Composition for preventing or treating muscle disease containing CXCL 14 as 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 muscle. Typically, the soleus muscle belongs to this type. On the other hand, "Type IIB muscle fibers" have a fast contraction speed and 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 muscles have the characteristic of being regenerated and maintained according to the environment, but these characteristics disappear as people age. As a result, not only does muscle mass decrease as aging progresses, but muscle strength is also lost.

[0005] Sarcopenia refers to a state in which the quantity and function of skeletal muscles decline. Sarcopenia is caused by various factors such as aging, hormonal abnormalities, nutritional deficiencies, lack of physical activity, inflammation, and degenerative diseases. Among them, cancer, aging, and sex hormone deficiency are known to be the main causes. With the development of medical technology and the development of various therapeutic drugs, the elderly population is increasing globally as the average life expectancy increases. Along with this, the demand for the treatment of sarcopenia is expected to continue to increase. In sarcopenia patients, the number of myoblasts decreases due to disorders in the recruitment, activity, or proliferation of satellite cells, which are the stem cells of myoblasts. The proliferation and differentiation of myoblasts decrease. As a result, the muscles of sarcopenia patients show symptoms of a decrease in the size and number of muscle fibers at the histological level and a decline in muscle function. In the past decade, research on the epidemiology of sarcopenia has been actively conducted mainly in the United States and Europe. In recent years, the interest in the clinical importance of sarcopenia has increased rapidly. In early studies, the mainstream result was that sarcopenia caused a decline in the quality of life due to general debility, activity impairment, and muscle strength reduction. However, a recently published study reported that in addition to the quality of life, the risk of osteoporotic fractures may increase significantly. Also, in sarcopenia patients, chronic diseases such as diabetes, metabolic syndrome, obesity, chronic renal failure, and chronic liver failure are caused, and ultimately, the mortality rate increases. Therefore, sarcopenia has attracted attention as a disease that should be appropriately treated. Recently, it has been reported in the United States that the possibility of physical disability occurring in sarcopenia patients increases by about 1.5 to about 3.5 times, causing a social cost of 18.5 billion USD per year. In Korea, according to the National Health and Nutrition Examination Survey, the prevalence of sarcopenia is 42.0% in men and 42.7% in women aged 60 and above, which is a very common disease. Especially in Korea, which has the fastest aging rate in the world, it is certain that it will become an important social problem in the future.

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a pharmaceutical composition for preventing or treating muscle diseases, which contains 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 preventing or improving muscle diseases, which contains CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient.

Means for Solving the Problems

[0008] To achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating muscle diseases, which contains CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient.

[0009] Further, the present invention provides a food composition for preventing or improving muscle diseases, which contains CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient.

Effects of the Invention

[0010] The present invention has confirmed that CXCL14 increases muscle differentiation and synthesis and increases muscle mass. In addition, in LPS-induced muscle atrophy or DEX-induced muscle atrophy, it has been confirmed that CXCL14 suppresses muscle loss, increases the expression of muscle synthesis and differentiation factors, and suppresses the expression of muscle degradation proteins. Further, since CXCL14 promotes muscle synthesis and differentiation and suppresses muscle degradation in vivo, it can be usefully utilized in related industries.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Embodiments 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 techniques known to those skilled in the art, the detailed description thereof can be omitted. Further, in describing the present invention, if it is determined that a specific description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof can be omitted. Furthermore, the terminology used in this specification is the terminology used to appropriately represent the preferred embodiments of the present invention, and this may vary depending on the intention of the user or operator, or the convention in the field to which the present invention belongs, etc.

[0013] Therefore, the definition of this term should be made based on the content throughout this specification. Throughout this specification, when a certain part says 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 acts of suppressing or delaying the progression of symptoms of a specific disease by administering the composition of the present invention.

[0016] The term "treatment" used in the present invention means all acts of improving or better changing 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 art 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 produced in a form in which an active ingredient is incorporated into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. The 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0019] The pharmaceutical composition of the present invention can be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, respectively, by ordinary methods.

[0020] When formulating, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. Such solid preparations can be prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the active ingredient. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., can be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used. As the base of suppositories, witepsol, tween 61, cocoa butter, laurin fat, glycerogelatin, etc. can be used.

