Application of arginine in repairing skeletal muscle injury caused by monensin poisoning

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of arginine in repairing skeletal muscle injury caused by monensin poisoning. It is found for the first time that arginine can remarkably recover the normal function of skeletal muscle mitochondria and the normal polymerization capacity of microtubules by up-regulating the expression level of mitochondrial oxidative phosphorylation (OXPHOS) protein complex and regulating microtubule polymerization kinetics and reversing monensin-induced skeletal muscle mitochondrial dysfunction and microtubule network disintegration; the method provides a theoretical basis for clinical transformation, and has good practical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of arginine in repairing skeletal muscle damage caused by monensin poisoning. BACKGROUND

[0002] Monensin, also known as rumen, monensin, monensin, and cinnamycin, is produced by Streptomyces cinnamoneus, and is an antibiotic used for preventing and treating animal coccidiosis and improving feed conversion rate of ruminants. Because it can improve energy utilization rate, reduce protein degradation, and thus improve feed utilization rate and animal weight gain, at present, more than 100 countries and regions in the world have widely used monensin for livestock and poultry breeding. As the number of contacts continues to increase, monensin poisoning patients have occurred in factory workshops with poor working conditions and poor labor protection, mainly caused by production contact and accidental ingestion.

[0003] The main target organs of monensin poisoning are skeletal muscle and heart. The toxicity of high-concentration monensin is first due to its ability to increase the aggregation of intracellular sodium ions. The increase in sodium ion concentration can secondarily cause the increase in calcium ions in the cytoplasm, which may be related to the exchange of Na + -Ca 2+ Pump exchange causes increased outflow of calcium from the endoplasmic reticulum and mitochondria and decreased outflow to the outside of the cell; and the poisoning level of calcium activates phospholipase and proteolytic enzyme, thereby causing damage to skeletal muscle and myocardial cells. After acute rhabdomyolysis and necrosis, myoglobin enters the blood, which can cause myoglobinuria, and severe cases can block the renal tubules to cause acute renal failure and even death. In addition, monensin can increase glycolysis to cause a sharp increase in lactic acid production in muscle, thereby causing different degrees of acidosis.

[0004] At present, the treatment of monensin poisoning mainly focuses on removing the poison, symptomatic and supportive therapy, and there is no specific antidote for treating monensin poisoning. Therefore, it is of great significance to develop an effective drug for treating monensin poisoning for clinical conversion treatment. SUMMARY

[0005] In view of the above problems, the purpose of the present application is to provide the application of arginine in repairing skeletal muscle damage caused by monensin poisoning, so as to solve the problems of the prior art.

[0006] The purpose of the present application can be realized by the following technical solutions: In a first aspect of the present application, the application of arginine in preparing a product for repairing skeletal muscle damage caused by monensin poisoning is provided.

[0007] Further, the active ingredient of the product is arginine.

[0008] Furthermore, the product functions to reverse the downregulation of monensin-induced expression levels of skeletal muscle mitochondrial oxidative phosphorylation protein complexes and microtubule network disintegration, improve mitochondrial function, and restore skeletal muscle microtubule polymerization.

[0009] This invention reveals for the first time that arginine can regulate microtubule polymerization kinetics by upregulating the expression level of the mitochondrial oxidative phosphorylation (OXPHOS) protein complex, reversing monensin-induced mitochondrial dysfunction and skeletal muscle microtubule network disintegration, significantly improving mitochondrial function and restoring skeletal muscle microtubule polymerization, providing a theoretical basis for clinical translation.

[0010] A second aspect of the invention provides the use of arginine in the preparation of formulations that improve mitochondrial function and promote skeletal muscle microtubule polymerization.

[0011] A third aspect of the present invention provides a pharmaceutical composition for repairing skeletal muscle damage caused by monensin poisoning, wherein the active ingredient is one of arginine or a pharmaceutically acceptable salt, ester, isomer, polymorph and solvate thereof.

[0012] Furthermore, the pharmaceutical composition uses arginine as the active ingredient.

