Composition for the prevention or treatment of muscle diseases, containing CXCL14 as the active ingredient

Abstract

To provide a composition for preventing or treating a muscle disease.SOLUTION: A pharmaceutical composition for preventing or treating a muscle disease is provided, comprising CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a composition for preventing or treating muscle diseases containing CXCL14 as an active ingredient.

Background Art

[0002] Muscle accounts for about 40% of the human body. To maintain the functional ability of the human body and prevent metabolic diseases, it is essential to ensure an appropriate muscle mass. It is roughly divided into smooth muscle, cardiac muscle, and skeletal muscle. Skeletal muscle occupies a considerable part of our entire body and promotes the movement of the skeleton. Skeletal muscle is the organ that occupies the largest part of the human body, accounting for 40 - 50% of the total body weight, and also plays an important role in various metabolic functions in the body such as energy homeostasis and heat generation. Human muscle decreases by more than 1% every year after the age of 40, and by about 50% of the maximum muscle mass by the age of 80. Sarcopenia in the elderly is recognized as the most important factor in reducing overall physical function.

[0003] The types of muscle fibers that make up muscle are mainly classified into Type I, Type IIA, and Type IIB according to the metabolic process that generates ATP and the contraction speed. "Type I muscle fibers" have a slow contraction speed, contain a large number of myoglobins and mitochondria, and are suitable for continuous and low-intensity aerobic activities. Type I muscle fibers are reddish and are also called red muscles. Typically, the soleus muscle belongs to this type. On the other hand, "Type IIB muscle fibers" have a fast contraction speed, are used for very short-term and high-intensity anaerobic exercises, have a low content of myoglobin, and are whitish. "Type IIA muscle fibers" have intermediate characteristics between the above two types of muscle fibers. As people age, not only does the composition of Type I and II muscle fibers by muscle site change, but all types of muscle fibers also decrease.

[0004] Skeletal muscle has the characteristic of regenerating and maintaining itself in response to the environment, but these characteristics disappear with age, resulting in not only a decrease in muscle mass but also a loss of muscle strength as aging progresses.

[0005] Myoblastosis is a condition characterized by a decrease in the volume and function of skeletal muscle. While various factors contribute to myoblastosis, including aging, hormonal imbalances, nutritional deficiencies, lack of physical activity, inflammation, and degenerative diseases, cancer, aging, and sex hormone deficiencies are known to be the primary causes. With advancements in medical technology and the development of various therapeutic drugs, average life expectancy is increasing worldwide, leading to a growing elderly population. Consequently, the demand for myoblastosis treatment is expected to continue increasing. In patients with myoblastosis, the number of myoblasts decreases due to impaired recruitment, activity, or proliferation of satellite cells (myoblast stem cells). This leads to reduced myoblast proliferation and differentiation, resulting in a histological decrease in muscle fiber size and number, and consequently, impaired muscle function. Over the past decade, epidemiological research on myoblastosis has been active, primarily in the United States and Europe, and in recent years, interest in the clinical importance of myoblastosis has surged. Early studies predominantly concluded that myocardial infarction (MMS) causes a decline in quality of life due to general weakness, reduced activity, and muscle loss. However, recent research has reported that, in addition to quality of life, MMS may also significantly increase the risk of osteoporotic fractures. Furthermore, MMS is associated with chronic diseases such as diabetes, metabolic syndromes, obesity, chronic renal failure, and chronic hepatic failure, ultimately leading to increased mortality. Therefore, MMS is attracting attention as a disease that should be appropriately treated. Recently in the United States, it has been reported that the likelihood of developing physical disability in MMS increases by approximately 1.5 to 3.5 times, resulting in an annual social cost of $18.5 billion USD. In South Korea, according to the National Health and Nutrition Survey, the prevalence of MMS is very high, at 42.0% for men and 42.7% for women over 60 years old. Given that South Korea has one of the fastest aging populations in the world, it is certain to become a significant social problem in the future. [Overview of the project] [Problems that the invention aims to solve]

[0006] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of muscle diseases, comprising the CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient.

