMI could restore mitochondrial morphology, promote the clearance of damaged mitochondria, and was more effective than administration alone. Figure 41 β-galactosidase staining and Giemsa staining results showed that, compared with the single-drug group, the combination-drug group had a lower proportion of senescent cells and stronger cell differentiation ability. Figures 42-43 The co-localization of MitoTracker and LysoTracker, as well as the co-localization of autophagy marker LC3 and mitochondrial marker TOMM20, were detected by immunofluorescence. The results showed that the co-localization was increased in the combined administration group and was superior to that in the single administration group, indicating that the combined administration promoted mitophagy and was beneficial for the clearance of damaged mitochondria. Figure 44 Immunofluorescence further confirmed that combined administration to C2C12 cells promoted the expression of the differentiation marker MyoD, and the effect was superior to that of administration alone. Figure 45 The results of DCFH-DA probe detection of ROS levels showed that compared with the NETs group, the ROS level in the single-drug group was lower, the ROS level in the Mdivi-1 group was higher, and the ROS level reduction in the combination drug group was more significant. Figure 46 ). Using Seahorse assays to detect glycolytic and mitochondrial stress, the results showed that treatment with NETs decreased the cell's OCR and increased its ECAR; the addition of DNase I and PMI increased the OCR-ECAR ratio. Figure 47 Western blot analysis of mitophagy and differentiation-related protein expression showed that the expression of LC3, Parkin, PINK, and MyoD proteins was upregulated in the combined drug administration group, indicating that mitophagy and cell differentiation were promoted. Figure 48 Cellular ATP, SOD, MDA, and GSH levels were measured, showing that the combined drug group had significantly increased ATP, SOD, and GSH levels and significantly decreased MDA levels, demonstrating a better improvement effect than the single drug group. Figure 49 ).

[0151] Example 6

[0152] The therapeutic effect of DNase I combined with PMI on a mouse model of sarcopenia

[0153] 1. D-galactose-induced sarcopenia model

[0154] Eight-week-old C57BL / 6 mice were randomly divided into four groups: Control, Model, DNase I, Mdivi-1, DNase I + Mdivi-1, PMI, and Combined Administration (DNase I + PMI), with six mice in each group. The modeling method followed the guidelines in Example 2, “2. D-galactose-induced sarcopenia mouse model.” Administration was performed via intraperitoneal injection every other day for four weeks. The model, DNase I, Mdivi-1, DNase I + Mdivi-1, PMI, and combined administration groups of C57BL / 6 mice were induced into a sarcopenia model by intraperitoneal injection of D-galactose (200 mg / kg, every other day for eight weeks; D-galactose was dissolved in physiological saline before injection). The control group of C57BL / 6 mice was intraperitoneally injected with an equal volume of physiological saline. After 4 weeks of D-galactose injection, mice in the DNase I group were intraperitoneally injected with DNase I (10 mg / kg, every other day for 4 weeks; DNase I was prepared with physiological saline before injection), the Mdivi-1 group (10 mg / kg, every other day for 4 weeks; Mdivi-1 was prepared with 1% DMSO + 0.9% physiological saline before injection), the DNase I + Mdivi-1 group (DNase I and Mdivi-1, 10 mg / kg each, every other day for 4 weeks; Mdivi-1 was prepared with 1% DMSO + 0.9% physiological saline before injection, and DNase I was prepared with physiological saline before injection), mice in the PMI group were intraperitoneally injected with PMI (10 mg / kg, every other day for 4 weeks; PMI was prepared with 5% DMSO + 40% PEG300 + 5% Tween-80 + 50% physiological saline before injection), and mice in the combined drug administration group were intraperitoneally injected with DNase I and PMI (DNase I + Mdivi-1). DNase I and PMI, 10 mg / kg each, every other day for 4 weeks; PMI was prepared by injection with 5% DMSO + 40% PEG300 + 5% Tween-80 + 50% saline, and DNase I was prepared by injection with saline. Testing was performed after the last dose in week 8.

[0155] 2. SAMP8 rapid aging model

[0156] Eight-month-old male SAMP8 mice were randomly divided into: a control group, a DNase I group (10 mg / kg), a PMI group (10 mg / kg), and a combined administration group (DNase I + PMI group, DNase I and PMI 10 mg / kg each). The administration method was the same as in "1. D-galactose-induced sarcopenia model" of this example, for a total of 4 weeks.

