A medicament for improving disuse muscle atrophy and use thereof

Abstract

This invention provides a drug for improving disuse muscle atrophy, comprising S-adenosylmethionine (SAM) and folic acid (FA). This drug effectively increases myoblast proliferation, significantly improving the atrophic phenotype of the soleus muscle, and specifically targets the soleus muscle. It features high biocompatibility and low cost, making it suitable for aerospace medicine and long-term bedridden individuals.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a drug for improving disuse muscle atrophy and its application. Background Technology

[0002] Spaceflight and prolonged bed rest can lead to disuse atrophy of skeletal muscles, resulting in skeletal muscle dysfunction and severely impacting astronauts' work capabilities and the prognosis of clinical patients. Damage to antigravity muscles such as the soleus muscle is most significant, manifesting as decreased muscle wet weight, reduced muscle fiber cross-sectional area, and impaired protein synthesis. Besides maintaining stability in upright posture and movement, the soleus muscle also promotes venous blood return from the lower limbs, hence its common nickname "second heart." Its physiological function not only relates to limb movement but also directly affects the body's circulatory homeostasis, making it a key muscle group for maintaining normal physiological function. Clinically, the structural integrity and normal function of the soleus muscle are crucial for preventing lower limb venous thrombosis, improving limb blood circulation, and avoiding orthostatic hypotension. Once the soleus muscle atrophies or its function is impaired, it can cause limb weakness and unsteady gait, directly obstructing venous return from the lower limbs, inducing hypotension and syncope. Mild cases may affect daily activities, while severe cases can lead to falls, thrombosis, and other serious adverse clinical events, significantly reducing quality of life and increasing the burden of clinical care. For astronauts, low blood pressure and fainting caused by poor soleus muscle function can directly threaten their life safety in orbit, significantly reduce their operational capabilities, and even interrupt space missions.

[0003] Currently, interventions for disuse muscular atrophy in aerospace medicine and ground-based clinical practice include exercise training, antioxidants, myosin antibodies, and irisin intervention. While these methods can alleviate muscular atrophy to some extent, they all have shortcomings such as poor adaptability, limited effectiveness, and significant side effects. They cannot meet the stringent requirements of long-term manned spaceflight and are also insufficient for the diagnostic and treatment needs of ground-based clinical practice. The main technical drawbacks include: 1) Most interventions only target protein degradation pathways (such as ubiquitin-proteasome and FOXO), failing to address the root cause of insufficient nucleotide and protein synthesis; 2) Existing drugs are costly and have unclear side effects, making them difficult to use long-term in aerospace or bedridden individuals; 3) Exercise therapy is time-consuming and energy-intensive, requiring strict control of exercise intensity, which takes up a significant amount of astronaut time and makes it difficult to promote in clinical practice. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a drug for improving disuse muscle atrophy in response to the shortcomings of the prior art. This drug can effectively increase the proliferation level of myoblasts, significantly improve the atrophic phenotype of soleus muscle, and has the characteristics of high biosafety and low cost, making it suitable for aerospace medicine and long-term bedridden people.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a drug for improving disuse muscle atrophy, the drug comprising: S-adenosylmethionine and folic acid, wherein the structural formula of S-adenosylmethionine is: .

[0006] Preferably, the ratio of S-adenosylmethionine to folic acid is 10 mg: 1.6 μg.

[0007] Preferably, the application includes one or more of the following applications: The drug is used to improve soleus muscle atrophy; The drug is used to increase the proliferation level of myoblasts.

[0008] Preferably, the dosage form of the drug is an oral tablet, oral liquid, capsule, injection, or powder.

[0009] Compared with the prior art, the present invention has the following advantages: 1. This invention is the first to disclose the use of SAM+folic acid as the core component to target the methionine cycle nucleotide metabolic axis to treat microgravity muscle atrophy. Supplementing with folic acid can reverse muscle atrophy by increasing THF and restoring nucleotide synthesis. The prepared drug is safe, low-cost, and can be taken orally, making it suitable for aerospace medicine and long-term bedridden patients.

[0010] 2. In this invention, when SAM and folic acid are used in combination for intervention, the proliferation level of myoblasts is significantly higher than that of intervention alone, and soleus muscle atrophy is significantly improved.

[0011] 3. The drug prepared by this invention specifically acts on the antigravity muscle (soleus muscle) and has no significant effect on the gastrocnemius muscle, thus exhibiting high safety.