[0007] Another object of the present invention is to provide a food composition for the prevention or improvement of muscle diseases, comprising the CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient. [Means for solving the problem]

[0008] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases, comprising CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient.

[0009] Furthermore, the present invention provides a food composition for the prevention or improvement of muscle diseases, comprising CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient. [Effects of the Invention]

[0010] This invention confirms that CXCL14 increases muscle differentiation and synthesis, thereby increasing muscle mass. Furthermore, it was confirmed that in LPS-induced or DEX-induced muscular atrophy, CXCL14 suppresses muscle loss, increases the expression of muscle synthesis and differentiation factors, and suppresses the expression of muscle-degrading proteins. Moreover, since CXCL14 promotes muscle synthesis and differentiation and suppresses muscle degradation in vivo, it can be usefully utilized in related industries. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows the quantification of myosin heavy chain expression in muscle fiber cells and muscle mass after CXCL14 treatment according to the present invention (A: results of immunofluorescence staining, B: quantification of muscle mass). [Figure 2] This figure shows the results of Western blotting analysis of muscle synthesis, differentiation, and degradation factor expression in muscle fiber cells after CXCL14 treatment according to the present invention. A: Confirmation of the AKT-S6K pathway up to 2 hours after CXCL14 treatment. B: Confirmation of FOXO1 / 3 and muscle degradation factor expression up to 2 hours after CXCL14 treatment. C: Confirmation of muscle synthesis, differentiation, and degradation factor expression 48 hours after CXCL14 treatment. [Figure 3] This figure shows the effect of CXCL14 treatment on suppressing muscle atrophy in muscle fiber cells that had been treated with LPS (A: Immunofluorescence staining results, B: Quantification of muscle mass). [Figure 4] This figure shows the expression of muscle synthesis, differentiation, and degradation factors in muscle fiber cells treated with CXCL14 after LPS-induced muscle atrophy, as analyzed by Western blotting (A: Confirmation of the AKT-S6K pathway, B: Confirmation of FOXO1 / 3 and muscle degradation factors). [Figure 5] This figure shows the effect of CXCL14 treatment on suppressing muscle atrophy in muscle fiber cells that had been treated with dexamethasone (A: Immunofluorescence staining results, B: Quantification of muscle mass). [Figure 6] This figure shows the expression of muscle synthesis, differentiation, and degradation factors in muscle fiber cells treated with CXCL14, as analyzed by Western blotting (A: Confirmation of the AKT-S6K pathway, B: Confirmation of FOXO1 / 3 and muscle degradation factors). [Figure 7] This is a schematic diagram of an experiment to confirm the muscle-increasing effect of the CXCL14 treatment of the present invention in vivo. [Figure 8] This figure shows the muscle-building effect of local injection of the CXCL14 expression vector of the present invention, confirmed by immunofluorescence staining and the CSA (Cross sectional area) index (A: Results of immunofluorescence staining, B: Quantification of the CSA index, C: Quantification of the CSA median). [Figure 9] This figure shows the results of a Western blot analysis of the expression of the AKT-S6K pathway and myolytic factors after local injection of the CXCL14 expression vector of the present invention. [Figure 10]This is a schematic diagram of an experiment to confirm the effect of local injection of the CXCL14 expression vector of the present invention on improving LPS-induced muscular atrophy. [Figure 11] This figure shows the effect of local injection of a CXCL14 expression vector on suppressing muscle atrophy in an LPS-induced muscular atrophy animal model, confirmed by immunofluorescence staining and the CSA (Cross sectional area) index (A: Immunofluorescence staining results, B: Quantification of the CSA index, C: Quantification of the CSA median). [Figure 12] This figure shows the results of Western blotting analysis of FOXO1 / 3 and myolytic factors expression after local injection of a CXCL14 expression vector in an LPS-induced muscular atrophy animal model. [Figure 13] This is a schematic diagram of an experiment to confirm the effect of local injection of the CXCL14 expression vector of the present invention on improving dexamethasone-induced muscular atrophy. [Figure 14] This figure shows the effect of local injection of a CXCL14 expression vector on suppressing muscle atrophy in a dexamethasone-induced muscular atrophy animal model, confirmed by immunofluorescence staining and the CSA (Cross sectional area) index (A: Immunofluorescence staining results, B: Quantification of the CSA index, C: Quantification of the CSA median). [Figure 15] This figure shows the results of a Western blot analysis of FOXO1 / 3 and myolytic factors expression after local injection of a CXCL14 expression vector in an animal model of dexamethasone-induced muscular atrophy. [Modes for carrying out the invention]

