Method for forming and converting skeletal muscle fiber types by using salvianic acid A sodium

Abstract

The invention relates to the field of biological pharmacy, and discloses a method for forming and transforming skeletal muscle fiber types by salvianic acid A sodium, which comprises the following steps: S1, small molecule drugs, S2, C2C12 cell culture and treatment, S3, mouse treatment, S4, measurement of grip strength and exercise tolerance, S5, evaluation of oxygen consumption, S6, RNA extraction and real-time fluorescent quantitative PCR (qPCR), S7, Westernblot, and S8, immunofluorescent staining. Sodium tanshinol adjusts expression of related genes of muscle fiber types, so that conversion of muscle fibers from glycolytic muscle fibers to oxidized muscle fibers is promoted, and understanding of sodium tanshinol on a molecular mechanism of muscle fiber type formation and conversion is deepened.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for sodium danshensu to form and transform skeletal muscle fiber types. Background Art

[0002] Skeletal muscle is composed of different types of muscle fibers. At present, the generally accepted classification of mammalian skeletal muscle fiber types is based on the difference in MyHC subtypes, which divides muscle fibers into four types, namely type I (slow oxidative muscle fibers), type IIa (fast oxidative muscle fibers), type IIb (fast glycolytic muscle fibers) and type IIx (intermediate muscle fibers), which express MyHC1 / slow, MyHC2a, MyHC2b and MyHC2x genes respectively. Oxidative muscle fibers contain more myoglobin and mitochondria, have higher oxidative capacity, and give the body stronger endurance, while people with a high proportion of fast glycolytic muscle fibers have a higher risk of obesity and related metabolic diseases, and their muscles are more susceptible to atrophy in old age. In animal husbandry production, meat yield and quality are directly related to the proportion of muscle fiber types in the muscle. Muscles with more slow oxidative muscle fibers contain a certain amount of intramuscular fat, have bright red color, and have higher scores; on the contrary, muscles with a high content of fast glycolytic muscle fibers have pale meat color, rapid pH drop after slaughter, and increased protein degradation, which will lead to the production of PSE meat. Therefore, elucidating the molecular mechanisms that regulate the formation of muscle fiber types is particularly important for the treatment of muscle-related diseases and the improvement of meat quality traits.

[0003] In recent years, in the study of the molecular regulatory mechanism of muscle fiber formation and transformation, many natural compounds have been identified that can regulate the formation or transformation of muscle fiber types and thus affect meat properties and muscle function. For example, after mice were treated with small molecule succinate, fast glycolytic muscle fibers were transformed into slow oxidative muscle fibers, and the mice's exercise endurance was enhanced; after mice were treated with quercetin, the expression of genes related to oxidative muscle fibers was upregulated, and muscle function was increased. Niacin supplementation significantly increased the proportion of oxidative muscle fibers in sheep, and improved the quality of Mianyang meat. By finding natural small molecule drugs that regulate muscle fiber types and transformation, supplementing this natural small molecule drug in daily diet can improve meat quality and enhance muscle function. Danshensu (DSS) is a water-soluble component extracted and separated from Danshensu, and its shown effects include coronary artery dilation and protection against myocardial ischemia-reperfusion injury. However, its effects on muscle fiber type formation and transformation and muscle function have not been reported. For this reason, the present invention proposes the effect of sodium danshensu (SDSS) on the formation and transformation of skeletal muscle fiber types. Summary of the invention

[0004] The present invention deepens the understanding of the molecular mechanism of sodium danshensu on the formation and transformation of muscle fiber types, and provides a theoretical basis for improving meat yield and meat quality in animal husbandry production and for medical treatment of muscle-related diseases.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for forming and transforming skeletal muscle fiber types by sodium danshensu, the method comprising the following steps:

[0006] S1, small molecule drugs;

[0007] S2, C2C12 cell culture and treatment

[0008] C2C12 myoblasts (ATCC) were cultured in high-glucose DMEM medium in 10% fetal bovine serum (FBS) at 37°C and 5% CO Cells were seeded in DMEM containing 10% FBS, and when cells reached 80% confluence, they were induced to differentiate for 4 days by adding 2% horse serum;

