Compound medicine for delaying skeletal muscle aging and application thereof

Abstract

The application relates to a compound medicine for delaying skeletal muscle aging and application thereof, and the medicinal active components in the compound medicine composition are composed of menadione bisulfite sodium and N-acetyl cysteine, and belong to the technical field of biological medicine. A novel drug combination mode is developed, an oxidant menadione bisulfite sodium (MBS) and an antioxidant N-acetyl-L-cysteine (NAC) are combined and given in different time periods to regulate the H2O2 circadian rhythm of the skeletal muscle of old mice, the changes of the muscle strength, histopathology and aging marker protein level of the skeletal muscle of the old mice are detected, and then the precise compound medicine capable of improving the skeletal muscle function of the old mice and delaying skeletal muscle aging is determined. The application provides an effective drug combination strategy for relieving or treating the delay of skeletal muscle aging, and has very significant meaning.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a compound drug for delaying skeletal muscle aging and its application. Background Technology

[0002] Skeletal muscle plays a crucial role in maintaining human health and daily function. With age, skeletal muscle mass and function gradually decline, a condition known as sarcopenia. This process not only affects the mobility of older adults but also significantly increases the risk of falls, chronic diseases, and death. Statistics show that up to 42% of older adults experience difficulties in daily life, 15%–30% are unable to lift or carry objects heavier than 4.5 kg, and over 30% face the risk of disability or even death. Furthermore, skeletal muscle, as one of the body's most important metabolic organs, regulates blood glucose and lipid metabolism. With decreased muscle mass, older adults face an increased risk of metabolic disorders and insulin resistance, raising the probability of developing type 2 diabetes. Therefore, delaying skeletal muscle aging and maintaining muscle health are crucial for the health and quality of life of older adults.

[0003] Although antioxidants have been studied and applied in improving age-related skeletal muscle function decline, their effectiveness remains controversial. Some studies have shown that mice overexpressing the human catalase gene can enhance mitochondrial antioxidant capacity and improve skeletal muscle function; resveratrol intake can improve mitochondrial function in mice and restore age-related physical decline. However, other antioxidants such as N-acetyl-L-cysteine ​​(NAC) are effective in extending the lifespan of mice, but have no significant effect on sarcopenia. Other studies have shown that while vitamin E and vitamin C supplementation reduces oxidative stress levels in aged rats, it has no effect on muscle mass or exercise capacity. Clinical trials have also shown that NAC supplementation disrupts skeletal muscle inflammation and repair capabilities; and that vitamin C and vitamin E supplementation has no significant effect on muscle strength in older adults. These results suggest that the use of antioxidants to treat skeletal muscle aging requires further discussion.

[0004] Furthermore, the importance of circadian rhythms in disease treatment is increasingly being recognized. Studies have shown that drug treatments are most effective at specific times. For example, diuretics are more effective when taken at night, while glucocorticoids are more effective when taken in the morning. This evidence suggests that synchronizing treatment regimens with the body's endogenous circadian rhythms can optimize efficacy. Research has shown that H2O2 levels in tissues and organs exhibit a diurnal rhythm; therefore, developing compound drugs to regulate the circadian rhythm of H2O2 in the skeletal muscle of aged mice may provide a new approach to delaying skeletal muscle aging. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide a compound drug for delaying skeletal muscle aging and its application. The pharmaceutically active components of the compound drug composition consist of sodium menadione bisulfite and N-acetylcysteine. This provides an effective drug combination strategy for alleviating or treating skeletal muscle aging, which is of great significance.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0007] This invention discloses the use of a combination pharmaceutical composition for preventing, improving, and / or treating skeletal muscle aging in the preparation of a medicament for preventing, improving, and / or treating skeletal muscle aging, characterized in that the combination pharmaceutical composition for preventing, improving, and / or treating skeletal muscle aging is composed of sodium menadione bisulfite and N-acetylcysteine.

[0008] Furthermore, the combined pharmaceutical composition is a combination of two separate formulations.

[0009] Furthermore, the two separate formulations are administered sequentially.

[0010] Furthermore, the two separate formulations were administered sequentially at different times to menadione sodium bisulfite and N-acetylcysteine.

