Application of L-lactic acid in preparation of medicine for promoting muscle injury repair and / or regeneration

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of L-lactic acid in preparation of a medicine for promoting muscle injury repair and / or regeneration. The L-lactic acid can significantly promote proliferation and differentiation of muscle satellite cells, and helps to improve some external stimulation factors, such as myasthenia, amyotrophy, poor muscle coordination and other skeletal muscle dysfunction diseases caused by aging, injury, disuse or some degenerative diseases, such as poor prognosis. The invention provides a new diagnosis and treatment strategy and an effective molecular treatment target for treatment and prognosis of skeletal muscle dysfunction diseases. And on the other hand, the L-lactic acid provided by the invention is a small molecule metabolite, is relatively low in economic cost and relatively good in treatment effect, and has a considerable clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the use of L-lactic acid in the preparation of a drug for promoting muscle injury repair and / or regeneration. Background Art

[0002] Skeletal muscle is a highly representative tissue in mammals, accounting for approximately 55% of an individual's body weight. See Schiaffino S, Reggiani C (2011) "Fiber types in mammalian skeletal muscles" Physiol Rev 91:1447-1531. It plays an extremely important role in aspects such as movement, posture maintenance, and energy metabolism. See Yadav, A. et al. (2021) "Magnoflorine prevent the skeletal muscle atrophy via Akt / mTOR / FoxO signal pathway and increase slow-MyHC production in streptozotocin-induced diabetic rats." J. Ethnopharmacol 2021, 267, 113510. Its main characteristic is the contraction of muscle fibers to generate relative acting forces to achieve body movement and stability. Muscle fibers have high adaptability and regenerative capacity. See Guilhot C et al. (2024) "The satellite cell in skeletal muscle: A story of heterogeneity." Curr Top Dev Biol. 2024;158:15-51. This characteristic is achieved by satellite cells responsible for skeletal muscle growth, repair, and maintenance. See Seale, P. et al. (2000). "Pax7 is required for the specification of myogenic satellite cells." Cell, 102, 777-786. Satellite cells are located between the sarcolemma and the basement membrane. This group of myogenic cells is generally in the mitotic quiescent phase, but when the body is induced by external stimuli such as injury, satellite cells will be activated (i.e., enter the cell cycle). See Masschelein et al. (2020). "Exercise promotes satellite cell contribution to myofibers in a load-dependent manner." Skeletal Muscle, 10(1), 21.

[0003] Skeletal muscle is mainly responsible for aspects such as the body's energy consumption, homeostasis regulation, and metabolic adaptation. See Plotkin, D.L. et al. (2021) "Muscle Fiber Type Transitions with Exercise Training: Shifting Perspectives." Sports 2021, 9, 127. It is the main site of glucose metabolism, energy utilization, and lactate production. See Tsukamoto S et al. (2018) "Lactate Promotes Myoblast Differentiation and Myotube Hypertrophy via a Pathway Involving MyoD In Vitro and Enhances Muscle Regeneration In Vivo." Int J Mol Sci. 2018 Nov 19; 19(11): 3649. The growth, development, and regulatory mechanisms of skeletal muscle are crucial for maintaining the protein pool required to resist disease conditions. See Tisdale MJ. "Cancer cachexia." Curr Opin Gastroenterol. 2009. It provides the body with a rich source of proteins needed to build immune defenses. See Mantovani G et al. (2001) "Managing cancer-related anorexia / cachexia." Drugs. 2001; 61: 499-514. And it is related to the survival rate of patients with several diseases including cancer. See Mantovani G et al. (2001) "Managing cancer-related anorexia / cachexia." Drugs. 2001; 61: 499-514. In addition, evidence from clinical trials of anabolic hormones shows that favorable changes in muscle size are accompanied by an increase in muscle strength. See Bhasin S (2005) et al. "Older men are as responsive as young men to the anabolic effects of graded doses of testosterone on the skeletal muscle." J Clin Endocrinol Metab. 2005; 90: 678-88.In the case of external stimuli such as aging, trauma, disuse, or certain degenerative diseases, see Bischoff, R. (1990) “Cell cycle commitment of rat muscle satellite cells.” The Journal of Cell Biology, 111(1), 201-207., a decrease in muscle mass and muscle strength may lead to skeletal muscle dysfunctional diseases, such as muscle atrophy, muscle weakness, poor muscle coordination, and other conditions with poor prognosis, see Manini TM, Clark BC. (2012) “Dynapenia and aging: an update.” J Gerontol A Biol Sci Med Sci. 2012 Jan;67(1):28-40.. And it is related to the occurrence and survival of chronic diseases including cancer, diabetes, and obesity, see Dutta, C. (1997) “Significance of sarcopenia in the elderly.” J. Nutrition 1997, 127, 992S-993S. and Karakelides, H.; Nair, K.S. (2005) “Sarcopenia of aging and its metabolic impact.” Curr. Top. Dev. Biol. 2005, 68, 123-148..

[0004] Lactic acid, scientifically named α-hydroxypropionic acid, with the molecular formula C2H5OCOOH, plays a role in various physiological processes. It is a naturally occurring organic acid that widely exists in humans, animals, plants, and microorganisms. Lactic acid is the smallest chiral molecule in nature. The carbon atom at the α-position of the carboxyl group in the molecule is an asymmetric carbon atom, with two configurations: L(+) and D(-). L-lactic acid is the left-handed type, and D-lactic acid is the right-handed type. L-lactic acid and D-lactic acid are chiral molecules and are optical isomers of each other. The equal proportion mixture of L-lactic acid and D-lactic acid is the racemic DL-type. Except for the optical rotation, the other physical and chemical properties of L-lactic acid and D-lactic acid are the same. The physical properties of DL-type lactic acid are slightly different, manifested in that its melting point and heat of fusion are lower than those of single D- or L-configured lactic acid.

[0005] The lactic acid provided by the present invention is L-lactic acid. L-lactic acid is an organic acid, mainly produced from corn starch through biotechnological fermentation, and is a lactic acid with high optical rotation (chirality). Due to its left-handed characteristic, it has good biocompatibility, can be compatible with mammals, can directly participate in human metabolism without any side effects, and is widely used in the fields of food, medicine, etc. The finished product is a colorless or light yellow clear viscous liquid with a slightly sour taste; it is hygroscopic, and its aqueous solution shows an acidic reaction. It is soluble in water and ethanol and insoluble in chloroform. Its chemical structural formula is shown as follows:

[0006]

[0007] L-lactic acid is the main form of lactic acid in humans and eukaryotes, and pyruvate is reduced to L-lactic acid by lactate dehydrogenase (LDH-A). L-lactic acid can be further oxidized to pyruvate in cells and then enter the mitochondria for tricarboxylic acid cycle metabolism. Although there have been studies reporting the mechanism of hypoxia-induced mitochondrial protein lactylation in muscle cells to prevent exercise-induced injury by inhibiting oxidative phosphorylation, see Mao Y et al. (2024) "Hypoxia induces mitochondrial protein lactylation to limit oxidative phosphorylation." Cell Res. 2024 Jan; 34(1): 13 - 30. However, there is very little research on the role of L-lactic acid in the development process of promoting muscle injury repair and / or regeneration.