[0009] S3. Mouse treatment

[0010] A total of 30 4-week-old male C57BL / 6J mice were obtained from the Model Animal Research Center of Nanjing University. Three to four mice were housed in each cage with a 12-h light / dark cycle and a temperature of 23±2°C. Water and food containing 10% kcal fat were freely available. After 7 days of adaptation, the mice were randomly divided into three groups with 10 replicates in each group. The mice were orally administered with 0 mg / kg, 5 mg / kg, and 10 mg / kg of sodium tanshinone for 8 weeks. Sodium tanshinone was dissolved in normal saline, and normal saline was used as a control. The mice were euthanized by cervical dislocation, and the tibialis anterior (TA), gastrocnemius (Gas), and quadriceps femoris (Qua) ​​muscles of the mice were collected and weighed, and the data were normalized to body weight (mg / g).

[0011] S4. Measurement of grip strength and exercise endurance

[0012] Mice were orally administered 0mg / kg, 5mg / kg and 10mg / kg of sodium tanshinone for 8 weeks. The grip strength of mice was tested using a grid strength meter. The mice were gently placed on the grip test grid, with the lever of the grip tester and the body of the mice in a vertical direction. After the test value was set to zero, the mice were quickly pulled back and the maximum grip strength of the mice was recorded. The test was performed five times, and the maximum strength was determined. The treadmill running test was performed on an XP-PT-10B animal treadmill.

[0013] S5. Assessment of oxygen consumption

[0014] Mice were orally administered 0mg / kg, 5mg / kg, and 10mg / kg of sodium danshensu for 8 weeks, and O2 consumption was detected using the TSE laboratory main system. Before the measurement, the mice were allowed to adapt for 24 hours, and the amount of oxygen consumed within 24 hours was measured;

[0015] S6. RNA extraction and real-time fluorescence quantitative PCR (qPCR)

[0016] Total RNA was isolated from cells or muscle tissue using Trizol reagent, RNA concentration was measured on NanoDrop 2000, and the integrity of RNA was checked by agarose gel electrophoresis. Total RNA (1 μg) was reverse transcribed using PrimeScript RT reagent;

[0017] S7, Western blot

[0018] According to the manufacturer's instructions, cells and tissues were lysed in RIPA buffer containing protease and phosphatase inhibitors, and protein lysates were heated at 95°C for 5 min in 5× sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDS-PAGE (30 μg per lane). Then, the gel was transferred to a PVDF membrane using a Mini Trans Blot Cell system, and the membrane was blocked with 5% skim milk for 1.5 h. The primary antibody was incubated at 4°C overnight, and the membrane was washed the next day and incubated with the secondary antibody at room temperature for 1 h. The membrane was visualized by ECL. The primary antibodies were MyHC1, MyHC2a, MyHC2b, MyHC 2x, PKM1, and β-actin, followed by IgG-HRP-conjugated antibodies. The bands on the membrane were observed by ECL;

[0019] S8. Immunofluorescence staining

[0020] The primary antibodies used in immunofluorescence staining were MyHC2a, MyHC2b, and MyHC1, and the secondary antibodies were anti-mouse FITC, anti-rabbit FITC. After the secondary antibody step, cell nuclei were stained with DAPI during incubation in the dark, and images were captured by confocal laser scanning microscopy.

[0021] Preferably, in step S2, sodium tanshinone is dissolved in PBS, the cells are treated with 20 μM sodium tanshinone, and PBS is used as a control.

[0022] Preferably, in step S3, all animal experiments are performed in accordance with the protocol approved by the Animal Welfare Committee of Zhoukou Normal University (license number ZKNU-2024009, license date March 5, 2022).

[0023] Preferably, in step S4, the awake mouse is placed on a treadmill with an initial speed of 10 m / min for 3 minutes, after which the speed is increased to 30 m / min to induce fatigue, and when the mouse can no longer stay on the treadmill, the total running distance up to this point is recorded.

[0024] Preferably, in step S5, for all experiments, mice were housed at 25°C with a 12-hour light / 12-hour dark cycle, with free access to food and water.

[0025] Preferably, in step S6, qPCR is performed using a LightCycler 96 system, and relative gene expression is analyzed using a Ct (2-ΔΔCt) method.