[0011] The present invention also discloses a combination pharmaceutical composition for preventing, improving and / or treating skeletal muscle aging, characterized in that it comprises active ingredients of sodium menadione bisulfite and N-acetylcysteine.

[0012] Furthermore, the above-described combination of pharmaceutical compositions is used in the preparation of medicaments for preventing, improving, and / or treating skeletal muscle aging.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0014] This invention is the first to propose regulating the diurnal rhythm of H2O2 in skeletal muscle of aged mice by administering the oxidant menadione bisulfite sodium (MBS) and the antioxidant N-acetyl-L-cysteine ​​(NAC) in combination at different time periods. The changes in muscle strength, histopathology, and aging marker protein levels in the skeletal muscle of aged mice were detected to identify the compound drug that can improve skeletal muscle function and delay skeletal muscle aging in aged mice and its application.

[0015] This invention is the first to propose a compound drug for delaying or improving skeletal muscle aging from the perspective of redox oscillation rhythm, emphasizing the importance of time. Different combinations of administration of oxidants and antioxidants at different times produced different effects on skeletal muscle aging in mice. Among them, the compound regimen Old-Plan1 (oxidant intake from ZT10.5 to ZT18, antioxidant intake from ZT18 to ZT10.5) showed the best effect on skeletal muscle in aged mice. Under this regimen, the forelimb muscle strength of aged mice increased, gait coordination improved, exercise endurance increased, and insulin sensitivity increased; simultaneously, the cross-sectional area of ​​skeletal muscle fibers increased, the degree of fibrosis decreased, the mitochondrial area decreased, and the expression of aging-related markers P21 and TNFα proteins decreased.

[0016] Overall, the time-limited intake of oxidants and antioxidants to regulate redox oscillation rhythms and delay skeletal muscle aging in this invention provides a new approach for future interventions in skeletal muscle aging, which can help improve the quality of life of older adults and slow the development of health problems related to aging skeletal muscle. Attached Figure Description

[0017] Figure 1 Analysis of H2O2 rhythm in skeletal muscle of young and old mice.

[0018] Figure 2 Different dosing regimens had varying effects on the H2O2 rhythm in mouse skeletal muscle. (A) Schematic diagram of the mouse dosing regimens. Sun: Illuminated environment, mouse resting period; Moon: Dark environment, mouse active period. The moment the light was turned on is denoted as ZT0. (B) Relative H2O2 levels at different time points in mice with different dosing regimens.

[0019] Figure 3 The dosing regimen of ZT10.5-ZT18, ingesting an oxidant and administering a reducing agent at the remainder of the time (OId-Plan 1), improved skeletal muscle function in aged mice. Specifically, (A, C) the pulling test assessed forelimb strength; (B, D) the running test assessed exercise endurance; and (C, E) the rotarod fatigue test assessed coordination and endurance.

[0020] Figure 4 The Old-Plan 1 dosing regimen improved the pathological state of skeletal muscle in aged mice. (A) H&E staining of muscle fibers to detect cross-sectional area of ​​muscle bundles. Representative figure (left), average area of ​​muscle fibers (middle), ratio of muscle fibers of different areas to the total number of fibers (right); (B) Sirius red staining to detect the degree of skeletal muscle fibrosis. Representative figure (left), ratio of fibrotic area of ​​skeletal muscle in each group of mice (right); (C) Transmission electron microscopy of mitochondria to detect mitochondrial area. Representative figure (left), average area of ​​mitochondria (middle), ratio of different mitochondrial numbers to the total number of mitochondria counted (right).

[0021] Figure 5 Effects of different time-limited dosing regimens on the expression of aging markers P21, P53, and TNFα in skeletal muscle of aged mice. A: Western blot representation of P21, P53, and TNFα proteins in mouse skeletal muscle; BD: Relative expression levels of P21 (B), TNFα (C), and P53 (D) proteins in mouse skeletal muscle, Young-Con group (n=4), Old-Con group (n=6), Old-Plan1 group (n=6), Old-Plan2 group (n=5), Old-Plan3 group (n=6). P Values ​​were derived using one-way ANOVA and corrected for Bonferroni. Data are expressed as mean ± standard deviation. ** represents P <0.01, *** represents P <0.001. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0023] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0024] Example

[0025] I. Experimental Materials and Methods.