[0008] Currently, lactic acid is considered a molecule with multiple functions such as metabolism, signal transduction, and acting as an epigenetic regulator, see Bartoloni B et al. (2024) "The Multiple Roles of Lactate in the Skeletal Muscle." Cells. 2024 Jul 10; 13(14): 1177. In skeletal muscle, lactic acid can be directly oxidized and taken up by organelles (such as mitochondria or peroxisomes), or secreted into the blood and used as an energy source by organs, see Brooks et al. (2022) "Lactate in contemporary biology: A phoenix risen. J. Physiol." 2022, 600, 1229 - 1251. As an energy source with high energy demand (such as during high-intensity exercise), lactic acid provides an effective compensatory mechanism for the regeneration of ATP, see Nalbandian et al. (2020) "Lactate Metabolism and Satellite" Cell Fate. Front. Physiol. 2020, 11, 610983.

[0009] At present, there is no effective drug treatment for muscle degenerative diseases. However, several drugs (such as testosterone, see Abellan van Kan G et al. (2009) “Carla Task Force on Sarcopenia: propositions for clinical trials.” J Nutr Health Aging. 2009; 13: 700-7.18., angiotensin-converting enzyme Bellofatto V. (2007) “Pyrimidine transport activities in trypanosomes.” Trends Parasitol. 2007; 23: 187-9. discussion 190., and myostatin inhibitors Sumukadas D, et al. (2008) “Ace inhibitors as a therapy for sarcopenia-evidence and possible mechanisms.” J Nutr Health Aging. 2008; 12: 480-5.) have the potential to improve muscle mass and contractile quality. Many of these drugs are still in the early stages of development, and the current status of diagnosis and treatment of skeletal muscle dysfunction diseases remains severe. Therefore, further research on muscle regulatory mechanisms will provide new molecular target perspectives for the development of new drugs to reduce the poor prognosis of muscle degenerative changes that occur in many diseases, reduce the occurrence of skeletal muscle dysfunction diseases caused by muscle mass loss and muscle strength weakness during the disease process, provide more effective prognostic treatment options for patients, and achieve the prerequisite for providing precise treatment for skeletal muscle dysfunction diseases. Summary of the Invention

[0010] The object of the present invention is to overcome the deficiencies in the prior art and provide an application of L-lactic acid in the preparation of a drug for promoting muscle injury repair and / or regeneration.

[0011] The technical solution adopted by the present invention to solve its technical problems is:

[0012] Application of L-lactic acid in the preparation of a drug for promoting muscle injury repair and / or regeneration.

[0013] Further, the muscle tissue is mammalian muscle tissue.

[0014] Further, the mammal is cattle or mice.

[0015] Further, the drug can improve the poor prognosis of skeletal muscle dysfunction diseases caused by aging, injury, disuse or degenerative diseases.

[0016] Furthermore, the skeletal muscle dysfunction diseases include muscle weakness, muscle atrophy, and poor muscle coordination.

[0017] Furthermore, the final concentration of L-lactic acid is 5 mmol / L.

[0018] Use of L-lactic acid in the preparation of a drug having any one or more of the following effects of promoting muscle injury repair and / or regeneration:

[0019] 1) Increasing muscle fiber area;

[0020] 2) Increasing muscle weight;

[0021] 3) Promoting the expression of myogenesis-related genes;

[0022] 4) Promoting the activation and / or proliferation of muscle stem cells;

[0023] 5) Enhancing the myogenic differentiation ability of muscle stem cells;

[0024] 6) Accelerating the signal transduction of the PI3K-AKT-mTOR signaling pathway.

[0025] The advantages and positive effects achieved by the present invention are as follows:

[0026] 1. In the present invention, L-lactic acid can significantly promote the proliferation and differentiation of satellite cells to regenerate muscle tissue and maintain muscle mass, and help improve the poor prognosis of skeletal muscle dysfunction diseases such as muscle weakness, muscle atrophy, and poor muscle coordination caused by certain external stimulating factors such as aging, injury, disuse, or certain degenerative diseases.

[0027] 2. The present invention provides a new diagnosis and treatment strategy and an effective molecular therapeutic target for the occurrence and / or maintenance, treatment, and prognosis of skeletal muscle dysfunction diseases.

[0028] 3. The present invention provides a molecular theoretical guidance for the research of lactate receptor inhibitors involved in the occurrence and / or maintenance of muscle diseases, which helps to improve the important role of lactate as a signaling molecule in pathology.

[0029] 4. The regulatory role of L-lactic acid in the process of myogenesis can be used as an evaluation index for exercise training design and nutritional supplements, providing an evaluation standard for the training tasks and rehabilitation programs of athletes and patients, and the energy supply situation of the body's energy system.

[0030] 5. Skeletal muscle is considered the main site of lactate production during muscle contraction, which is related to poor muscle oxygenation and fatigue. The present invention can provide a reference for related research.

[0031] 6. On the other hand, the L-lactic acid involved in the present invention is a small molecule metabolite, with low economic cost and good therapeutic effect, and has promising clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is the absorbance value diagram of cells after treating bovine muscle satellite cells with L-lactic acid in the present invention;

[0033] Figure 2 It is the cell survival rate diagram of cells after treating bovine muscle satellite cells with L-lactic acid in the present invention;

[0034] Figure 3 It is the influence diagram of 5 mmol / L L-lactic acid on the proliferation of bovine muscle satellite cells in the present invention; wherein, Figure 3 A is the statistical chart of qRT-PCR results of the influence of 5 mmol / L L-lactic acid on the proliferation of bovine muscle satellite cells; Figure 3 B is Figure 3 the Western blot result diagram of A; Figure 3 C is Figure 3 the bar chart of A;

[0035] Figure 4 It is the influence diagram of 5 mmol / L L-lactic acid on the proliferation of bovine muscle satellite cells in the present invention; wherein, Figure 4 A is the representative picture of the EdU positive cell count for detecting the influence of 5 mmol / L L-lactic acid on the proliferation of bovine muscle satellite cells by the EdU cell proliferation method in the present invention; Figure 4 B is the statistical result diagram of the EdU labeling index;

[0036] Figure 5 It is the light microscope picture (100X) of cells in the differentiation stage after treating bovine muscle satellite cells with 5 mmol / L L-lactic acid in the present invention;