[0013] Furthermore, the pharmaceutical composition also includes at least one other non-pharmaceutical active ingredient.

[0014] Furthermore, the other non-pharmaceutical active ingredients include pharmaceutically acceptable carriers, excipients, and / or diluents. Examples include pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and nonionic surfactants or lipids, pharmacologically harmless salts such as sodium chloride, flavoring agents, vitamins such as vitamin A or vitamin E, tocopherols or provitamins, antioxidants such as ascorbic acid, and stabilizers and / or preservatives for prolonging the use and shelf life of pharmaceutical active ingredients or formulations, and other commonly used non-pharmaceutical active ingredients or adjuvants and additives known in the art, as well as mixtures thereof.

[0015] Furthermore, the dosage form of the pharmaceutical composition is an oral formulation, a pulmonary inhalation formulation, a mucosal delivery formulation, or an injection.

[0016] Furthermore, the oral preparation is selected from granules, powders, pills, tablets, capsules, or liquid preparations.

[0017] Furthermore, the solvent used in the liquid formulation is one of sterile water, water-propylene glycol solution, or ethylene glycol aqueous solution.

[0018] Furthermore, the liquid formulation includes an active ingredient, a colorant, a flavoring agent, a stabilizer, and a thickener.

[0019] In the applications described above, the arginine is L-arginine, D-arginine, or DL-arginine. In some embodiments of the present invention, the arginine is L-arginine.

[0020] The present invention also provides a method for treating or repairing skeletal muscle damage caused by monensin poisoning, the method comprising: administering an effective dose of arginine or the above-mentioned pharmaceutical composition to a subject.

[0021] The subject refers to an animal, including humans and non-human animals, that is already the object of treatment, observation, or experimentation. The "therapeutic effective amount" refers to an amount of the active compound or agent, including the compound of the present invention, that can elicit a biological or medical response in an organ system, animal, or human sought by the researcher, veterinarian, physician, or other medical professional. This includes the reduction or partial reduction of symptoms of the treated disease, syndrome, symptom, or disorder. It must be recognized that the optimal dosage and interval of the active ingredient described in this invention are determined by its properties and external conditions such as the form, route, and site of administration, and the specific animal being treated, and this optimal dosage can be determined using conventional techniques. It must also be recognized that the optimal course of treatment, i.e., the daily dose of the compound over a specified period, can be determined using methods known in the art.

[0022] Compared with existing technologies, the above technical solutions have the following advantages: This invention reveals for the first time that arginine can regulate microtubule polymerization kinetics by upregulating the expression level of the mitochondrial oxidative phosphorylation (OXPHOS) protein complex, reversing monensin-induced mitochondrial dysfunction and skeletal muscle microtubule network disintegration, significantly improving mitochondrial function and restoring skeletal muscle microtubule polymerization, providing a theoretical basis for clinical translation. Attached Figure Description