[0026] 1. Correlation between H2O2 rhythm and skeletal muscle aging: The correlation between H2O2 rhythm and skeletal muscle aging was analyzed by detecting H2O2 rhythm in the skeletal muscle of young and old mice.

[0027] Male C57BL / 6J mice (n=54 per age) aged 3 months and 18–20 months were purchased from Beijing Weishang Lide Biotechnology Co., Ltd., and housed in an SPF-grade environment. Mice were subjected to a 12:12h light / dark cycle (light intensity of 200 Lux during the light period), with the light-on time designated as ZT0 and the light-off time as ZT12. They were allowed free access to food and water for at least 2 weeks to acclimatize. After the acclimatization period, skeletal muscle tissue samples were collected from young and aged mice starting at time point ZT0. Samples were collected every 3 hours for 24 hours (ZT0, 3, 6, 9, 12, 15, 18, 21, 24, n=6 mice per group at each time point). All tissues were collected, flash-frozen in liquid nitrogen, and stored at -80℃ after collection. Hydrogen peroxide concentration in skeletal muscle of young and aged mice at various time points was determined using the Amplex® Red Hydrogen Peroxide / Peroxidase Assay Kit (cat# A22188, Fisher Scientific, Waltham, MA, USA). Specifically, 15 mg of mouse skeletal muscle was added to 0.6 mL of assay buffer and homogenized using a tissue homogenizer. The homogenate was then sonicated at 4°C (30% power, 15 s on, 45 s off, 10 min), followed by centrifugation at 12000 x g to remove debris. 50 μL of sample and ADHP / HRP working solution were added to each well of a microplate. After incubation at room temperature in the dark for 30 min, readings were taken using a fluorescent microplate with excitation wavelengths of 530–560 nm and emission wavelengths of 590 nm. The peroxide concentration in the sample was calculated by comparing the relative fluorescence units of each sample with a simultaneously prepared standard curve.

[0028] 2. Efficacy of the dosing regimen in intervening in skeletal muscle H2O2 rhythm: The H2O2 rhythm of mouse skeletal muscle was detected by administering a combination of oxidants and antioxidants at different times of the same day.

[0029] Ninety-six 10-month-old male C57BL / 6 mice were synchronized for two weeks in a 12-hour light-dark cycle environment. Following different time-limited protocols, they were given oral solutions containing the oxidant menadione bisulfite. Mice were divided into four groups for administration of sodium (MBS) and / or the antioxidant N-acetyl-L-cysteine ​​(NAC): (1) Water group: water only for 24 hours; (2) Plan 1 group: MBS (0.8 g / L) was given to mice from ZT10.5 to ZT18, and NAC (13.4 g / L) was given to mice from ZT18 to ZT10.5; (3) Plan 2 group: MBS (7.2 g / L) was given to mice from ZT2.5 to ZT10, and NAC (6.2 g / L) was given to mice from ZT10 to ZT2.5; (4) Plan 3 group: MBS (1.6 g / L) was given to mice from ZT18.5 to ZT2, and NAC (8.4 g / L) was given to mice from ZT2 to ZT18.5. To ensure consistent administration, the concentration of each drug was adjusted according to the difference in water consumption at different times of the day. Skeletal muscle tissue was collected at different time points after 4 weeks of continuous drug administration, and the H2O2 level in mouse skeletal muscle was detected using the Amplex® Red Hydrogen Peroxide / Peroxidase Assay Kit.

[0030] 3. Precision dosing regimen screening: By administering a combination of oxidants and antioxidants at different times of the same day, we screened for precision dosing regimens that could significantly improve skeletal muscle function decline in aging mice.

[0031] Subsequently, we exposed 60 healthy male aged C57BL / 6 mice aged 20-23 months and 20 aged 3 months to a 12-hour light-dark cycle environment for two weeks. Then, the aged mice that drank the oxidant MBS and / or the antioxidant NAC were randomly divided into six groups according to different time limits: (1) Old-Con group: water only for 24 hours; (2) Old-MBS group: MBS for 24 hours; (3) Old-NAC group: NAC for 24 hours; (4) Old-Plan1 group: MBS for ZT10.5-ZT18, NAC for ZT18-ZT10.5; (5) Old-Plan2 group: MBS for ZT2.5-ZT10, NAC for ZT10-ZT2.5; (6) Old-Plan3 group: MBS for ZT18.5-ZT2, NAC for ZT2-ZT18.5. Meanwhile, 3-month-old male C57BL / 6 mice were used as the control group (Young-Con group: water only for 24 hours). The medication was administered for a total of 90 days; the specific administration regimen is shown in Table 1.