[0037] Figure 6 It is the influence diagram of L-lactic acid on the differentiation of bovine muscle satellite cells in the present invention; wherein, Figure 6 A is the statistical chart of RT-PCR results of the influence of 5 mmol / L L-lactic acid on the differentiation of bovine muscle satellite cells; Figure 6 B is Figure 6 the Western blot result diagram of A; Figure 6 C is Figure 6 the bar chart of A;

[0038] Figure 7 It is the influence diagram of L-lactic acid on the PI3K / Akt / mTOR signaling pathway during the proliferation stage of treating bovine muscle satellite cells in the present invention; wherein, Figure 7Western blot detection of the expression of related proteins in bovine muscle satellite cells treated with 5 mmol / L L-lactic acid during the proliferation period and its effect on the PI3K / Akt / mTOR signaling pathway Figure 7 B is Figure 7 Quantitative analysis chart of A;

[0039] Figure 8 It is a diagram showing the effect of L-lactic acid on the PI3K / Akt / mTOR signaling pathway in bovine muscle satellite cells during the differentiation period in the present invention; among them, Figure 8 A is the Western blot detection of the expression of related proteins in bovine muscle satellite cells treated with 5 mmol / L L-lactic acid during the differentiation period and its effect on the PI3K / Akt / mTOR signaling pathway, Figure 8 B is Figure 8 Quantitative analysis chart of A;

[0040] Figure 9 It is a comparison diagram of the gastrocnemius muscle isolated from L-lactic acid-treated mice in the present invention; among them, Figure 9 A is the gastrocnemius muscle isolated from L-lactic acid-treated mice, with the left being the control group and the right being the L-lactic acid injection group, Figure 9 B is Figure 9 The wet weight of the gastrocnemius muscle of A;

[0041] Figure 10 It is a diagram showing the detection results of the gastrocnemius muscle of L-lactic acid-treated mice in the present invention; among them, Figure 10 A is a section of the gastrocnemius muscle of L-lactic acid-treated mice, Figure 10 B is Figure 10 The fiber diameter of A, Figure 10 C is Figure 10 The cross-sectional area of the muscle fibers of A;

[0042] Figure 11 It is a diagram showing the effect of L-lactic acid on mouse muscle development in the present invention; among them, Figure 11 A is a statistical chart of the qRT-PCR results of the effect of L-lactic acid on mouse muscle development, Figure 11 B is Figure 11 Quantitative analysis chart of A;

[0043] Figure 12 It is a diagram showing the effect of L-lactic acid on mouse muscle development in the present invention; among them, Figure 12 A is the Western blot results and quantitative analysis chart of the effect of L-lactic acid on mouse muscle development, Figure 12 B is Figure 12 Quantitative analysis chart of A;

[0044] Figure 13 It is a diagram showing the effect of L-lactic acid on the PI3K / Akt / mTOR signaling pathway in mouse skeletal muscle in the present invention; among them,Figure 13 Effect of 5 mmol / L L-lactic acid on mouse skeletal muscle on PI3K / Akt / mTOR signaling pathway. Expression levels of proteins in PI3K / Akt / mTOR signaling pathway were detected by Western blot. Figure 13 B is Figure 13 Quantitative analysis chart of A. Specific implementation manners

[0045] The present invention will be further described below in conjunction with embodiments. The following embodiments are narrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0046] All kinds of experimental operations involved in the specific embodiments are conventional techniques in the art. For parts not specifically noted herein, those of ordinary skill in the art can refer to various common reference books, scientific and technological literatures, or relevant specifications, manuals, etc. before the filing date of the present invention application for implementation.

[0047] Application of L-lactic acid in the preparation of a drug for promoting muscle injury repair and / or regeneration.

[0048] Preferably, the muscle tissue is muscle tissue of a mammal.

[0049] Preferably, the mammal is cattle or mouse.

[0050] Preferably, the drug can improve the poor prognosis of skeletal muscle dysfunction diseases caused by aging, injury, disuse or degenerative diseases.

[0051] Preferably, the skeletal muscle dysfunction diseases include muscle weakness, muscle atrophy and poor muscle coordination.

[0052] Preferably, the final concentration of the L-lactic acid is 5 mmol / L.

[0053] Application of L-lactic acid in the preparation of a drug having any one or more of the following promoting muscle injury repair and / or regeneration effects:

[0054] 1) Increasing muscle fiber area;

[0055] 2) Increasing muscle weight;

[0056] 3) Promoting the expression of myogenesis-related genes;

[0057] 4) Promoting the activation and / or proliferation of muscle stem cells;

[0058] 5) Improving the myogenic differentiation ability of muscle stem cells;

[0059] 6) Accelerating the signal transduction of PI3K-AKT-mTOR signaling pathway.

[0060] Specifically, the related preparation and detection are as follows:

[0061] Materials:

[0062] 1. Bovine muscle satellite cells were independently isolated, identified and preserved by the Tianjin Key Laboratory of Animal Breeding and Healthy Aquaculture. They are a kind of publicly available cells. Specifically, please refer to the patent: A method for promoting myogenic differentiation of bovine skeletal muscle satellite cells (Patent No.: CN201911180725.2).

[0063] 2. The complete medium contains 10% fetal bovine serum (FBS) purchased from Gibco; 100 U / ml Penicillin purchased from Gibco; 100 U / ml Streptomycin purchased from Gibco; DMEM / F12 medium (Dulbecco’s modified Eagle’s medium / F12) purchased from Gibco.

[0064] 3. The differentiation medium contains 2% horse serum (HS) purchased from Gibco; 100 U / ml Penicillin purchased from Gibco; 100 U / ml Streptomycin purchased from Gibco; DMEM / F12 medium (Dulbecco’s modified Eagle’s medium / F12) purchased from Gibco.

[0065] 4. L-lactic acid was purchased from Sigma-Aldrich.

[0066] 5. Cell Counting Kit-8 (CCK-8) was purchased from Ribobio Co., Ltd.

[0067] 6. Cell-Light TM EdU Apollo 567 In Vitro Kit was purchased from Ribobio Co., Ltd.

[0068] 7. Reverse transcription reagents were purchased from ComWin Biotech Co., Ltd.

[0069] 8. BCA Protein Concentration Assay Kit was purchased from ComWin Biotech Co., Ltd.

[0070] 9. Tissue / Cell High Efficiency RIPA Lysis Buffer (PMSF) was purchased from Solarbio.

[0071] 10. Super-Sensitive ECL Chemiluminescence Solution was purchased from Solarbio.

[0072] 11. The PBS buffer was purchased from Solarbio.

[0073] 12. The skim milk powder was purchased from Solarbio.

[0074] 13. The 4% paraformaldehyde was purchased from Solarbio.