[0012] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0013] Figure 1 Figure 1 shows the results of SMG cells and mice treated with SAM and folic acid in Example 1 of this invention; Figure A shows relative cell proliferation; Figure B shows newly synthesized RNA in myoblasts; and Figure C shows the soleus muscle atrophy phenotype.

[0014] Figure 2 The figures shown are the results of SAM treatment alone in SMG mice in Comparative Example 1 of this invention; wherein, Figure A is the wet weight of soleus muscle; Figure B is the SAM content; and Figure C is H&E staining.

[0015] Figure 3Figure 1 shows the results of folic acid treatment alone in SMG mice in Comparative Example 2 of this invention; Figure A shows the FH4 content in soleus muscle; Figure B shows the ADP content in soleus muscle; Figure C shows the GMP content in soleus muscle; Figure D shows the IMF content in soleus muscle; and Figure E shows the newly synthesized RNA in cells. Detailed Implementation

[0016] Example 1

[0017] This example demonstrates the combined use of SAM and folic acid to improve disuse muscle atrophy.

[0018] The chemical formula of S-adenosylmethionine in this embodiment is C. 15 H 23 N6O5S + The structural formula is: The abbreviation is SAM, purchased from New England Biolabs, Inc (NEB), item number B9003S.

[0019] The folic acid in this example was purchased from MCE, catalog number HY-16637.

[0020] The mouse myoblasts C2C12 in this embodiment were purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-505; Cell model: A disuse muscular atrophy cell model was constructed using mouse C2C12 myoblasts. Cells were cultured in DMEM containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. When the C2C12 confluence reached 10%, the cell culture flasks were filled with complete culture medium, air bubbles were carefully removed, and the flasks were sealed. The cells were then fixed onto a 2D rotator, which was then placed in the cell culture incubator and cultured at 37°C, 5% CO2, and 25 rpm for 3 days.

[0021] After modeling, the cells were cultured under normal conditions and then divided into four groups after digestion: SMG group, SMG-SAM group (SAM added only), SMG-F group (folic acid added only), and SMG-SAM / F group (SAM and folic acid added). Cell viability was detected by CCK8 assay.

[0022] Mouse tail-hanging model: C57BL / 6J mice (8 weeks old, average weight range: 20-23g) were purchased from the Experimental Animal Center of Air Force Medical University (Xi'an, China). They were housed under ambient temperature (22±1℃), humidity (60%±10%), and light / dark cycle (12 hours / 12 hours). One week before tail hanging, they were transferred to tail-hanging cages to acclimatize. The tail-hanging process was as follows: The mice were secured using a restraint device. The tails were wiped clean with alcohol. After the alcohol had completely evaporated, benzoin tincture was applied to the tails to reduce skin irritation during the tail-hanging process. After the benzoin tincture dried, rosin was applied to the tails to increase friction with the bandages. After drying, the tails were wrapped with medical bandages and adhesive tape. A paperclip was embedded in the tape at the end of the tail to connect to the tail-hanging chain. The mouse's posture was adjusted by the height of the paperclip on the chain, ensuring that its hind limbs were slightly off the cage floor, its body formed a 30° angle with the ground, and its forelimbs could move freely and access food and water.

[0023] Intraperitoneal injection of SAM + folic acid: Mice were randomly divided into five groups: Ctrl group (ground control), SMG group (tail-suspended control), SMG-SAM group (tail-suspended plus SAM), SMG-F group (tail-suspended plus folic acid), and SMG-SAM / F group (tail-suspended plus SAM and folic acid). Except for the Ctrl group, the other groups used the tail-suspended method to establish a disuse muscular atrophy model. The mice were provided with adequate water and food by bottled water and feed placed at the bottom of the cages. At the same time, the mice in each group were given the corresponding drugs via intraperitoneal injection every two days. The concentrations of SAM 10 mg / kg and folic acid 1.6 μg / g were administered every two days for two weeks. The maximum intraperitoneal volume was 100 μL.

[0024] After two weeks of tail-suspended administration, all mice were anesthetized with isoflurane and weighed. They were then perfused with PBS at room temperature, and the soleus muscle tissue was dissected as soon as possible to observe the effect of drug intervention on the soleus muscle phenotype. For the tissue neoRNA synthesis detection experiment, 5-ethynyl-uridine (EU) was injected intraperitoneally 4 hours before tissue collection. EU was used as a uridine substitute to incorporate newly generated RNA for subsequent detection.