[0032] Table 1 Dosing Regimen

[0033] .

[0034] We used a grip test (to detect changes in forelimb muscle strength in mice), a running test, and a tumbler fatigue test (to assess exercise endurance in mice) to clarify the effects of different drug administration regimens on skeletal muscle function in mice. To investigate the pathological and molecular effects of different drug administration regimens on mouse skeletal muscle, we used hematoxylin-eosinstaining (H&E) staining to observe the cross-sectional area of ​​skeletal muscle fibers, Sirius red staining to observe the degree of muscle fiber fibrosis, transmission electron microscopy to observe the morphology and size of mitochondria, and Western blot to detect the expression levels of aging-related P21, P53, and TNFα proteins.

[0035] 4. Grip test.

[0036] Four weeks after drug administration, the animals were removed from their enclosures four hours in advance to allow them to acclimatize to the experimental environment and reduce stress and environmental disturbances. The grip strength tester was calibrated to ensure accurate and reliable measurements. The mouse was grasped by the tail, and its forelimbs were placed on the mesh of the tester, allowing them to grip the mesh. The mouse was then slowly and evenly pulled backward until it left the mesh. Each mouse was measured three times, and data were recorded with at least 15 minutes between each measurement.

[0037] 5. Running experiment.

[0038] Six weeks after drug administration, a running experiment was conducted. During the training phase, on days 1 and 2, mice were removed from their cages and allowed to move freely on a track for 10 minutes. No training was conducted on day 3. On day 4, mice were placed on a treadmill and ran at a speed of 10 m / min for 20 minutes. No training was conducted on day 5, and the training from day 4 was repeated on day 6. In the formal experiment, mice were removed 4 hours in advance to acclimatize. On a 15° inclined treadmill, mice first warmed up at a speed of 5 m / min for 5 minutes. Then, the treadmill speed was gradually increased by 1 m / min until it reached 20 m / min, and maintained at this speed for 5 minutes. The speed was then increased to 21 m / min and maintained for 10 minutes, finally increasing to 22 m / min until the mouse reached exhaustion. Exhaustion was defined as a mouse that did not continue running within 10 seconds of electrical stimulation. The running distance was the total distance the mouse ran before exhaustion.

[0039] 6. Rotating rod fatigue test.

[0040] The experiment was conducted 6 weeks after drug administration. Mice were trained for two days prior to the formal experiment, with the rotation speed at 10 rpm / min for 5 minutes. During the formal experiment, the rotation speed of the rod was set to 10 rpm / min and uniformly accelerated for 5 minutes to 25 rpm / min, then maintained at a constant speed of 25 rpm / min for another 5 minutes. The latency period (the time the mouse remained on the rotating rod) was recorded during this time. If the mice successfully completed the entire test cycle, the longest recorded time was 600 seconds. The experiment was repeated 3 times, with at least 15 minutes between each attempt.

[0041] 7. Stain the sections.

[0042] Paraffin embedding and H&E staining of mouse skeletal muscle tissue were performed by GigaDevice Biotechnology Co., Ltd. Sirius red staining was performed using a modified Sirius red staining kit (G1472, Beijing Solarbio Science & Technology Co., Ltd., China), and the specific steps are as follows:

[0043] (1) Sectioning: Fix the paraffin block on the microtome and section the thickness to 4 μm;

[0044] (2) Baking: Remove the slices from the 37°C water bath and bake them in a 60°C oven for 60 minutes;

[0045] (3) Hydration: Xylene I 5 min, Xylene II 5 min, Xylene III 5 min, anhydrous ethanol 1 min, 95% ethanol 1 min, 75% ethanol 1 min, wash with distilled water for 5 min;

[0046] (4) Staining: Prepare iron hematoxylin staining solution, add staining solution for 5-10 min, wash with distilled water for 10-20 s to remove excess staining solution, rinse with running water for 5 min, add Sirius red staining solution for 15-30 min, and rinse slightly with running water.