[0012] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, for well-known technologies known to those skilled in the art, detailed descriptions thereof can be omitted. Further, in describing the present invention, if it is determined that specific descriptions of related known functions or configurations may unnecessarily obscure the gist of the present invention, detailed descriptions thereof can be omitted. Furthermore, the terminology used in this specification is the terminology used to appropriately represent desirable embodiments of the present invention, and this may vary depending on the intentions of users and operators, or the conventions of the field to which the present invention belongs.

[0013] Therefore, the definition of this term should be based on the content throughout this specification. Throughout this specification, when a certain part states that a certain component "includes", unless otherwise specified, this does not exclude other components, but means that other components can be further included.

[0014] The present invention provides a pharmaceutical composition for preventing or treating muscle diseases containing CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient.

[0015] The term "prevention" used in the present invention means all actions that suppress the symptoms of a specific disease or delay its progression by administering the composition of the present invention.

[0016] The term "treatment" used in the present invention means all actions that improve or better change the symptoms of a specific disease by administering the composition of the present invention.

[0017] The pharmaceutical composition of the present invention can further contain an adjuvant in addition to the active ingredient. Any adjuvant known in the relevant technical field can be used without limitation, but for example, by further containing Freund's complete adjuvant or incomplete adjuvant, its effect can be enhanced

[0018] The pharmaceutical composition according to the present invention can be manufactured in a form in which the active ingredient is mixed with a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0019] Each of the pharmaceutical compositions of the present invention can be formulated by conventional methods into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, or sterile injection solutions, and used in those forms.

[0020] When formulation, the drug can be prepared using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules. Such solid formulations can be prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Water-insoluble solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. The base for the suppositories can be witepsol, tween 61, cocoa butter, lauric acid butter, glycerol gelatin, etc.

[0021] The pharmaceutical composition according to the present invention can be administered to an individual by various routes. All possible administration methods are conceivable, but for example, it can be administered orally, intravenously, intramuscularly, subcutaneously, or intraperitoneally by injection.

[0022] The dosage of the pharmaceutical composition according to the present invention is selected considering the individual's age, weight, sex, physical condition, etc. It is clear that the concentration of the active ingredient contained in the pharmaceutical composition can be selected in various ways depending on the target, and preferably it is contained in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, there is a possibility that no pharmaceutical activity will be exhibited, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0023] Furthermore, the CXCL14 protein contained in the pharmaceutical composition of the present invention includes proteins having substantially equivalent physiological activity to the aforementioned protein. The CXCL14 proteins having substantially equivalent physiological activity include the aforementioned protein and its functional equivalents and functional derivatives. A "functional equivalent" refers to an amino acid sequence variant in which some or all of the amino acids of the native protein are substituted, or some amino acids are deleted or added, and which has substantially equivalent physiological activity to the native CXCL14 protein. A "functional derivative" refers to a protein that has been modified to increase or decrease the physicochemical properties of the CXCL14 protein and which has substantially equivalent physiological activity to the native CXCL14 protein.

[0024] According to one embodiment of the present invention, the CXCL14 protein may contain the amino acid sequence of SEQ ID NO: 1.

[0025] According to one embodiment of the present invention, the composition may further comprise a CXCL14 expression vector, and the CXCL14 expression vector may be a plasmid comprising the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 3.

[0026] Plasmid expression vectors are FDA-approved gene delivery methods that can be used in humans to directly deliver plasmid DNA to human cells (Nable, EG, et al., Science, 249:1285-1288, 1990). Plasmid DNA has the advantage of being able to be uniformly purified, unlike viral vectors. Mammalian expression plasmids known in the art can be used as plasmid expression vectors in this invention. Examples include, but are not limited to, pRK5 (European Patent No. 307,247), pSV16B (International Patent Publication No. 91 / 08291), and pVL1392 (PharMingen).