[0075] 14. The 10× electroporation buffer was purchased from Solarbio.

[0076] 15. The 5× Tris-glycine electrophoresis buffer was purchased from Solarbio.

[0077] 16. The 20× TBS was purchased from Solarbio.

[0078] 17. The 10% PAGE gel rapid preparation kit was purchased from YaMei Biotechnology Co., Ltd.

[0079] 18. The GAPDH Antibody was purchased from Zhongshan Golden Bridge.

[0080] 19. The goat anti-mouse IgG / horseradish peroxidase conjugate was purchased from Zhongshan Golden Bridge.

[0081] 20. The goat anti-rabbit IgG / horseradish peroxidase conjugate was purchased from Zhongshan Golden Bridge.

[0082] 21. The Tubulin Antibody was purchased from Sanying Biotechnology Co., Ltd.

[0083] 22. The MyHC Antibody was purchased from Bioscience Biotechnology Co., Ltd.

[0084] 23. The MyOG Antibody (DSHB) was purchased from Bioscience Biotechnology Co., Ltd.

[0085] 24. The Pax7 Antibody was purchased from Bioscience Biotechnology Co., Ltd.

[0086] 25. The antibodies related to the PI3K / Akt / mTOR signaling pathway were purchased from Abcam (Shanghai) Trading Co., Ltd.

[0087] 26. The absolute ethanol was purchased from Solarbio.

[0088] 27. The hematoxylin staining solution was purchased from Solarbio.

[0089] 28. The xylene was purchased from Solarbio.

[0090] 29. The eosin staining solution was purchased from Solarbio.

[0091] 30. The tissue embedding cassettes were purchased from Jiangsu Shitai Company.

[0092] 31. The paraffin was purchased from Shanghai Huayong Company.

[0093] 32. The neutral balsam was purchased from Dezhou Runxin Company.

[0094] 33. C57B6 / L male mice were purchased from the National Institutes for Food and Drug Control (Daxing).

[0095] Example 1: Culture and induced differentiation of primary bovine muscle satellite cells

[0096] A cell model was constructed using the revived primary bovine muscle satellite cells.

[0097] 1. Preparation of the culture medium

[0098] (1) Preparation of the proliferation medium: 20% fetal bovine serum (FBS) was mixed evenly with 80% DMEM / F12 medium in equal proportions, and then 1% penicillin-streptomycin was added, which was the proliferation medium.

[0099] (2) Preparation of the differentiation medium: 2% horse serum (HS) was mixed evenly with 98% DMEM / F12 medium in equal proportions, and then 1% penicillin-streptomycin was added, which was the differentiation medium.

[0100] 2. Culture and induced differentiation of bovine muscle satellite cells

[0101] (1) Cell resuscitation: The cryopreserved primary bovine muscle satellite cells were taken out from the liquid nitrogen tank and quickly placed in a 37°C constant temperature water bath. Shake well until the cell solution becomes pink and jelly-like. Immediately after melting, an equal volume of pre-warmed growth medium (DMEM containing 1% penicillin-streptomycin-amphotericin triple antibiotic solution and 10% fetal bovine serum) at 37°C was added, and pipetted and mixed well. The cell-containing liquid was transferred to a centrifuge tube and centrifuged at 1000 rpm / min for 10 min. After centrifugation, the supernatant was carefully discarded, and the cell pellet was gently resuspended with the proliferation medium. The cells were evenly inoculated into the corresponding culture dishes according to the cell amount during separation and cryopreservation, and cultured in a 37°C, 5% CO2 incubator.

[0102] (2) Cell passage: When the cells reached more than 80% confluence, passage operation was carried out. Aspirate the culture medium, add an appropriate amount of 0.25% trypsin, and place it in a 37°C incubator for digestion. When most cells detached under the microscope, immediately add an equal volume of proliferation medium to neutralize, terminate digestion, and centrifuge at 1000 rpm / min for 10 min. Discard the supernatant, and transfer the cell solution to a cell culture plate for continued culture according to the above operation to construct a cell proliferation model.

[0103] (3) Cell differentiation: When the cell fusion reaches over 80%, aspirate and discard the culture medium, add pre-warmed differentiation medium (DMEM containing 2% pregnant mare serum) to induce differentiation, and construct a cell differentiation model.

[0104] Example 2: Effects of different concentrations of L-lactic acid on the viability of bovine muscle satellite cells

[0105] Screen the concentration of L-lactic acid with the maximum cell viability

[0106] 1. CCK-8 assay

[0107] (1) Cell seeding: Passage the cells into a 96-well culture plate for culture.

[0108] (2) Drug treatment: Set the concentration gradients of L-lactic acid as 0 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, and 6 mmol / L.

[0109] (3) Cell culture: After the cells adhere, discard the original culture medium, add the mixed solution of L-lactic acid medium with different concentrations to the 96-well plate, 100 μl per well, and place the 96-well plate in an incubator at 37 °C for 24 h.

[0110] (4) Absorbance measurement and result analysis: Mix the CCK-8 enhanced solution and the growth medium at a volume ratio of 1:9, prepare it immediately before use, and store it in the dark.

[0111] (5) Discard the original culture medium, add 100 μl of CCK-8 mixture to each well, shake gently, and operate in the dark. Place it in an incubator at 37 °C for 1.5 h.

[0112] (6) Set the parameters of the microplate reader and measure the absorbance at a wavelength of 450 nm. Note that there should be no bubbles in the wells.

[0113] (7) Data analysis of the CCK-8 screening concentration is performed using one-way ANOVA; cell counting is performed using Image J software; the positive cell rate of EdU is statistically analyzed using the chi-square test; the IC50 value of L-lactic acid is calculated using GraphPad software, and the results are as Figure 1 shown.

[0114] The effects of different concentrations of L-lactic acid on cell viability were detected by the CCK-8 method. The microplate reader detected the absorbance value of the cells at a wavelength of 450 nm. The cell viability is proportional to the absorbance value, as shown in Table 1.

[0115] Table 1 Absorbance values and viability of cells at 450 nm after treatment with different concentrations of L-lactic acid

[0116] L-lactic acid concentration (mmol / L) <![CDATA[D 450 value]]> Cell viability 0 0.3415±0.0265 100％ 1 0.371±0.0050 110％ 2 0.3795±0.0075 113％ 3 0.4075±0.0035* 122％ 4 0.4125±0.0035* 124％ 5 0.4550±0.0037** 138％ 6 0.389±0.0090 116％

[0117] Compared with the control group, when the concentration of L-lactic acid was 3 mmol / L, the absorbance value increased significantly, and the cell survival rate also increased significantly (P < 0.05); when the concentration of L-lactic acid was 5 mmol / L, the absorbance value and the cell survival rate increased extremely significantly (P < 0.01). The results are as Figure 2 shown.