[0025] The results are as follows Figure 1 As shown in Figure A, the proliferation levels of myocytes in all four groups increased over time, and the differences between groups gradually widened with increasing days. The relative proliferation level of cells in the SMG-SAM / F group was significantly higher than that in the other three groups, and the difference reached a highly significant level on days 3 and 4 (p < 0.005). Figure 1 As shown in B, compared with the SMG group, the drug-treated groups showed increased neoRNA synthesis, with the double-drug combination group showing a more significant increase in neoRNA synthesis compared to the other two groups; phenotypic observation of the soleus muscle tissue yielded the following results. Figure 1 As shown in C, compared with the Ctrl group, the SMG group and the SMG-SAM / F group showed significant muscle atrophy, but the SMG-SAM / F group showed an improved muscle atrophy phenotype compared with the SMG group.

[0026] Comparative Example 1 This comparative example demonstrates the use of S-adenosylmethionine alone to improve disuse muscle atrophy.

[0027] Mouse tail-hanging model: C57BL / 6J mice (8 weeks old, average weight range: 20-23g) were purchased from the Experimental Animal Center of Air Force Medical University (Xi'an, China). They were housed under ambient temperature (22±1℃), humidity (60%±10%), and light / dark cycle (12 hours / 12 hours). One week before tail-hanging, the mice were transferred to tail-hanging cages to acclimatize. The tail-hanging process was as follows: the mice were fixed in place using a restraint device, and their tails were wiped with alcohol to ensure cleanliness. After the alcohol had completely evaporated, the mice were allowed to... Apply benzoin tincture to the rat's tail to reduce skin irritation from tail hanging. After the benzoin tincture dries, apply rosin to the tail to increase friction between it and the bandage. Once dry, wrap the tail with medical bandage and adhesive tape, and embed a paperclip in the tape at the end of the tail to connect it to the tail hanging chain. Adjust the rat's posture by the height of the paperclip on the tail hanging chain, so that its hind limbs are slightly off the bottom of the cage, and its body forms a 30° angle with the ground, allowing its forelimbs to move freely and obtain food and water.

[0028] Intraperitoneal injection of SAM treatment: The successfully modeled mice were randomly divided into two groups of 6 mice each. The control group (SMG group) used the tail-hanging method to construct the disuse muscle atrophy model, and the mice were given sufficient water and food by bottled water and feed placed at the bottom of the cage. The experimental group (SMG-SAM group) was treated the same as the control group, and the mice in the experimental group were supplemented with SAM by intraperitoneal injection every day at a concentration of 10 mg / kg for 2 weeks. The maximum intraperitoneal injection volume was 200 μL. The ground control group (Ctrl group) was selected from the same batch of mice that had not been modeled.

[0029] After two weeks of tail suspension / administration, all mice were anesthetized with isoflurane and weighed, then perfused with PBS at room temperature, and subsequently dissected for soleus muscle.

[0030] The results are as follows Figure 2 As shown in Figure A, compared with the Ctrl group, the wet weight of soleus muscle decreased in both the SMG and SMG-SAM groups, but compared with the SMG group, the wet weight of soleus muscle in the SMG-SAM group increased significantly; Figure 2 As shown in B, compared with the SMG group, the SAM content in the soleus muscle of the SMG-SAM group was restored; Figure 2The CH&E results showed that, compared with the Ctrl group, the cross-sectional area of ​​muscle fibers in both the SMG group and the SMG-SAM group was significantly reduced, but compared with the SMG group, the cross-sectional area of ​​muscle fibers in the SMG-SAM group was significantly increased (scale bar is 100μm). This indicates that SAM can be used to improve disuse muscle atrophy in mice and increase the wet weight of soleus muscle and the cross-sectional area of ​​muscle fibers.

[0031] Comparative Example 2 This comparative example is folic acid used alone to improve disuse muscle atrophy.