[0027] Plasmid expression vectors containing polynucleotides according to the present invention can be introduced into cells by methods known in the art, for example, but not limited to transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known methods for introducing DNA into cells (Wu et al., J. Bio. Chem., 267:963-967, 1992; Wu and Wu, J. Bio. Chem., 263:14621-14624, 1988).

[0028] The vector capable of expressing CXCL14 can be administered by known methods. For example, it can be administered topically by parenteral, oral, nasal, intravenous, intramuscular, subcutaneous, or other appropriate means.

[0029] According to one embodiment of the present invention, the CXCL14 may be a substance that increases muscle mass, and the substance that increases muscle mass may be a substance that increases the number of myosin heavy chain-positive cells.

[0030] According to one embodiment of the present invention, CXCL14 may increase the expression of muscle synthesis and differentiation factors, and the muscle synthesis and differentiation factors may be AKT (Protein kinase B)-S6K (Ribosomal protein S6 kinase) pathway factors, and the AKT-S6K pathway factors may be selected from the group consisting of p-ATK (T308), p-AKT (S473), total AKT, p-S6K (T389), total S6K, p-mTOR (Mammailan target of rapamycin), and total mTOR.

[0031] According to one embodiment of the present invention, the muscle synthesis and differentiation factor may be (Forkhead box protein O1) or FOXO3 (Forkhead box protein O3).

[0032] According to one embodiment of the present invention, the CXCL14 may suppress the expression of a muscle-degrading factor, and the muscle-degrading factor may be atrogin-1 or MuRF1 (Muscle Ring-finger protein-1).

[0033] According to one embodiment of the present invention, the muscle disease may be a disease selected from the group consisting of muscular atrophy, myopathy, muscle degeneration, myasthenia, muscular injury, dystrophinopathy, myopathy, muscular dystrophy, cachexia, and sarcopenia, and is preferably myrcopenia or muscular atrophy, but is not limited to these.

[0034] According to one embodiment of the present invention, the muscle disease may be induced by a bacterial infection or steroids.

[0035] Furthermore, the present invention provides a food composition for the prevention or improvement of muscle diseases, comprising CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient.

[0036] As used in this invention, the term "improvement" means all actions that at least reduce the parameters related to the condition being treated, such as the severity of symptoms.

[0037] In addition to containing the active ingredient of the present invention, the food composition of the present invention may also contain various flavorings or natural carbohydrates as additional ingredients, as is the case with ordinary food compositions.

[0038] Examples of natural carbohydrates mentioned above include monosaccharides, such as glucose and fructose; disaccharides, such as maltose and sucrose; and common sugars such as polysaccharides, such as dextrin and cyclodextrin; as well as sugar alcohols such as xylitol, sorbitol, and erythritol. The aforementioned flavoring agents can effectively utilize natural flavoring agents (thaumatin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Examples of foods to which the composition of the present invention can be added include beverages, meats, chocolates, food products, confectionery, pizzas, ramen, other noodles, gums, candies, ice creams, alcoholic beverages, vitamin complexes, and health supplements.

[0039] Furthermore, in addition to the active ingredient extract, the food composition may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (for cheese, chocolate, etc.), pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. In addition, the food composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages.