[0023] Figure 1 The images show the Western blot and quantitative analysis of oxidative phosphorylation (OXPHOS) in the control group and the multinucleated myotube mitochondria treated with 0.5 μM monensin in this embodiment of the invention; where A is the protein blot of oxidative phosphorylation (OXPHOS) and B is the bar chart of quantitative analysis. Figure 2 The images show Western blotting and quantitative analysis of polymerized and free α-Tubulin microtubules from the control group and the group treated with 0.5 μM monensin in this embodiment of the invention; where A is the protein blot map of polymerized and free α-Tubulin microtubules, and B is the bar chart of quantitative analysis. Figure 3The images shown are super-resolution microscopy observation diagrams of microtubule networks and bar charts of abnormal microtubule statistics before and after treatment with 1.25mM arginine in the embodiments of the present invention; wherein, A is a super-resolution microscopy microtubule network observation diagram and B is a bar chart of abnormal microtubule statistics. Figure 4 The images show Western blotting and quantitative analysis of mitochondrial oxidative phosphorylation (OXPHOS) before and after treatment with 1.25 mM arginine in the control group of this invention; where A is the protein blot of oxidative phosphorylation (OXPHOS) and B is the bar chart of quantitative analysis. Figure 5 The figures shown are Western blot and quantitative analysis diagrams of polymeric and free α-Tubulin in the control group and before and after treatment with 1.25 mM arginine in this embodiment of the invention; wherein, A is the protein blot diagram of polymeric and free α-Tubulin, and B is the bar chart of quantitative analysis. Figure 6 This is a quantitative analysis of the total distance traveled by control mice and mice poisoned with 10 mg / kg*d monensin in this embodiment of the invention. Figure 7 This is a quantitative analysis of serum creatine kinase (CK) and aspartate aminotransferase (ASL) levels in control mice and mice poisoned with 10 mg / kg*d monensin in this embodiment of the invention; wherein, A is a bar chart of the quantitative analysis results of serum creatine kinase (CK), and B is a bar chart of the quantitative analysis results of aspartate aminotransferase (ASL). Figure 8 The images show the pathological phenotypes of skeletal muscle stained with HE and NADH and the statistical graph of abnormal muscle fibers stained with NADH in the control group mice and mice poisoned with 10 mg / kg*d monensin in this invention. Among them, A is the pathological phenotype of HE and NADH staining with a scale bar of 40 μm, and B is the bar chart of the statistical results of abnormal muscle fibers stained with NADH. Figure 9 The images show Western blotting and quantitative analysis of mitochondrial oxidative phosphorylation (OXPHOS) in skeletal muscle of control mice and mice poisoned with 10 mg / kg*d monensin in this invention embodiment; where A is the protein blot of oxidative phosphorylation (OXPHOS) and B is the bar chart of quantitative analysis. Figure 10 The images show Western blotting and quantitative analysis of polymerized and free α-Tubulin microtubules in skeletal muscle of control mice and mice poisoned with 10 mg / kg*d monensin in this invention embodiment; where A is the protein blot map of polymerized and free α-Tubulin microtubules, and B is the bar chart of quantitative analysis. Detailed Implementation

[0024] Terminology Explanation Oxidative phosphorylation Oxidative phosphorylation (OXPHOS) is a core process in eukaryotic mitochondria that generates energy through the electron transport chain coupled with ATP synthesis. The OXPHOS protein complex is a key functional unit involved in this process, mainly comprising complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome bc1 complex), complex IV (cytochrome c oxidase), and complex V (ATP synthase). These complexes are systematically located within the inner mitochondrial membrane, driving the formation of a proton transmembrane gradient through electron transport. Ultimately, complex V utilizes the proton gradient potential energy to catalyze the phosphorylation of ADP to generate ATP, providing the primary energy source for cellular life activities. Abnormalities in this system are closely associated with energy metabolism-related diseases and are a core focus of research in bioenergy metabolism. α-Tubulin α-Tubulin is a core protein subunit constituting microtubules. It forms a heterodimer with β-Tubulin via non-covalent bonds, serving as a basic structural unit for microtubule assembly. This protein is widely distributed in eukaryotic cells and its expression exhibits high stability. Western blotting (WB) technology can be used to detect the content of α-Tubulin in both free and polymerized microtubules, enabling quantitative analysis of both. This allows for precise assessment of the equilibrium state of intracellular microtubule dynamic assembly and depolymerization, providing an important molecular marker for studying microtubule function and dynamic changes in the cytoskeleton.

[0025] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0027] The arginine used in the following examples is L-arginine, CAS number 74-79-3, molecular formula C6H 14 N4O2, with a molecular weight of 174.2, has the following chemical structural formula.