[0047] (5) Dehydration and transparency: 75% ethanol for 1 min, 95% ethanol for 1 min, anhydrous ethanol for 1 min, xylene 3 times, 1~2 min each time;

[0048] (6) Sealing: Seal with neutral resin.

[0049] 8. Mitochondrial electron microscopy.

[0050] Skeletal muscle tissue blocks (1mm×1mm×1mm) were placed in pre-cooled electron microscopy fixative at 4℃ for 2–4 hours, and the morphology and size of mitochondria were observed under an electron microscope. Mitochondria were grouped by size, and the proportion of mitochondria in each size category relative to the total number of mitochondria was calculated.

[0051] 9. Western blot.

[0052] (1) Extraction of total protein from tissues.

[0053] Weigh 25 mg of skeletal muscle sample into a grinding tube, and add the corresponding lysis buffer at a ratio of 500 µL of RIPA lysis buffer per 25 mg of tissue (RIPA: PMSF = 100:1). After grinding, sonicate until the solution is clear, and centrifuge at 12000 x g, 4 °C for 10 min. Transfer the supernatant to a new RNase-free EP tube, add 6 × Loading Buffer, mix well, boil in 95 °C water for 10 min, briefly centrifuge, and store the sample at -80 °C for later use.

[0054] (2) Gel preparation.

[0055] Prepare a 12% lower layer adhesive according to the formula shown in Table 2, and prepare a 5% thicker adhesive according to the formula shown in Table 3.

[0056] Table 2 12% separating gel (15 mL)

[0057] .

[0058] Table 3 5% Stacking Gel (3mL)

[0059] .

[0060] (3) Sample loading and electrophoresis.

[0061] Slowly add 30µg of protein sample into the well, connect the power supply, and adjust the voltage to 80V constant voltage electrophoresis. After the sample enters the separating gel and the marker separates, adjust the voltage to 120V until the bromophenol blue migrates to the bottom of the gel.

[0062] (4) Transfer membrane.

[0063] Prepare the transfer solution according to Table 4, and transfer the membrane at a constant current of 300mA for 1 hour.

[0064] Table 4 Transfer Buffer (1×)

[0065] .

[0066] (5) Closed.

[0067] After the transfer was completed, the PVDF membrane was removed and washed three times with TBST (TBS buffer containing 0.05% Tween-20) for 5 min each time. Then the membrane was placed in TBST buffer containing 5% skim milk powder and blocked at room temperature for 60 min.

[0068] (6) Antibody hybridization.

[0069] Remove the blocked PVDF membrane, wash it three times with TBST for 10 min each time, dilute the primary antibody according to the dilution ratio shown in Table 5, and incubate overnight at 4°C. The next day, remove the PVDF membrane. Wash it three times with TBST for 10 min each time, then add a secondary antibody of the same species as the primary antibody (5% skim milk: secondary antibody = 10000:1), and incubate slowly on a shaker at room temperature for 60 min. After the secondary antibody incubation, wash it three times with PBST for 10 min each time.

[0070] Table 5 Primary antibody dilution ratio

[0071] .

[0072] (7) Development.

[0073] An appropriate amount of developer was dropped onto the PVDF membrane, and the membrane was developed and photographed using a chemiluminescence imaging system. The images were then analyzed using ImageJ software.

[0074] 10. Statistical data analysis.

[0075] All statistical results in this paper were analyzed using GraphPad Prism 8.0.2 software. Data are expressed as mean ± standard error. One-way ANOVA was used for one-way ANOVA, and two-way ANOVA was used for two-way ANOVA. P A value <0.05 was considered statistically significant. The diurnal rhythm was analyzed using JTK_Cycle. P <0.05 indicates rhythm.

[0076] II. Experimental Results.

[0077] 1. Disruption of H2O2 rhythm in skeletal muscle of aged mice.

[0078] ADHP probes were used to detect H2O2 content in the skeletal muscle of young and old mice. The H2O2 level in the skeletal muscle of young mice showed a rhythmic oscillation. P <0.05, Figure 1 In older mice, the H2O2 rhythm was lost and its content increased in skeletal muscle. The H2O2 rhythm in the skeletal muscle of older mice was disordered. P A value <0.05 indicates rhythm.

[0079] 2. Different dosing regimens have varying effects on the H2O2 rhythm of mouse skeletal muscle.