[0026] Preferably, in step S8, the protein expression levels of MyHC2a, MyHC2b and MyHC1 are determined by the ratio of the number of cell nuclei to the total number of nuclei in MyHC subtype-positive myotubes.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. Using mouse myoblast C2C12 and C57BL mice as models, the molecular mechanism of tanshinone sodium regulating the formation and transformation of muscle fiber types was explored at the cellular and individual levels. Male mice were gavaged with tanshinone sodium, and grip strength, running, Western blot, qPCR and immunofluorescence staining were measured. It was found that the muscle mass, percentage of slow oxidative muscle fibers, muscle endurance, oxygen consumption and MyHC1 and MyHC2a gene expressions of mice treated with tanshinone sodium increased, while the expression of MyHC2b gene and the percentage of glycolytic muscle fibers decreased.

[0029] 2. The experimental results show that Danshensu sodium promotes the transformation of muscle fibers from glycolytic muscle fibers to oxidative muscle fibers by regulating the expression of muscle fiber type-related genes;

[0030] 3. The present invention deepens the understanding of the molecular mechanism of sodium danshensu on the formation and transformation of muscle fiber types, and provides a theoretical basis for improving pork production and pork quality in animal husbandry and for the treatment of muscle-related diseases in medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a graph showing the expression of myofiber-related genes in C2C12 myoblasts of the present invention;

[0032] Figure 2 This is a graph showing the muscle mass and endurance test of mice of the present invention;

[0033] Figure 3 This is a detection diagram of the mouse skeletal muscle fiber type-related genes of the present invention;

[0034] Figure 4 This is a diagram of the mouse metabolism detection of the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] The present invention provides a method for forming and transforming skeletal muscle fiber types using sodium danshensu, the method comprising the following steps:

[0037] S1, small molecule drugs;

[0038] S2, C2C12 cell culture and treatment

[0039] C2C12 myoblasts (ATCC) were cultured in high-glucose DMEM medium in 10% fetal bovine serum (FBS) at 37°C and 5% CO Cells were seeded in DMEM containing 10% FBS, and when cells reached 80% confluence, they were induced to differentiate for 4 days by adding 2% horse serum;

[0040] S3. Mouse treatment

[0041] A total of 30 4-week-old male C57BL / 6J mice were obtained from the Model Animal Research Center of Nanjing University. Three to four mice were housed in each cage with a 12-h light / dark cycle and a temperature of 23±2°C. Water and food containing 10% kcal fat were freely available. After 7 days of adaptation, the mice were randomly divided into three groups with 10 replicates in each group. The mice were orally administered with 0 mg / kg, 5 mg / kg, and 10 mg / kg of sodium tanshinone for 8 weeks. Sodium tanshinone was dissolved in normal saline, and normal saline was used as a control. The mice were euthanized by cervical dislocation, and the tibialis anterior (TA), gastrocnemius (Gas), and quadriceps femoris (Qua) ​​muscles of the mice were collected and weighed, and the data were normalized to body weight (mg / g).

[0042] S4. Measurement of grip strength and exercise endurance

[0043] Mice were orally administered 0mg / kg, 5mg / kg, and 10mg / kg of sodium tanshinone for 8 weeks. The grip strength of mice was tested using a grid strength meter. The mice were gently placed on the grip test grid, with the lever of the grip tester and the body of the mice in a vertical direction. After the test value was set to zero, the mice were quickly pulled back and the maximum grip strength of the mice was recorded. The test was performed five times, and the maximum strength was determined. The treadmill running test was performed on an XP-PT-10B animal treadmill.

[0044] S5. Assessment of oxygen consumption

[0045] Mice were orally administered 0mg / kg, 5mg / kg, and 10mg / kg of sodium danshensu for 8 weeks, and O2 consumption was detected using the TSE laboratory main system. Before the measurement, the mice were allowed to adapt for 24 hours, and the amount of oxygen consumed within 24 hours was measured;

[0046] S6. RNA extraction and real-time fluorescence quantitative PCR (qPCR)

[0047] Total RNA was isolated from cells or muscle tissue using Trizol reagent, RNA concentration was measured on NanoDrop 2000, and the integrity of RNA was checked by agarose gel electrophoresis. Total RNA (1 μg) was reverse transcribed using PrimeScript RT reagent;