[0157] 3. Detection indicators

[0158] Hind limb leg circumference and grip strength; high-resolution ultrasound detection of quadriceps muscle thickness; serum NE and MPO-DNA levels; gastrocnemius muscle H&E staining; immunofluorescence detection of LC3 and MyoD expression; Western blot detection of MyoD protein expression; serum ATP level.

[0159] The results are as follows Figures 50-59 As shown. The leg circumference and grip strength of mice in the combined drug group were similar to those in the control group and superior to those in the single drug group (as shown). Figures 50-51 Ultrasound imaging in small animals showed that the quadriceps muscle thickness in the combined drug group was significantly greater than that in the model group, and the improvement effect was better than that in the single drug group. Figures 52-53 The detection of NETs biomarkers MPO-DNA and NE levels showed that the combined drug administration group had significantly lower NETs levels. Figures 54-55 HE staining of the gastrocnemius muscle showed decreased muscle fiber density and widened inter-fiber spaces in the model group, accompanied by increased inflammatory cell infiltration. These pathological changes were restored after combined treatment with DNase I and PMI. Figure 56 Immunofluorescence assays further revealed that the combined-drug group showed increased expression of the autophagy marker LC3 in the gastrocnemius muscle tissue, which was superior to the single-drug group. Figure 57 Further protein analysis using Western blot revealed increased levels of differentiation-related proteins after combined treatment, indicating that the combined administration effectively promotes myosatellite cell differentiation. Figure 58 Serum assay results showed that the ATP level in the combination therapy group was significantly higher than that in the single-drug group. Figure 59 ).

[0160] The embodiments merely illustrate several implementations of the present invention. While the related descriptions are detailed, they should not be construed as limiting the scope of protection of this invention. It should be understood that any modifications and improvements made by those skilled in the art without departing from the overall concept of the invention fall within the scope of protection of this invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A pharmaceutical composition for treating sarcopenia, characterized in that: The pharmaceutical composition comprises DNase I and PMI.

2. The pharmaceutical composition according to claim 1, characterized in that: The mass ratio of DNase I to PMI is 1:0.075 to 1:

1.

3. The pharmaceutical composition according to claim 1, characterized in that: The mass ratio of DNase I to PMI is 1:

1.

4. The pharmaceutical composition according to claim 1, characterized in that: The pharmaceutical composition comprises DNase I and PMI as active ingredients, and also includes pharmaceutically acceptable excipients.

5. The use of the pharmaceutical composition of claim 1 in the preparation of a medicament for treating sarcopenia.

6. The application according to claim 5, characterized in that: The use of the pharmaceutical composition in the preparation of a medicament for treating sarcopenia by performing at least one of the following actions: (1) Synergistically clear the extracellular trap of neutrophils accumulated in skeletal muscle and enhance mitochondrial autophagy in muscle satellite cells; (2) Restore mitochondrial morphology and membrane potential of muscle satellite cells and promote the clearance of damaged mitochondria; (3) Reduces muscle satellite cell senescence and oxidative stress; (4) Increase the ratio of skeletal muscle cell oxygen consumption rate to extracellular acidification rate and improve mitochondrial energy metabolism; (5) Promotes myogenic differentiation of myosatellite cells and upregulates the expression of MyoD, LC3, Parkin and PINK1 proteins.

7. The application according to claim 5, characterized in that: The drug is available in the form of an injection, a skeletal muscle-targeting delivery system, or a non-injection route.

8. The application according to claim 7, characterized in that: The injectable formulations include solution-type injectables, in-situ gels, injectable microspheres, and lyophilized powder injections; the delivery system targeting skeletal muscle includes skeletal muscle-targeting ligand-modified liposomes or lipid nanoparticles, exosomes, or engineered extracellular vesicles; and the dosage forms via non-injection routes include oral nanodelivery systems, pulmonary inhalers, and soluble microneedle patches.

9. The application according to claim 8, characterized in that: The in-situ gel is an in-situ thermosensitive hydrogel. Application of 10.2-DG in the preparation of drugs for treating sarcopenia.

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