[0080] Based on the H2O2 rhythm characteristics of skeletal muscle in young and old mice, as well as the mice's daily routine and routine drug administration, a combined use regimen of oxidant (MBS) and antioxidant (NAC) was designed. Figure 2A). In all three administration regimens, the H2O2 content in skeletal muscle was lower when mice drank NAC and higher when they drank MBS, and the H2O2 level in the skeletal muscle of mice in Plan 1 and Plan 3 groups showed a diurnal rhythm. P <0.05, Figure 2 B).

[0081] 3. The dosing regimen of ingesting oxidants during ZT10.5-ZT18 and ingesting antioxidants at the rest of the time (Old-Plan 1) improved skeletal muscle function in aged mice.

[0082] In grip strength, running, and twirling fatigue tests, neither the Old-NAC group nor the Old-MBS group showed significant improvement. Figure 3 AC, only the Old-Plan1 group mice showed significantly higher forelimb muscle strength and exercise endurance than the Old-Con group ( P <0.05, Figure 3 DF), while the Old-Plan 2 and Old-Plan 3 groups also showed no significant effect ( Figure 3 DF).

[0083] 4. The Old-Plan1 dosing regimen improves the pathological state of skeletal muscle in aged mice.

[0084] Compared to the Old-Con group, the cross-sectional area of ​​the muscle bundles in the Old-Plan1 group was larger. P <0.05, Figure 4 A), decreased degree of fibrosis ( P <0.05, Figure 4 B), the area of ​​mitochondria decreases ( P <0.05, Figure 4 C) Neither Old-Plan 2 nor Old-Plan 3 showed significant improvement (results). Figure 4 AB).

[0085] 5. Effects of time-limited intake of oxidants and antioxidants on skeletal muscle aging markers in aged mice.

[0086] Behavioral and pathological results both indicated that Old-Plan1 could improve the skeletal muscle status of aged mice. To clarify whether this dosing regimen could improve the expression levels of aging markers in the skeletal muscle of aged mice, we used Western blot to detect the expression levels of aging markers P21, P53, and TNFα proteins. Statistical results showed that compared with the Young-Con group, the expression levels of P21 and TNFα proteins in the skeletal muscle of mice in the Old-Con group were significantly increased (…). P <0.001, Figure 5Compared to the Old-Con group, the expression levels of P21 and TNFα proteins in the skeletal muscle of mice in the Old-Plan1 group were significantly reduced (B, C). P <0.01, Figure 5 In mice in groups B and C, the expression level of P21 protein in skeletal muscle of Old-Plan2 or Old-Plan3 mice showed no significant change. Figure 5 B). Compared with the Young-Con group, the expression level of p53 protein in the skeletal muscle of Old-Con group mice did not change significantly ( Figure 5 D), but there is an increasing trend, while the trend of Old-Plan1 reducing skeletal muscle P53 protein levels in aged mice is not significant ( Figure 5 D). This suggests that the expression levels of P21 and TNFα proteins in the skeletal muscle of older mice are increased compared to younger mice. The time-limited dosing regimen Old-Plan 1 can reduce the expression of P21 and TNFα proteins in older mice, while Old-Plan 2 and Old-Plan 3 have no significant effect on the expression of P21 and TNFα proteins.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of a combination pharmaceutical composition for preventing, improving, and / or treating skeletal muscle aging in the preparation of a medicament for preventing, improving, and / or treating skeletal muscle aging, characterized in that, The combined pharmaceutical composition for the prevention, improvement and / or treatment of skeletal muscle aging consists of menadione sodium bisulfite and N-acetylcysteine, wherein menadione sodium bisulfite is administered during ZT10.5 to ZT18, and N-acetylcysteine ​​is administered during ZT18 to ZT10.5, wherein ZT0 represents the light start time under a 12-hour light / 12-hour dark light-dark cycle, and ZT12 represents the light stop time.

2. The application according to claim 1, characterized in that, The combined pharmaceutical composition is a combination of two separate formulations.

3. The application according to claim 2, characterized in that, The two separate formulations were administered sequentially.

4. The application according to claim 2, characterized in that, The two separate formulations were administered sequentially at different times to menadione sodium bisulfite and N-acetylcysteine.

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