[0048] S7, Western blot

[0049] According to the manufacturer's instructions, cells and tissues were lysed in RIPA buffer containing protease and phosphatase inhibitors, and protein lysates were heated at 95°C for 5 min in 5× sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDS-PAGE (30 μg per lane). Then, the gel was transferred to a PVDF membrane using a Mini Trans Blot Cell system, and the membrane was blocked with 5% skim milk for 1.5 h. The primary antibody was incubated at 4°C overnight, and the membrane was washed the next day and incubated with the secondary antibody at room temperature for 1 h. The membrane was visualized by ECL. The primary antibodies were MyHC1, MyHC2a, MyHC2b, MyHC 2x, PKM1, and β-actin, followed by IgG-HRP-conjugated antibodies. The bands on the membrane were observed by ECL;

[0050] S8. Immunofluorescence staining

[0051] The primary antibodies used in immunofluorescence staining were MyHC2a, MyHC2b, and MyHC1. The secondary antibodies were anti-mouse FITC, anti-rabbit FITC, and after the secondary antibody step, cell nuclei were stained with DAPI during incubation in the dark, and images were captured by confocal laser scanning microscopy.

[0052] In this embodiment: in step S2, sodium tanshinone is dissolved in PBS, and cells are treated with 20 μM sodium tanshinone, and PBS is used as a control.

[0053] In this embodiment: In step S3, all animal experiments were performed in accordance with the protocol approved by the Animal Welfare Committee of Zhoukou Normal University (license number ZKNU-2024009, license date March 5, 2022).

[0054] In this embodiment: In step S4, the awake mouse is placed on a treadmill with an initial speed of 10 m / min for 3 minutes, after which the speed is increased to 30 m / min to induce fatigue, and when the mouse can no longer stay on the treadmill, the total running distance up to this point is recorded.

[0055] In this embodiment: In step S5, for all experiments, mice were housed at 25°C with a 12-hour light / 12-hour dark cycle, with free access to food and water.

[0056] In this embodiment: in step S6, qPCR was performed using the LightCycler 96 system, and the Ct (2-ΔΔCt) method was used to analyze relative gene expression.

[0057] In this embodiment: in step S8, the protein expression levels of MyHC2a, MyHC2b and MyHC1 are determined by the ratio of the number of cell nuclei to the total number of nuclei in the MyHC subtype-positive myotubes.

[0058] 2. Results and Analysis

[0059] 2.1 Danshensu sodium upregulates the expression of genes related to oxidative muscle fibers

[0060] C2C12 myoblasts were treated with 20 μM sodium tanshinone, and the expression of myofiber-related genes was detected at the transcriptional level, protein level and in situ level by qPCR, WB and immunofluorescence. The qPCR results showed that compared with the control group, the expression of MyHC1, MyHC2a and PGC1α mRNA increased significantly, the levels of MyHC2b and Tnni2 mRNA decreased significantly, and the expression of MyHC2x and Tnni1 did not change significantly ( Figure 1 A). Western blot and immunofluorescence results showed that 20 μM sodium tanshinone significantly increased the expression of MyHC1 and MyHC2a proteins, and decreased the expression of MyHC2b protein ( Figure 1 BD). These research results show that sodium tanshinone can up-regulate the expression of genes related to oxidative muscle fibers and down-regulate the expression of genes related to glycolytic muscle fibers.

[0061] 2.2 Danshensu sodium enhances muscle mass and muscle endurance

[0062] In order to study the effect of sodium tanshinone on skeletal muscle function, we measured the muscle weight, exercise capacity and grip strength of mice, and treated the mice with 5mg / kg and 10mg / kg sodium tanshinone by gavage for 8 weeks (the control group was treated with normal saline). The experimental results showed that compared with the control group, the body weight of the two groups of mice treated with sodium tanshinone (5mg / kg and 10mg / kg) did not change significantly ( Figure 2 A), the weight of subcutaneous fat and inguinal fat tissue of mice was significantly reduced ( Figure 2 B and C), while the weight of gastrocnemius (Gas) and tibialis anterior (TA) muscles increased significantly ( Figure 2 D and E), there was no significant change in the weight of the quadriceps (Qua) ​​( Figure 2 F). The results of the measurement of mouse motor ability and grip strength showed that there was no significant difference in grip strength among the three groups ( Figure 2 G). The results of the mouse running experiment showed that compared with the control group, the 10mg / kg sodium tanshinone treatment group ran a longer distance before exhaustion ( Figure 2 H). The lactic acid content in the muscles was then measured, and the results showed that compared with the control group, the lactic acid content in the muscles of mice treated with sodium danshensu was significantly reduced ( Figure 2 I). The above research results show that Danshensu sodium can increase muscle weight, reduce muscle fatigue, and enhance muscle endurance. Figure 2 These are graphs showing muscle mass and endurance testing of mice. Notes: A is weight of mice; B is weight of subcutaneous fat (sWAT) of mice; C is weight of inguinal fat (iWAT) of mice; D is weight of gastrocnemius muscle (Gas) of mice; E is weight of tibialis anterior muscle (TA) of mice; F is weight of quadriceps femoris muscle (Qua) ​​of mice; G is grip strength testing of mice; H is endurance testing of mice; I is muscle lactate content of mice.