[0118] 2. EDU experiment

[0119] (1) Cell seeding: Transfer the cells to a 96-well culture plate for culture.

[0120] (2) Drug treatment: Set the concentration gradient of L-lactic acid to 0 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, and 6 mmol / L.

[0121] (3) Cell culture: After the cells adhered, discard the original culture medium, add the mixed solution of L-lactic acid culture medium with different concentrations to the 96-well plate, 100 μl per well, and place the 96-well plate in an incubator at 37 °C for 24 h.

[0122] (4) Incubation: Discard the original culture medium, add 100 μl of EdU culture medium diluted to 50 μmol / L to each well and incubate in an incubator for 2 h. Discard the culture medium and wash twice with sterile PBS preheated at 37 °C;

[0123] (5) Fixation: Fix the cells with PBS containing 4% paraformaldehyde for 30 minutes and discard the liquid;

[0124] (6) Incubation: Add 50 μl of glycine solution with a concentration of 2 mg / mL to each well, incubate on a shaker for 10 minutes, discard the liquid, and wash twice with preheated sterile PBS;

[0125] (7) Permeabilization: Add 100 μl of permeabilizing agent (PBS containing 0.5% Triton X-100) to each well, incubate on a shaker for 10 minutes, discard the waste liquid, and wash twice with sterile PBS;

[0126] (8) Apollo staining: Perform Apollo staining under light-proof conditions. After 30 minutes, discard the liquid, incubate with the permeabilizing agent 2 - 3 times, and wash with PBS;

[0127] (9) DNA staining: Perform DNA staining under light-proof conditions. After 30 minutes, discard the liquid and wash with PBS.

[0128] Finally, randomly select 3 fields of view in each well, take pictures with a fluorescence inverted microscope, perform cell counting, and calculate the EdU positive cell rate. Compared with the control group, the expression level of Pax7 in the L-lactic acid group at the RNA level increased significantly (P < 0.01), and at the same time, the expression level of Ki67 also increased significantly (P < 0.05). At the protein level, the expression level of Pax7 in the L-lactic acid group increased significantly (P < 0.05), and the number of EdU positive cells increased extremely significantly (P < 0.01), indicating that L-lactic acid has a promoting effect on the proliferation of bovine muscle satellite cells. The results are as Figure 4 shown.

[0129] Example 3: Effect of L-lactic acid on the proliferation of bovine muscle satellite cells

[0130] Treat bovine muscle satellite cells with 0 mmol / L and 5 mmol / L L-lactic acid, and detect the changes in cell proliferation ability.

[0131] 1. RNA extraction and reverse transcription

[0132] (1) After treating bovine muscle satellite cells seeded in 12-well plates with 5 mmol / L L-lactic acid and culturing them to the proliferation and differentiation stages, discard the culture medium, wash twice with cold enzyme-free PBS buffer, and add 350 μL of RLT lysis buffer to each well for cell lysis; quickly collect the mixture into an enzyme-free Ep tube, label it, and store it briefly on ice;

[0133] (2) After all samples are collected, centrifuge and transfer them all to a DNA removal column, centrifuge at 13000 rpm for 1 minute, and retain the filtrate;

[0134] (3) Estimate the volume of the filtrate and add 70% ethanol equal to the volume of the filtrate, mix well, transfer to an RNA adsorption column, and centrifuge at 13000 rpm for 0.5 min, discard the waste liquid;

[0135] (4) Add 700 μL of deproteinization solution, let it stand for 1 minute, centrifuge at 12000 rpm / min for 0.5 min, and discard the liquid;

[0136] (5) Add 500 μL of protein wash solution, centrifuge at 12000 rpm / min for 0.5 min, discard the liquid, and repeat the above operation once.

[0137] (6) After centrifuging at 13000 rpm / min for 2 min, place the column in a new Ep tube, estimate the RNA yield, add 40 μL of enzyme-free water to the membrane at the center of the column, let it stand for 1 minute, centrifuge at 12000 rpm for 1 minute, and the filtrate is the extracted RNA.

[0138] (7) Aliquot a portion of the obtained RNA for concentration determination, and calculate the amount of RNA template based on the RNA concentration. Prepare the reverse transcription system according to Table 2:

[0139] Table 2

[0140]

[0141]

[0142] (8) Place the prepared system into a PCR instrument, set the program to 42°C for 15 min, 85°C for 5 min and run it. The obtained product is cDNA.

[0143] 2. Real-time fluorescence quantitative PCR

[0144] (1) Prepare the real-time fluorescence quantitative PCR reaction system according to Table 3.

[0145] Table 3 Real-time fluorescence quantitative PCR reaction system

[0146] Reagent Volume (μL) Forward Primer 0.5 Reverse Primer 0.5 cDNA 2 Mix 10 RNase free water 7

[0147] (2) React the prepared system according to the program shown in Table 4.

[0148] Table 4 Real-time fluorescence quantitative PCR reaction program

[0149]

[0150] (3) Information on partial primer sequences is shown in Table 5.

[0151] Table 5 Information on real-time fluorescence quantitative PCR primer sequences

[0152]

[0153]

[0154] 3. Protein extraction, concentration determination and denaturation treatment

[0155] (1) Treat bovine muscle satellite cells seeded in a 6-well cell culture plate with 5 mmol / L L-lactic acid.

[0156] (2) Prepare a protein lysate containing a protease inhibitor in advance when harvesting proteins. Discard the culture medium, wash twice with RNase Free PBS, and discard the liquid.

[0157] (3) Add 200 μL of lysate to each well and lyse at 4°C for 10 min. Collect the mixture into an RNase Free tube and label it.

[0158] (4) Set the speed of the low-temperature centrifuge to 12,000 rpm and centrifuge for 10 min. Carefully aspirate the supernatant, which is the required protein.

[0159] (5) Use the BCA method to measure the protein concentration.

[0160] (6) Add 100 μL of the standard product to well A of the 96-well plate, add 100 μL of the standard product and 100 μL of PBS to well B, and also add 100 μL of PBS to wells C, D, E, F, and G.

[0161] (7) Thoroughly pipette and mix the mixture in well B, aspirate 100 μL into well C, continue to pipette and aspirate, aspirate 100 μL of the mixture into well D, and add successively to well F. Aspirate 100 μL and discard it, so that the standard product concentrations from well A to well G are 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0 mmol / L in sequence. Then, transfer each concentration of the standard product to 3 wells respectively, 25 μL per well.

[0162] (8) Prepare the BCA working solution by mixing solution A and solution B in a volume ratio of 50:1. The total volume of the BCA working solution = (7 standards × 3 + number of samples × 2) × 200 μL.