[0040] The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, rounds, etc., for the purpose of preventing or treating muscle diseases. In the present invention, "health functional food composition" refers to a food manufactured and processed using raw materials and components that have useful functions for the human body as defined in Act No. 6727 on Health Functional Foods, and means that it is taken for the purpose of obtaining effects useful for health purposes, such as regulating nutrients in relation to the structure and function of the human body or physiological effects. The health functional food of the present invention may contain ordinary food additives, and unless otherwise specified, the suitability as a food additive is determined by the standards and criteria for the item in question, based on the general provisions and general test methods of the Food Additives Code approved by the Korea Food and Drug Administration. Examples of items listed in the aforementioned "Food Additives Codex" include chemically synthesized substances such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as sweet coloring, licorice extract, crystalline cellulose, sorghum coloring, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar dye preparations. For example, a tablet-type functional food can be produced by granulating a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, and other additives in a conventional manner, then adding a lubricant and compressing it, or by directly compressing the mixture. The tablet-type functional food can also contain flavoring agents as needed. Among capsule-type functional foods, hard capsules can be produced by filling a conventional hard capsule with a mixture of the active ingredient of the present invention with excipients and other additives, while soft capsules can be produced by filling a capsule base such as gelatin with a mixture of the active ingredient of the present invention with excipients and other additives. The soft capsules may optionally contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc. Round health functional foods can be prepared by molding a mixture of the active ingredients of the present invention with excipients, binders, disintegrants, etc., using conventionally known methods, and may optionally be coated with sucrose or other coating agents, or the surface may be coated with a substance such as starch or talc.Granular health functional foods can be manufactured in granular form by conventionally known methods by mixing the active ingredients of the present invention with excipients, binders, disintegrants, etc., and may contain flavoring agents, taste enhancers, etc., as needed.

[0041] The present invention will be described in more detail below with reference to examples. These examples are merely for the purpose of illustrating the present invention more concretely, and it will be obvious to those with ordinary skill in the art that the scope of the present invention is not limited to these examples.

[0042] <Example 1> Confirmation of increased muscle fiber cell mass in CXCL14 The effect of CXCL14, a novel protein of the present invention, on increasing muscle fiber cell mass was confirmed. Specifically, mouse muscle cell line C2C12 cells were cultured in a DMEM / FBS 10% culture medium and increased to 1 × 10⁶ cells. 5 After inoculating each well of a collagen-coated 12-well plate with individual cells, the cells were cultured for 24 hours until confluent. The cell culture medium was replaced with DMEM / 2% horse serum culture medium, and the cells were cultured for a further 4 days to generate muscle fiber cells through induced muscle differentiation. The culture medium of the obtained muscle fiber cells was again replaced with DMEM / 10% FBS to prevent further muscle differentiation, and then the cells were treated with recombinant CXCL14 (CXC motif chemokine ligand 14) protein at concentrations of 20 or 100 ng / ml and cultured for 48 hours. After the culture period, the cells were fixed, and the expression of myosin heavy chain protein was confirmed using immunofluorescence staining. A negative control group (Control) treated with the same amount of PBS was used as a control group, and the amino acid sequence of the recombinant CXCL14 protein of the present invention (SEQ ID NO: 1) is shown in Table 1 below.

[0043] [Table 1]

[0044] As a result, as shown in Figure 1, we confirmed that the thickness of muscle fiber cells increased in a concentration-dependent manner in the group treated with CXCL14 compared to the control group (Figure 1A), and we confirmed that CXCL14 increased the mass index of muscle fiber cells based on the quantitative value of fluorescence expression in muscle fiber cells (Figure 1B).

[0045] <Example 2> Confirmation of the regulation of muscle fiber cell synthesis and differentiation factor expression in CXCL14 We confirmed the regulation of muscle fiber cell synthesis and differentiation factor expression by CXCL14, the recombinant protein of the present invention. Specifically, after culturing C2C12 cells in the same manner as in Example 1, the expression of muscle fiber cell synthesis and differentiation factors up to 2 hours after CXCL14 treatment, and the expression of factors at 48 hours after treatment, were analyzed by Western blotting. As muscle fiber cell synthesis factors, Total AKT (protein kinase B) and AKT activation in the AKT-S6K pathway were confirmed by phosphorylation of T308 (threonine 308) and S473 (serine 473), and Total S6K (Ribosomal protein S6 kinase) and S6K (T389) were confirmed by phosphorylation. Furthermore, we confirmed the expression of the proteolytic regulators FOXO1 (Forkhead box protein O1) and FOXO3 (Forkhead box protein O3), and analyzed the expression of the muscle proteolytic enzymes MuRF1 (Muscle RING-finger protein-1) and Atrogin-1 (also known as F-box only protein 32, FBXO32).