[0032] Mouse tail-hanging model: C57BL / 6J mice (8 weeks old, average weight range: 20-23g) were purchased from the Experimental Animal Center of Air Force Medical University (Xi'an, China). They were housed under ambient temperature (22±1℃), humidity (60%±10%), and light / dark cycle (12 hours / 12 hours) conditions. One week before tail-hanging, the mice were transferred to tail-hanging cages to acclimatize. The tail-hanging process was as follows: the mice were fixed in place using a restraint device, and their tails were wiped with alcohol to ensure cleanliness. After the alcohol had completely evaporated... Apply benzoin tincture to the rat's tail to reduce skin irritation from tail hanging. After the benzoin tincture dries, apply rosin to the tail to increase friction between it and the bandage. Once dry, wrap the tail with medical bandage and adhesive tape, and embed a paperclip in the tape at the end of the tail to connect it to the tail hanging chain. Adjust the rat's posture by the height of the paperclip on the tail hanging chain, so that its hind limbs are slightly off the bottom of the cage, and its body forms a 30° angle with the ground, allowing its forelimbs to move freely and obtain food and water.

[0033] Intraperitoneal injection of folic acid: Mice were randomly divided into three groups of six each: ground control (Ctrl), tail-suspended control (SAM or SMG-O), and tail-suspended drug-treated group (SMG-F or SMG-FH4). Except for the ground control group, the other groups used the tail-suspended method to establish a disuse muscular atrophy model. The mice were provided with adequate water and food by bottled water and feed placed at the bottom of the cages. At the same time, the tail-suspended drug-treated group mice were given folic acid intraperitoneally every two days at a concentration of 1.6 mg / kg for 2 weeks. The maximum intraperitoneal administration volume was 100 μL.

[0034] After two weeks of tail-suspended administration, all mice were anesthetized with isoflurane and weighed. They were then perfused with PBS at room temperature, and the soleus muscle was subsequently dissected for tissue nucleotide content detection. For the detection of novel RNA synthesis in tissues, 5-ethynyl-uridine (EU) was injected intraperitoneally four hours before tissue collection. This EU was used as a uridine substitute to incorporate into newly generated RNA for subsequent detection.

[0035] The results are as follows Figure 3As shown in Figure A, compared with the Ctrl group, the tetrahydrofolate (FH4) content in the soleus muscle of the tail-hanging group (SMG) was lower, while the FH4 content in the drug-treated group (SMG-F) was restored; Figure 3 As shown in B-D, compared with the Ctrl group, the content of ADP / GMP / IMP nucleotides in the soleus muscle of the tail-hanging group (SMG) was lower, while the content of these three nucleotides in the drug-treated groups (SMG-F) recovered to some extent; Figure 3 As shown in E, compared with the Ctrl group, the SMG and SMG-F groups had relatively low levels of new RNA synthesis, while the SMG-F group had increased new RNA synthesis compared with the SMG group (**p<0.01) (scale bar is 100μm).

[0036] In summary, this study explored a drug and its application strategy for improving disuse atrophy. The study found that the levels of SAM, FH4, and RNA synthesis-related nucleotides were decreased in the atrophied soleus muscle. It is speculated that disuse atrophy may affect the efficiency of methionine cycle metabolism by reducing SAM levels, thereby reducing the one-carbon cycle transport mainly carried by FH4, thus affecting the levels of nucleotide synthesis initiation molecules ADP, GMP, and IMP, ultimately leading to a decrease in overall RNA synthesis levels. Through cellular and animal experiments, it was found that supplementation with SAM and FH4 separately, as well as the combination of two drugs, effectively improved the atrophic phenotype of soleus muscle and enhanced the level of new RNA synthesis. This confirms that both drugs have a positive effect on improving disuse atrophy, and the combination of two drugs is more effective than single drugs, suggesting a potential new method for preventing and improving disuse atrophy.

[0037] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A drug for improving disuse muscle atrophy, characterized in that, The drug comprises: S-adenosylmethionine and folic acid, wherein the structural formula of S-adenosylmethionine is: 。 2. The drug for improving disuse muscle atrophy according to claim 1, characterized in that, The ratio of S-adenosylmethionine to folic acid is 10 mg: 1.6 μg.

3. The application of a medicament for improving disuse muscle atrophy as described in claim 1 or 2, characterized in that, The application includes one or more of the following: The drug is used to improve soleus muscle atrophy; The drug is used to increase the proliferation level of myoblasts.

4. The application according to claim 3, characterized in that, The dosage form of the drug is oral tablets, oral liquids, capsules, injections, or powders.

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