[0063] 2.3 Danshensu sodium regulates the transformation of skeletal muscle fiber types in vivo

[0064] In order to determine the effect of sodium tanshinone on the transformation of mouse muscle fiber types, we performed immunofluorescence staining on the gastrocnemius muscle. The percentage of slow oxidative muscle fibers increased in the 5mg / kg and 10mg / kg sodium tanshinone treatment groups, while the percentage of fast glycolytic muscle fibers decreased significantly ( Figure 3 ). Western blot results showed that compared with the control group, the expression levels of MyHC1 and MyHC2a proteins in the mice treated with sodium tanshinone significantly increased, while the expression level of MyHC2b protein significantly decreased ( Figure 3 ). The Western blot results were further confirmed by immunofluorescence detection and qPCR detection ( Figure 3 ). These results indicate that sodium tanshinone promotes the transformation of muscle fiber type from fast glycolytic type to slow oxidative type.

[0065] 2.4 Danshensu sodium promotes skeletal muscle oxidative metabolism

[0066] In order to explore the effect of sodium tanshinone on skeletal muscle metabolic capacity, the oxygen consumption and respiratory entropy (RQ) of mice were measured. Changes in skeletal muscle fiber type lead to changes in metabolic function. Oxidative muscle fibers are rich in mitochondria, have high oxidative capacity, and require more oxygen. The oxygen consumption of mice in the control and experimental groups was measured in metabolic cages. The results showed that in the 12-hour light / dark cycle, the total O 2 Consumption increased significantly ( Figure 4 A), respiratory entropy (RQ) decreased only during the 12-h dark cycle ( Figure 4 B). These results indicate that sodium tanshinone increased the energy expenditure of mice. Figure 4 These are metabolic test diagrams for mice. Note: (A) Oxygen consumption curve; (B) Respiratory entropy (RQ) curve.

[0067] The above results show that sodium danshensu can increase the muscle mass of mice, increase the proportion of slow oxidative muscle fibers in the muscles, and increase muscle endurance and oxygen consumption of mice. At the molecular level, sodium danshensu promotes the expression of genes related to slow oxidative muscle fibers and inhibits the expression of genes related to glycolytic muscle fibers. The present invention deepens the understanding of the molecular mechanism of sodium danshensu regulating the formation and transformation of muscle fiber types, and provides a theoretical basis for improving meat production and meat quality in animal husbandry production and for the treatment of muscle-related diseases in medicine.