[0046] As a result, as shown in Figure 2, compared to the control group, the group treated with CXCL14 (100 ng / ml) showed a time-dependent increase in AKT-S6K activity and increased phosphorylation of FOXO1 / 3, a protein degradation regulator, up to 2 hours after CXCL14 treatment. Furthermore, a significant decrease in the expression of the muscle protein-degrading enzymes MuRF1 and Atrogin-1 was confirmed (Figures 2A and 2B). Moreover, at 48 hours after CXCL14 treatment, the activity of AKT-S6K and phosphorylation of FOXO1 / 3 increased in a concentration-dependent manner, the activity of FOXO1 and FOXO3 increased, and the expression of MuRF1 and Atrogin-1 also decreased in a concentration-dependent manner (Figure 2C). This confirmed that the recombinant CXCL14 protein induces an increase in muscle fiber cell mass by increasing protein synthesis within muscle fiber cells and inhibiting protein degradation.

[0047] <Example 3> Confirmation of the effect of CXCL14 on suppressing LPS-induced muscle atrophy To confirm the effect of CXCL14 in the present invention on improving muscle atrophy caused by bacterial infection, we confirmed its effect of suppressing LPS (Lipopolysaccharide)-induced muscle atrophy. Specifically, C2C12 cells were cultured in the same manner as in Example 1, then treated with LPS at a concentration of 100 ng / ml, followed by treatment with CXCL14 recombinant protein at 100 ng / ml, and then cultured for 48 hours. After the culture period, myosin heavy chain protein expression was analyzed by immunofluorescence staining, and muscle synthesis and differentiation factors and Total mTOR expression in Example 2 were analyzed by Western blotting. As control groups, an untreated control group (Control), an LPS group in which muscle atrophy was induced with LPS, and a group treated only with CXCL14 were used.

[0048] As a result, as shown in Figure 3, myosin heavy chain expression was significantly reduced in the LPS group compared to the control group, but it was confirmed that the myosin heavy chain expression that was reduced in LPS was significantly increased by treatment with CXCL14.

[0049] Furthermore, as shown in Figure 4, when we examined the expression of muscle synthesis and differentiation factors, we found that the activity of AKT-S6K-related factors, FOXO1, and FOXO3 was significantly reduced in the LPS-treated group compared to the control group. However, we confirmed that CXCL14 significantly increased the activity of AKT-S6K-related factors, FOXO1, and FOXO3 that had been reduced by LPS. In addition, LPS treatment increased the expression of the muscle proteolytic enzymes MuRF1 and Atrogin-1, but CXCL14 treatment significantly reduced the increased proteolytic enzymes, confirming that CXCL14 improves muscle atrophy induced by bacterial infection.

[0050] <Example 4> Confirmation of the dexamethasone-induced muscle atrophy inhibitory effect of CXCL14 To confirm the effect of CXCL14 on improving steroid-induced muscle atrophy, the inhibitory effect on dexamethasone (DEX)-induced muscle atrophy was investigated. Specifically, C2C12 cells were cultured in the same manner as in Example 1, then treated with 10 μM dexamethasone, followed by 100 ng / ml of recombinant CXCL14 protein, and cultured for 48 hours. After the culture period, myosin heavy chain protein expression was analyzed by immunofluorescence staining, and muscle synthesis and differentiation factors and Total mTOR expression in Example 2 were analyzed by Western blotting. The control groups included an untreated control group, a DEX group in which muscle atrophy was induced with dexamethasone, and a group treated only with CXCL14.

[0051] As a result, as shown in Figure 5, myosin heavy chain expression was significantly reduced in the DEX group compared to the control group, but it was confirmed that the myosin heavy chain expression that was reduced in the DEX group was significantly increased by treatment with CXCL14.