[0163] (9) Add 200 μL of the working solution to each well, wrap it with tin foil to avoid light, incubate in a 37°C incubator for half an hour, preheat the microplate reader, measure the OD562 value, plot the standard curve, and calculate the sample concentration.

[0164] (10) Mix the protein sample, 4× buffer, and 1× buffer, then boil at 100°C for 10 min to denature the protein, and store it at -20°C.

[0165] 4. Western blot experiment

[0166] (1) SDS PAGE Electrophoresis: First, clean the long and short glass plates with clear water and let them dry. Align the long and short glass plates and clamp them with a splint, then fix the splint on the rack. Add an appropriate amount of absolute ethanol between the glass plates and check for liquid leakage between the glass plates and the rack. If there is leakage, remove the glass plates, clean and dry them again, repeat the previous steps, and check for leakage again until there is no liquid leakage. Then discard the absolute ethanol, prepare the lower-layer gel solution and the lower-layer gel buffer in a volume ratio of 1:1, mix them well by pipetting, add 4 mL of the lower-layer gel to the middle of the clamped glass plates, quickly add absolute ethanol to flatten it, and let it stand for 15 min. Wait for the gel to solidify, pour out the ethanol, and let it stand for 1 - 2 min to allow the ethanol to evaporate. Prepare the upper-layer gel according to the volume ratio of upper-layer gel solution: upper-layer gel buffer = 1:1, fill it up with a pipette, insert a comb, and let it stand for half an hour. Wait for the gel to solidify, take out the glass plates and place them in the electrophoresis tank, pour electrophoresis buffer into the electrophoresis tank to exceed two-thirds of its volume, perform electrophoresis at 80 V for 30 min, and then change to 120 V for 45 min.

[0167] (2) Transfer: After electrophoresis, take out the gel, cut off the excess gel, cut the filter paper and PVDF membrane according to the size of the remaining gel, and activate the PVDF membrane with methanol for standby. Note that the size of the PVDF membrane is slightly larger than that of the filter paper, and the size of the filter paper is slightly larger than that of the gel. Clamp them in the order of sponge - filter paper - gel - PVDF membrane - filter paper - sponge. Fix it in the electrotransfer tank, add more than two-thirds of the electrotransfer buffer, put an ice pack to keep the electrotransfer buffer at a lower temperature, and adjust the power supply to 300 mA for electrotransfer for 2 h.

[0168] (3) Blocking: Dissolve non-fat milk powder with TBST (35 mL TBS + 1.65 mL 20% Tween 20 + 665 mL ultrapure water) to prepare a blocking solution. After electrotransfer, put the PVDF membrane into the blocking solution and block it on a constant-temperature shaker for 2 hours. After blocking, wash it 3 times with TBST, 10 minutes each time.

[0169] (4) Incubate with Primary Antibody: Cut out the desired band according to the marker, put it into an antibody incubation bag, pour in enough primary antibody to remove air bubbles, and incubate it at 4℃ overnight.

[0170] (5) Incubate with Secondary Antibody: Take out the band, wash it 3 times with TBST, 10 minutes each time. Dilute the corresponding secondary antibody with TBST, add 6 mL of the secondary antibody to each band, and incubate it on a shaker for 1 h.

[0171] (6) Exposure: Wash it 3 times with TBST on a shaker, 10 min each time, to wash away the excess secondary antibody. Prepare the enhanced chemiluminescence solution in a volume ratio of 1:1, image it with a protein exposure imager and save it.

[0172] (7) The results of qRT-PCR and Western blot were analyzed using the t-test; among them, significant differences (P < 0.05) were indicated by "*", extremely significant differences (P < 0.01) were indicated by "**", and non-significant differences (P > 0.05) were indicated by "N.S.", and the results were as Figure 3 shown in A, 3B, and 3C.

[0173] Cells were treated with 5 mmol / L L-lactic acid, and RNA and proteins in the proliferative phase were harvested after 24 hours. The expression levels of the proliferation marker factors Pax7 and Ki67 were detected by real-time fluorescence quantitative PCR (qRT-PCR) respectively, and the expression level of the proliferation marker factor Pax7 was detected by Western blot. According to Figure 3 and Figure 4 the results, compared with the control group, the expression level of Pax7 in the L-lactic acid group was significantly increased at the RNA level (P < 0.01), and at the same time, the expression level of Ki67 was also significantly increased (P < 0.05). At the protein level, the expression level of Pax7 in the L-lactic acid group was significantly increased (P < 0.05), and the number of EdU-positive cells was extremely significantly up-regulated (P < 0.01), indicating that L-lactic acid has a promoting effect on the proliferation of bovine satellite cells, and the results were as Figure 4 shown in A, 4B.

[0174] Example 4: Effect of L-lactic acid on the differentiation of bovine satellite cells

[0175] Bovine satellite cells were treated with 0 mmol / L and 5 mmol / L L-lactic acid, and the changes in cell differentiation ability were detected.

[0176] (1) Bovine satellite cells were treated with 5 mmol / L L-lactic acid and induced to differentiate. RNA and proteins on the third day of differentiation were harvested, and the expression levels of the differentiation marker factors MyOD, MyOG, and MyHC were detected by real-time fluorescence quantitative PCR and Western blot respectively. The cell morphology was as Figure 5 shown.

[0177] Compared with the control group, there were no significant changes in the mRNA expression levels of MyOD, MyOG, and MyHC (P > 0.05); there was no significant change in the protein expression level of MyOG (P > 0.05), and the expression level of MyHC was extremely significantly up-regulated (P < 0.01). In summary, it was shown that L-lactic acid promoted the differentiation of bovine satellite cells, and the results were as Figure 6 shown in A, 6B, and 6C.

[0178] Example 5: Effect of L-lactic acid on the PI3K / Akt / mTOR signaling pathway in bovine satellite cells during the proliferative phase

[0179] Treat bovine muscle satellite cells with 0 mmol / L and 5 mmol / L L-lactic acid, and detect the effect of cells on the PI3K / Akt / mTOR signaling pathway during the proliferation period.

[0180] (1) Treat the cells with 5 mmol / L L-lactic acid, and collect the proteins in the proliferation period after 24 hours for Western blot to detect the expression levels of proteins related to the PI3K / Akt / mTOR signaling pathway.

[0181] Compared with the control group, after treatment with 5 mmol / L L-lactic acid, the phosphorylation level of mTOR was extremely significantly up-regulated (P<0.01), the phosphorylation level of PI3K was significantly up-regulated (P<0.05), while there was no significant difference in the expression levels of Akt, p-Akt, mTOR, and PI3K (P>0.05), indicating that lactic acid promotes cell proliferation by activating the PI3K / Akt / mTOR signaling pathway. The results are as Figure 7 shown in Figures A and 7B.