[0028]

[0029] Example 1 (1) Construction and validation of an in vitro monensin poisoning skeletal muscle injury model A drug-induced myotube injury model was established by differentiating C2C12 myotubes in vitro and treating them with monensin to simulate skeletal muscle injury caused by monensin poisoning. Mouse myoblast line C2C12 (Pronosai, CL-0044) was cultured in DMEM high-glucose medium containing 10% fetal bovine serum until confluence reached 80%-90%. The medium was then replaced with high-glucose DMEM differentiation medium containing 2% horse serum to induce differentiation for 7 days, forming multinucleated myotubes. The differentiated myotubes were divided into a control group, an injury group, and a treatment group. All three groups were starved for 6 hours in DMEM high-glucose medium without L-arginine. Subsequently, the injury group and the treatment group were treated with 0.5 μM monensin (40000× stock solution prepared by dissolving in anhydrous ethanol and stored at -20°C; diluted to 1× with DMEM high-glucose medium without L-arginine according to the dosage before use, prepared fresh each time) for 24 hours. The control group received an equal volume of DMEM high-glucose medium without L-arginine. Total protein was extracted from myotubules in both the control and damaged groups, and the expression level of mitochondrial oxidative phosphorylation (OXPHOS) protein complex was detected by Western blotting. Free microtubules and polymerized microtubules were extracted from myotubules in both the control and damaged groups, and the α-Tubulin content was quantified by Western blotting. The proportion of polymerized microtubules (polymerized microtubules / total microtubules × 100%) was calculated.

[0030] The results are as follows Figure 1 As shown, treatment of multinucleated myotubes with 0.5 μM monensin significantly downregulated the expression level of the mitochondrial oxidative phosphorylation (OXPHOS) complex, thereby inducing mitochondrial dysfunction and leading to mitochondrial damage; Figure 2 As shown, treatment of multinucleated myotubules with 0.5 μM monensin can significantly reduce the proportion of polymerized microtubules, interfere with the normal polymerization process of microtubules, and ultimately induce microtubule network structure disorder.

[0031] (2) L-arginine treatment and efficacy evaluation system After monensin treatment for 24 hours, the medium was replaced with DMEM high-glucose medium without L-arginine. The treatment group was given DMEM high-glucose medium containing a final concentration of 1.25 mM L-arginine (HY-N0455) (100× stock solution was prepared by dissolving in ddH2O and stored at -20℃; before use, it was diluted to 1× with L-Arg-free medium according to the dosage and prepared fresh). The control group and the damaged group were given an equal volume of L-arginine-free DMEM high-glucose medium and cultured for another 24 hours. Microtubule structures in myotube cells were stained with microtubule-specific fluorescent dyes. The morphology of the microtubule network in the control, damaged, and treated groups was observed and analyzed using super-resolution fluorescence microscopy. Total protein was extracted from myotubes in the control, damaged, and treated groups, and the expression level of the mitochondrial oxidative phosphorylation (OXPHOS) protein complex was detected by Western blotting. Free and polymerized microtubules were extracted from myotubes in the control, damaged, and treated groups, and the α-Tubulin content was quantified by Western blotting. The percentage of polymerized microtubules (polymerized microtubules / total microtubules × 100%) was calculated.

[0032] The results are as follows Figure 3 As shown, treatment with 1.25 mM L-arginine significantly reduced the proportion of abnormal microtubules, repaired damaged microtubule structures in myotubules, and maintained the stability of the microtubule network within myotubules; Figure 4 As shown, treatment with 1.25 mM arginine significantly upregulated the expression level of the mitochondrial oxidative phosphorylation (OXPHOS) complex, improved mitochondrial function, and reversed mitochondrial dysfunction; Figure 5 As shown, treatment with 1.25 mM L-arginine significantly restored microtubule polymerization function, repaired damaged microtubules in myotubules, and maintained the stability of the microtubule network in myotubules.