[0052] Furthermore, as shown in Figure 6, when we examined the expression of muscle synthesis and differentiation factors, we found that the activity of AKT-S6K-related factors, FOXO1, and FOXO3 was significantly reduced in the DEX-treated group compared to the control group. However, we confirmed that CXCL14 significantly increased the activity of AKT-S6K-related factors, FOXO1, and FOXO3 that had been reduced by DEX. In addition, DEX treatment increased the expression of the muscle proteolytic enzymes MuRF1 and Atrogin-1, but CXCL14 treatment significantly reduced the increased proteolytic enzymes, confirming that CXCL14 improves steroid-induced muscle atrophy.

[0053] <Example 5> Confirmation of in vivo muscle growth due to local expression of CXCL14 This study investigated whether local expression of CXCL14 increases muscle mass. Specifically, two types of human CXCL14 gene expression plasmid DNA (CXCL14-Myc; SEQ ID NO: 2 or HA-CXCL14; SEQ ID NO: 3) were injected into the tibialis anterior muscle (TA) of C57BL / 6 mice by electroporation and stabilized for 3 weeks. Subsequently, the tibialis anterior muscle was excised by humanely sacrificing the mice, and transverse paraffin sections of the muscle were prepared. A control group injected with a mock plasmid (empty vector) was used as the control group, and the entire experimental process is shown in Figure 7. Subsequently, CXCL14 protein expression was confirmed by immunofluorescence staining along with laminin protein expression. For CXCL14-Myc, which has a Myc-epitope tag at the C-terminal, an anti-Myc antibody was used, and for HA-CXCL14, which has an HA (Hemagglutinin)-epitope tag at the N-terminal, an anti-HA antibody was used. Furthermore, the ImageJ program was used to measure the cross-sectional area (CSA) of muscle fiber cells, which was then used as a measure of muscle fiber cell mass.

[0054] Furthermore, Western blot analysis was used to measure the expression and activation levels of major factors involved in protein synthesis and degradation processes in muscle tissue lysates.

[0055] As a result, as shown in Figure 8, we confirmed that CXCL14 is located in the interstitial space of muscle fiber cells, and that the laminin protein is expressed in the basal lamina surrounding muscle fiber cells. Furthermore, we confirmed that the group injected with CXCL14 plasmid DNA showed increased CSA levels and a significant increase in the median CSA level compared to the control group (Figures 8B and 8C).

[0056] Furthermore, as shown in Figure 9, we confirmed that CXCL14 expression increases the activity of AKT-S6K signaling. In addition, we confirmed that it decreases the expression of the muscle proteases Atrogin-1 and MuRF1.

[0057] <Example 6> Confirmation of suppression of LPS-induced muscular atrophy by local expression of CXCL14 This study investigated whether local expression of CXCL14 suppresses LPS-induced muscle atrophy in vivo. Specifically, a CXCL14 gene expression plasmid (20 μg) was injected into the TA muscle of C57BL / 6 mice by electroporation and stabilized for 3 weeks. Subsequently, muscle atrophy was induced by a single intraperitoneal injection of LPS (1 mg / kg of body weight). Two days after LPS injection, the mice were humanely sacrificed, the TA muscle was excised, and transverse paraffin sections of the muscle were prepared. The entire experimental process is shown in Figure 10. Subsequently, CXCL14 protein and laminin protein expression were confirmed by immunofluorescence staining. In addition, the cross-sectional area (CSA) of muscle fiber cells was measured using the ImageJ program and used as a measure of muscle fiber cell mass.

[0058] Furthermore, Western blot analysis was used to measure the expression and activation levels of major factors involved in protein synthesis and degradation processes in muscle tissue lysates.

[0059] As a result, as shown in Figure 11, the group injected with LPS showed a significant decrease in CSA levels and CSA median levels compared to the control group, while the group injected with the CXCL14 plasmid showed a significant increase in CSA levels and CSA median levels, confirming that CXCL14 overexpression suppresses LPS-induced muscle atrophy.

[0060] Furthermore, as shown in Figure 12, we confirmed that the expression of CXCL14 increases the activity of FOXO1 and FOXO3, and decreases the expression of the muscle protein-degrading enzymes Atrogin-1 and MuRF1.