[0182] Example 6: Effect of L-lactic acid on the PI3K / Akt / mTOR signaling pathway in bovine muscle satellite cells during the differentiation period

[0183] Treat bovine muscle satellite cells with 0 mmol / L and 5 mmol / L L-lactic acid, and detect the effect of cells on the PI3K / Akt / mTOR signaling pathway during the differentiation period.

[0184] (1) Treat the cells with 5 mmol / L L-lactic acid, and collect the proteins in the differentiation period after three days for Western blot to detect the expression levels of proteins related to the PI3K / Akt / mTOR pathway.

[0185] Compared with the control group, after treatment with L-lactic acid, there was no significant change in the relative protein expression levels of Akt, mTOR, and PI3K (P>0.05), and the phosphorylation levels of Akt, mTOR, and PI3K were all significantly down-regulated (P<0.05), indicating that lactic acid promotes the differentiation of satellite cells by inhibiting the activation of the PI3K / Akt / mTOR signaling pathway. The results are as Figure 8 shown in Figures A and 8B.

[0186] Example 7: Construction of an L-lactic acid mouse model

[0187] Construct an L-lactic acid mouse model using C57BL / 6 male mice.

[0188] 1. Experimental design

[0189] (1) Adaptively raise 7 mice for a period of time to avoid stress behavior in mice that may affect the experimental results.

[0190] (2) By referring to relevant literature and based on the body weight of mice, the skeletal muscles of mice were injected with L-lactic acid diluent at a dose of 20 mg / kg to simulate the generation of lactic acid under physiological conditions.

[0191] (3) The left and right leg skeletal muscles of each mouse were used as a control group. Among them, the right leg was defined as the L-lactic acid group, and the gastrocnemius muscle was injected with L-lactic acid diluent every two days during the experiment; the left leg was the control group, and an equal amount of normal saline was injected each time.

[0192] (4) Weigh the mice before each injection to monitor their growth. After two weeks, the mice were sacrificed by cervical dislocation, and the gastrocnemius muscles were separated for standby.

[0193] 2. Reagent preparation

[0194] (1) Weigh 0.02 g of L-lactic acid and dissolve it in 100 mL of normal saline to prepare a 0.2 mg / mL L-lactic acid diluent, which is stored at 4 °C for standby.

[0195] (2) 70% alcohol: Add 30 mL of pure water to 70 mL of absolute ethanol to prepare 100 mL of 70% alcohol.

[0196] (3) 80% alcohol: Add 20 mL of pure water to 80 mL of absolute ethanol to prepare 100 mL of 80% alcohol.

[0197] (4) 90% alcohol: Add 10 mL of pure water to 90 mL of absolute ethanol to prepare 100 mL of 90% alcohol.

[0198] (5) 95% alcohol: Add 5 mL of pure water to 95 mL of absolute ethanol to prepare 100 mL of 95% alcohol.

[0199] (6) 1% hydrochloric acid alcohol: Add 1 mL of concentrated hydrochloric acid to 99 mL of 75% alcohol.

[0200] 2. Sampling of muscle specimens

[0201] (1) After the mice completed all injections, they were sacrificed by cervical dislocation. The gastrocnemius muscles of the control group and the L-lactic acid group were separated, weighed, and recorded respectively.

[0202] (2) Divide each muscle into three equal parts, which are used to extract tissue RNA, protein, and make tissue sections respectively. The samples used to extract RNA and protein are stored in a liquid nitrogen tank, and the samples used to make tissue sections are immersed in 4% paraformaldehyde and stored in the dark in a cool place.

[0203] 3. Preparation of tissue sections

[0204] (1) Prepare the samples: Take out the gastrocnemius muscle from 4% paraformaldehyde, blot the residual liquid, cut it into pieces about 5 mm thick, and put them into a tissue embedding cassette;

[0205] (2) Dealdehyde removal: Rinse the tissue embedding cassette under running water for at least 6 hours;

[0206] (3) Dehydration: Immerse the tissue in ethanol solutions of different concentrations for stepwise dehydration, with each level of ethanol dehydration for 40 minutes;

[0207] (4) Clearing: Immerse in xylene Ⅰ and xylene Ⅱ sequentially for 20 minutes;

[0208] (5) Wax infiltration: Immerse in low and high melting point wax blocks sequentially for 1 hour;

[0209] (6) Embedding: Place in a metal embedding cassette and pour in melted wax;

[0210] (7) Sectioning: Fix the wax block on a microtome, with the wax block section parallel to the knife edge, and the section thickness about 4 μm;

[0211] (8) Spreading: Gently pick up the wax section with forceps and place it in a spreading box with the water temperature around 45 °C;

[0212] (9) Picking up sections: Attach the section to a glass slide and pick up the section;

[0213] (10) Baking sections: Air-dry the sections slightly and bake in an oven at 60 °C for 2 hours;

[0214] (11) Dewaxing: Immerse the sections in xylene Ⅰ and xylene Ⅱ sequentially for 3 minutes;

[0215] (12) Rehydration: Stain with 100%, 90%, 80%, 70% alcohol for 1 minute each, and finally rinse with tap water for 1.5 minutes;

[0216] (13) Staining: Place the paraffin sections in hematoxylin solution for staining for 10 minutes, rinse with tap water for half a minute, differentiate with 1% hydrochloric acid alcohol for 3 s, rinse with tap water for half a minute, blue with 1% ammonia aqueous solution for 1 minute, rinse with tap water for half a minute, place in eosin aqueous solution for staining for 1.5 minutes, and rinse with tap water for half a minute;

[0217] (14) Dehydration: Place the paraffin sections in 80%, 90%, 95% Ⅰ, 95% Ⅱ, 100% Ⅰ, 100% Ⅱ alcohol for 30 s each;

[0218] (15) Clearing: Immerse in xylene Ⅰ and xylene Ⅱ sequentially for 3 minutes;

[0219] (16) Sealing: Drop neutral balsam on the glass slide and cover with a coverslip.

[0220] 4. Total RNA extraction from tissues and reverse transcription

[0221] (1) Wrap the forceps and an appropriate amount of ceramic beads with tin foil in advance and sterilize them in an oven at 160 °C for 6 h.

[0222] (2) Turn on the tissue grinder 10 min in advance before the experiment starts, set the temperature of the grinding chamber to -40 °C for pre-cooling. Take out the muscle sample from the liquid nitrogen tank and quickly transfer it to a 1.5 mL enzyme-free centrifuge tube. Use forceps to pick up 5 - 6 ceramic beads and put them into the centrifuge tube, add an appropriate amount of lysis buffer, place it in the grinding chamber of the tissue grinder and grind at 50 Hz for 45 s, pause for 15 s, and grind several times at this frequency until there are no obvious tissue blocks in the tube.