[0068] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0069] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for forming and transforming skeletal muscle fiber types using sodium danshensu, characterized in that: The method comprises the following steps: S1, small molecule drugs; S2, C2C12 cell culture and treatment C2C12 myoblasts (ATCC) were cultured in high-glucose DMEM medium in 10% fetal bovine serum (FBS) at 37°C and 5% CO Cells were seeded in DMEM containing 10% FBS, and when cells reached 80% confluence, they were induced to differentiate for 4 days by adding 2% horse serum; S3. Mouse treatment A total of 30 4-week-old male C57BL / 6J mice were obtained from the Model Animal Research Center of Nanjing University. Three to four mice were housed in each cage with a 12-h light / dark cycle and a temperature of 23±2°C. Water and food containing 10% kcal fat were freely available. After 7 days of adaptation, the mice were randomly divided into three groups with 10 replicates in each group. The mice were orally administered with 0 mg / kg, 5 mg / kg, and 10 mg / kg of sodium tanshinone for 8 weeks. Sodium tanshinone was dissolved in normal saline, and normal saline was used as a control. The mice were euthanized by cervical dislocation, and the tibialis anterior (TA), gastrocnemius (Gas), and quadriceps femoris (Qua) ​​muscles of the mice were collected and weighed, and the data were normalized to body weight (mg / g). S4. Measurement of grip strength and exercise endurance Mice were orally administered 0mg / kg, 5mg / kg and 10mg / kg of sodium tanshinone for 8 weeks. The grip strength of mice was tested using a grid strength meter. The mice were gently placed on the grip test grid, with the lever of the grip tester and the body of the mice in a vertical direction. After the test value was set to zero, the mice were quickly pulled back and the maximum grip strength of the mice was recorded. The test was performed five times, and the maximum strength was determined. The treadmill running test was performed on an XP-PT-10B animal treadmill. S5. Assessment of oxygen consumption Mice were orally administered 0mg / kg, 5mg / kg, and 10mg / kg of sodium danshensu for 8 weeks, and O2 consumption was detected using the TSE laboratory main system. Before the measurement, the mice were allowed to adapt for 24 hours, and the amount of oxygen consumed within 24 hours was measured; S6. RNA extraction and real-time fluorescence quantitative PCR (qPCR) Total RNA was isolated from cells or muscle tissue using Trizol reagent, RNA concentration was measured on NanoDrop 2000, and the integrity of RNA was checked by agarose gel electrophoresis. Total RNA (1 μg) was reverse transcribed using PrimeScript RT reagent; S7, Western blot According to the manufacturer's instructions, cells and tissues were lysed in RIPA buffer containing protease and phosphatase inhibitors, and protein lysates were heated at 95°C for 5 min in 5× sodium dodecyl sulfate (SDS) sample buffer and separated by 10% SDS-PAGE (30 μg per lane). Then, the gel was transferred to a PVDF membrane using a Mini Trans Blot Cell system, and the membrane was blocked with 5% skim milk for 1.5 h. The primary antibody was incubated at 4°C overnight, and the membrane was washed the next day and incubated with the secondary antibody at room temperature for 1 h. The membrane was visualized by ECL. The primary antibodies were MyHC1, MyHC2a, MyHC2b, MyHC 2x, PKM1, and β-actin, followed by IgG-HRP-conjugated antibodies. The bands on the membrane were observed by ECL; S8. Immunofluorescence staining The primary antibodies used in immunofluorescence staining were MyHC2a, MyHC2b, and MyHC1, and the secondary antibodies were anti-mouse FITC, anti-rabbit FITC. After the secondary antibody step, cell nuclei were stained with DAPI during incubation in the dark, and images were captured by confocal laser scanning microscopy.

2. The method for forming and transforming skeletal muscle fiber types by sodium danshensu according to claim 1, characterized in that: In step S2, sodium tanshinone was dissolved in PBS, and cells were treated with 20 μM sodium tanshinone, and PBS was used as a control.

3. The method for forming and transforming skeletal muscle fiber types by sodium danshensu according to claim 1, characterized in that: In step S3, all animal experiments were performed in accordance with the protocol approved by the Animal Welfare Committee of Zhoukou Normal University (license number ZKNU-2024009, license date March 5, 2022).

4. The method for forming and transforming skeletal muscle fiber types by sodium danshensu according to claim 1, characterized in that: In step S4, the awake mouse is placed on a treadmill with an initial speed of 10 m / min for 3 minutes, after which the speed is increased to 30 m / min to induce fatigue, and when the mouse can no longer stay on the treadmill, the total running distance up to this point is recorded.

5. The method for forming and transforming skeletal muscle fiber types by sodium danshensu according to claim 1, characterized in that: In step S5, for all experiments, mice were housed at 25°C with a 12-hour light / 12-hour dark cycle with free access to food and water.

6. The method for forming and transforming skeletal muscle fiber types using sodium danshensu according to claim 1, characterized in that: In step S6, qPCR was performed using the LightCycler 96 system, and the Ct (2-ΔΔCt) method was used to analyze relative gene expression.

7. The method for forming and transforming skeletal muscle fiber types using sodium danshensu according to claim 1, characterized in that: In step S8, the protein expression levels of MyHC2a, MyHC2b and MyHC1 are determined by the ratio of the number of cell nuclei to the total number of nuclei in the MyHC subtype-positive myotubes.

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