[0061] <Example 7> Confirmation of suppression of dexamethasone-induced muscular atrophy by local expression of CXCL14 This study investigated whether local expression of CXCL14 suppresses dexamethasone (DEX)-induced muscle atrophy in vivo. Specifically, a CXCL14 gene expression plasmid (20 μg) was injected into the TA muscle of C57BL / 6 mice by electroporation and stabilized for 3 weeks. Subsequently, DEX (20 mg / kg of body weight) was administered intraperitoneally once daily for 6 days to induce muscle atrophy. One week after DEX injection, mice were humanely sacrificed, the TA muscle was excised, and transverse paraffin sections of the muscle were prepared. The entire experimental process is shown in Figure 13. Subsequently, CXCL14 protein and laminin protein expression were confirmed by immunofluorescence staining. In addition, the cross-sectional area (CSA) of muscle fiber cells was measured using the ImageJ program and used as a measure of muscle fiber cell mass.

[0062] Furthermore, Western blot analysis was used to measure the expression and activation levels of major factors involved in protein synthesis and degradation processes in muscle tissue lysates.

[0063] As a result, as shown in Figure 14, the group injected with DEX showed a significant decrease in CSA levels and CSA median levels compared to the control group, while the group injected with the CXCL14 plasmid showed a significant increase in CSA levels and CSA median levels, confirming that CXCL14 overexpression suppresses dexamethasone-induced muscle atrophy.

[0064] Furthermore, as shown in Figure 15, we confirmed that the expression of CXCL14 increases the activity of FOXO1 and FOXO3, and decreases the expression of the muscle protein-degrading enzymes Atrogin-1 and MuRF1.

[0065] Therefore, the present invention has confirmed that CXCL14 increases muscle differentiation and synthesis, and increases muscle mass. Furthermore, it has been confirmed that in LPS-induced amyotrophy or DEX-induced amyotrophy, it suppresses muscle loss, increases the expression of muscle synthesis and differentiation factors, and suppresses the expression of muscle-degrading proteins. In addition, it has been confirmed that CXCL14 promotes muscle synthesis and differentiation and suppresses muscle degradation in vivo.

Claims

1. Contains CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient. A pharmaceutical composition for the prevention or treatment of muscle diseases selected from the group consisting of muscular atrophy, myopathy, muscle degeneration, myasthenia, muscle injury, dystrophinopathy, muscular dystrophy, cachexia, and sarcopenia.

2. The composition according to claim 1, wherein the CXCL14 protein comprises the amino acid sequence of SEQ ID NO:

1.

3. The composition according to claim 1, further comprising a CXCL14 expression vector.

4. The composition according to claim 3, wherein the CXCL14 expression vector is a plasmid containing the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

5. The composition according to claim 1, wherein the CXCL14 increases muscle mass.

6. The composition according to claim 5, wherein the substance that increases muscle mass is the substance that increases the number of myosin heavy chain-positive cells.

7. The composition according to claim 1, wherein the CXCL14 increases the expression of muscle synthesis and differentiation factors.

8. The composition according to claim 7, wherein the muscle synthesis and differentiation factor is an AKT (protein kinase B)-S6K (Ribosomal protein S6 kinase) pathway factor.

9. The composition according to claim 8, wherein the AKT-S6K pathway factor is selected from the group consisting of p-ATK(T308), p-AKT(S473), total AKT, p-S6K(T389), total S6K, p-mTOR, and total mTOR.

10. The composition according to claim 7, wherein the muscle synthesis and differentiation factor is FOXO1 (Forkhead box protein O1) or FOXO3 (Forkhead box protein O3).

11. The composition according to claim 1, wherein the CXCL14 suppresses the expression of muscle-degrading factors.

12. The composition according to claim 11, wherein the muscle-degrading factor is atrogin-1 or MuRF1 (Muscle ring-finger protein-1).

13. The composition according to claim 1, wherein the muscle disease is induced by bacterial infection or steroids.

14. Contains CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient. A food composition for the prevention or improvement of muscle diseases selected from the group consisting of muscular atrophy, myopathy, muscle degeneration, myasthenia, muscle injury, dystrophinopathy, muscular dystrophy, cachexia, and sarcopenia.

Images