[0223] (3) Vortex the homogenized sample and incubate it at low temperature for 5 minutes for sufficient lysis;

[0224] (4) Centrifuge at 12000 rpm at 4 °C for 10 min and take the supernatant;

[0225] (5) Add chloroform at 1 / 5 of the volume of the lysis buffer, vortex vigorously for 15 s, and incubate at room temperature for 3 min;

[0226] (6) Centrifuge at 12000 rpm at 4 °C for 10 min and aspirate the aqueous phase;

[0227] (7) Add absolute ethanol at 1 / 2 of the volume of the aqueous phase, mix well and transfer it to the adsorption column RA, centrifuge at 12000 rpm for 30 s, and discard the liquid;

[0228] (8) Aspirate 500 μL of protein removal solution and add it to the column, centrifuge at 12000 rpm for half a minute, and discard the liquid;

[0229] (9) Aspirate 500 μL of wash solution and add it to the column, centrifuge at 12000 rpm for half a minute, discard the liquid, and repeat this step once more;

[0230] (10) Centrifuge the adsorption column RA at 12000 rpm for 2 min to remove the wash solution to the greatest extent;

[0231] (11) Place the adsorption column into a new enzyme-free tube, add 60 μL of enzyme-free water to the adsorption membrane in the middle of the column, let it stand for 2 min, and centrifuge at 12000 rpm for 1 min. The obtained liquid is RNA.

[0232] 5. Real-time fluorescence quantitative PCR

[0233] Table 6 Primer sequence information for mouse real-time fluorescence quantitative PCR

[0234]

[0235] 6. Extraction of tissue protein, determination of protein concentration and denaturation treatment

[0236] (1) Wrap the forceps and an appropriate amount of ceramic beads with tin foil in advance and sterilize them in an oven at 160 °C for 6 hours.

[0237] (2) Turn on the tissue grinder 10 minutes in advance before the experiment starts, set the temperature of the grinding chamber to -40 °C for pre-cooling. Take out the muscle samples from the liquid nitrogen tank and quickly transfer them to 1.5 mL enzyme-free centrifuge tubes. Use forceps to pick up 5 - 6 ceramic beads and put them into the centrifuge tubes. Prepare an appropriate amount of lysis buffer according to the volume ratio of RIPA lysis buffer:PMSF = 100:1. Add 200 μL of lysis buffer to each tube, place it in the grinding chamber of the tissue grinder and grind at 50 Hz for 45 s, pause for 15 s, and grind several times at this frequency until there are no obvious tissue blocks in the tube.

[0238] (3) Centrifuge the homogenized samples at 12,000 rpm / min for 10 min at 4 °C, and take the supernatant, which is the extracted protein.

[0239] (4) The tissue section images were collected using the Zhiwei Image.lnk system. The muscle fiber diameter and cross-sectional area were measured using Image J. The data of gastrocnemius muscle wet weight, muscle fiber diameter and cross-sectional area were all analyzed by independent sample T-test using SPSS 18.0.

[0240] Isolate the gastrocnemius muscle of mice, weigh and record it, and make skeletal muscle tissue sections. The experimental results are shown in Figure 9 Figures A and 9B. Grind the muscle tissue to obtain tissue RNA and protein, detect the expression levels of the marker factors Pax7, Ki67, MyOD, MyOG and MyHC mRNA levels by real-time fluorescence quantitative PCR, and detect the expression levels of proteins by Western blot. The results are shown in Figure 11 、 Figure 12 Figure. The results showed that the muscle wet weight and muscle fiber diameter in the L-lactic acid group were significantly higher than those in the control group (P < 0.01), and the cross-sectional area of muscle fibers was also significantly larger than that in the control group (P < 0.01). The results are shown in Figure 10 Figures A, 10B, and 10C. Compared with the control group, the expression level of Pax7 at the RNA level was significantly increased after injecting L-lactic acid (P < 0.01), and the expression levels of Ki67, MyOD, MyOG and MyHC did not change significantly (P > 0.05). At the protein level, the expression levels of Pax7 and MyOG were significantly up-regulated (P < 0.05), and the expression level of MyHC did not change significantly (P > 0.05). In summary, L-lactic acid has a significant promoting effect on the development of mouse skeletal muscle.

[0241] Extract tissue proteins and detect the expression levels of proteins related to the PI3K / Akt / mTOR pathway by Western blot. The results are shown in Figure 13 Figure A, Figure 13As shown in Figure B. It can be seen from the figure that compared with the control group, the expression levels of PI3K, p-PI3K, Akt, and mTOR showed no significant changes (P>0.05); the expression levels of p-Akt and p-mTOR were both significantly decreased (P<0.05), indicating that L-lactic acid can regulate the development of mouse skeletal muscle by inhibiting the PI3K / Akt / mTOR signaling pathway.

[0242] As can be seen from the above embodiments, the present invention provides the application of L-lactic acid in the preparation of drugs for promoting muscle injury repair and / or regeneration. The addition of L-lactic acid has a significant promoting effect on the proliferation and differentiation of bovine muscle satellite cells, thus enabling L-lactic acid to play a key role in the clinical treatment of skeletal muscle dysfunction diseases. At the same time, it helps to improve some adverse prognostic conditions such as muscle weakness, muscle atrophy, and poor muscle coordination caused by external stimulating factors such as aging, injury, disuse, or certain degenerative diseases. And it provides new diagnosis and treatment strategies and effective molecular treatment targets for the treatment and prognosis of skeletal muscle dysfunction diseases.

[0243] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art can understand that various substitutions, changes, and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. Use of L-lactic acid in the preparation of a drug for promoting muscle injury repair and / or regeneration.

2. The application according to claim 1, wherein: The muscle tissue is mammalian muscle tissue.

3. The application according to claim 2, wherein: The mammals are cattle and mice.

4. The application according to claim 1, characterized in that: The drug can improve the poor prognosis of skeletal muscle dysfunction diseases caused by aging, injury, disuse or degenerative diseases.

5. The application according to claim 4, wherein: The skeletal muscle dysfunction diseases include muscle weakness, muscle atrophy, and poor muscle coordination.

6. The application according to any one of claims 1 to 5, characterized in that: The final concentration of the L-lactic acid is 5 mmol / L.

7. Use of L-lactic acid in the preparation of a drug having the effect of promoting muscle injury repair and / or regeneration as described in any one or more of the following 1) to 6); 1) Increasing the myofiber area; 2) Increasing muscle weight; 3) Promoting the expression of myogenesis-related genes; 4) Promoting the activation and / or proliferation of muscle stem cells; 5) Enhancing the myogenic differentiation ability of muscle stem cells; 6) Accelerating the signal transduction of the PI3K-AKT-mTOR signaling pathway.

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