[0033] Example 2 Construction and validation of an in vivo animal model of monensin poisoning A monensin poisoning animal model was established by gavage. Eight-week-old C57BL / 6 mice, with a male-to-female ratio of 1:1, were selected. The experimental group was administered 10 mg / kg monensin daily by gavage (10× stock solution was prepared by dissolving in anhydrous ethanol and stored at -20°C; before use, it was diluted with corn oil (HY-Y1888) to a 1× suspension according to the dosage, and thoroughly vortexed before use; it was prepared fresh each time). The gavage volume was controlled at 100 μL / 10 g body weight (i.e., 10 μL / g). The control group was administered the same volume (10 μL / g body weight) of solvent control solution daily by gavage. The composition of the control solution was consistent with the solvent system of the 1× suspension in the experimental group, specifically anhydrous ethanol and corn oil mixed at a volume ratio of 1:9. The intervention was continued for 6 weeks. Six weeks later, exercise exhaustion experiments were conducted on mice in both the control and experimental groups to measure and analyze the total distance traveled. Blood samples were collected from both groups, and serum was separated to detect and quantify the levels of creatine kinase (CK) and aspartate aminotransferase (ASL). Mouse muscle tissue was treated with HE and NADH staining to observe its pathological phenotype and to quantitatively count the proportion of myofibrils with disordered microtubule network structure. Total protein was extracted from myofibrils in both the control and injured groups, and the expression level of mitochondrial oxidative phosphorylation (OXPHOS) protein complex was detected by Western blotting. Free and polymerized microtubules were extracted from mouse muscle, and α-Tubulin content was quantified by Western blotting to calculate the proportion of polymerized microtubules (polymerized microtubules / total microtubules × 100%).

[0034] The results are as follows Figure 6 As shown, mice in the experimental group treated with monensin for 6 weeks exhibited a significantly shortened total distance traveled, indicating a decline in exercise endurance; Figure 7 As shown, mice treated with monensin for 6 weeks exhibited significantly elevated serum creatine kinase (CK) and aspartate aminotransferase (ASL) levels, indicating muscle tissue damage; Figure 8 As shown in Figure 9, in mice treated with monensin for 6 weeks, the proportion of abnormally stained muscle fibers increased after NADH staining, indicating disordered and damaged microtubule network structure within the muscle fibers. As also shown in Figure 9, the expression level of mitochondrial oxidative phosphorylation (OXPHOS) protein complex in the muscle tissue of mice treated with monensin for 6 weeks was significantly downregulated, leading to mitochondrial dysfunction and impaired mitochondrial function. Figure 10 As shown, after 6 weeks of treatment with monensin, the proportion of muscle fibers with disordered microtubule network structure increased, while the proportion of polymerized microtubules decreased significantly, indicating that monensin had a significant destructive effect on the microtubule network.

[0035] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of arginine in the preparation of products for repairing skeletal muscle damage caused by monensin poisoning.

2. The application according to claim 1, characterized in that, The product functions to reverse the downregulation of monensin-induced expression levels of oxidative phosphorylation protein complexes in skeletal muscle mitochondria and microtubule network disintegration, improve mitochondrial function, and restore skeletal muscle microtubule polymerization.

3. Application of arginine in the preparation of formulations that improve mitochondrial function and promote skeletal muscle microtubule polymerization.

4. A pharmaceutical composition for repairing skeletal muscle damage caused by monensin poisoning, characterized in that, The drug has arginine or one of its pharmaceutically acceptable salts, esters, isomers, polymorphs and solvates as its active ingredient.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition uses arginine as its active ingredient.

6. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition also includes at least one other non-pharmaceutical active ingredient.

7. The pharmaceutical composition according to claim 6, characterized in that, The other non-pharmaceutical active ingredients include pharmaceutically acceptable carriers, excipients, and / or diluents.

8. The pharmaceutical composition according to claim 6, characterized in that, Other non-pharmaceutical active ingredients include pharmaceutically compatible inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and nonionic surfactants or lipids, and pharmacologically harmless salts.

9. The pharmaceutical composition according to claim 4, characterized in that, The dosage form of the pharmaceutical composition is an oral preparation, a pulmonary inhalation preparation, a mucosal delivery preparation, or an injection.

10. The pharmaceutical composition according to claim 9, characterized in that, Oral preparations are selected from granules, powders, pills, tablets, capsules, or liquid preparations.

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