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

[0022] The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, gender, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient contained in the pharmaceutical composition can be variously selected according to the subject, and preferably it is contained in the pharmaceutical composition at a concentration of 0.01 - 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, the pharmaceutical activity may not appear, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0023] In addition, the CXCL14 protein contained in the pharmaceutical composition of the present invention includes proteins having substantially the same physiological activity as the said protein. The CXCL14 proteins having substantially the same physiological activity include the said protein and its functional equivalents and functional derivatives. The "functional equivalent" refers to an amino acid sequence variant in which some or all of the natural-type protein amino acids are substituted, or some amino acids are deleted or added, and which has substantially the same physiological activity as the natural-type CXCL14 protein. The "functional derivative" means a protein to which a modification has been made to increase or decrease the physicochemical properties of the CXCL14 protein and which has substantially the same physiological activity as the natural-type 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 contain a CXCL14 expression vector, and the CXCL14 expression vector may be a plasmid containing the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 3.

[0026] The plasmid expression vector is a method of directly delivering plasmid DNA to human cells by an FDA-approved gene delivery method for use in humans (Nable, E.G., et al., Science, 249:1285-1288, 1990). Plasmid DNA has the advantage that it can be purified uniformly, unlike viral vectors. As the plasmid expression vector that can be used in the present invention, mammalian expression plasmids known in the art can be used. For example, but not limited to, pRK5 (European Patent No. 307,247), pSV16B (International Patent Publication No. 91 / 08291), and pVL1392 (PharMingen) are representative.

[0027] The plasmid expression vector containing the polynucleotide according to the present invention can be introduced into cells by methods known in the art, such as, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation method, 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 the CXCL14 can be administered by known methods. For example, it can be administered locally parenterally, orally, nasally, intravenously, intramuscularly, subcutaneously, or by other appropriate means.

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

[0030] According to an embodiment of the present invention, the CXCL14 may increase the expression of muscle synthesis and differentiation factors, 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 an embodiment of the present invention, the muscle synthesis and differentiation factors may be (Forkhead box protein O1) or FOXO3 (Forkhead box protein O3).

[0032] According to an embodiment of the present invention, the CXCL14 may suppress the expression of muscle catabolic factors, and the muscle catabolic factors may be atrogin-1 or MuRF1 (Muscle RING-finger protein-1).

[0033] According to an 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, preferably sarcopenia or muscular atrophy, but not limited thereto.

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

[0035] Furthermore, the present invention provides a food composition for preventing or improving muscle diseases, which contains CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient.

[0036] The term "improvement" used in the present invention means all acts that at least reduce parameters related to the state to be treated, such as the degree of symptoms.

[0037] In addition to containing the active ingredient of the present invention, the food composition of the present invention can contain various flavoring agents or natural carbohydrates, etc. as additional ingredients, like ordinary food compositions.

[0038] Examples of the aforementioned natural carbohydrates include general sugars such as monosaccharides, for example, glucose, fructose, etc.; disaccharides, for example, maltose, sucrose, etc.; and polysaccharides, for example, dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. Examples of the aforementioned flavoring agents can effectively use natural flavoring agents (thaumatin), stevia extracts (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention is formulated in the same manner as the pharmaceutical composition and can be 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, for example, beverages, meats, chocolates, foods, confectioneries, pizzas, ramen, other noodles, gums, candies, ice creams, alcoholic beverages, vitamin complexes, and health supplements, etc.

[0039] In addition to the extract as the active ingredient, the food composition can contain various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (for cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. Further, the food composition of the present invention can contain natural fruit juices, fruit juice beverages, and pulp for the production of 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, pills, etc. for the purpose of preventing or treating muscle diseases. In the present invention, the "health functional food composition" refers to a food manufactured and processed using raw materials and ingredients having useful functions for the human body according to the Law No. 6727 on Health Functional Foods, and means ingesting it for the purpose of regulating nutrients with respect to the structure and function of the human body and obtaining useful effects for health purposes such as physiological actions. The health functional food of the present invention can contain ordinary food additives, and the conformity as a food additive is determined according to the standards and criteria for the item based on the general rules and general test methods of the Food Additive Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Examples of the items listed in the "Food Additive Codex" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, cinnamic acid; natural additives such as sweeteners, licorice extracts, crystalline cellulose, cochineal pigments, guar gum; and mixed preparations such as sodium L-glutamate preparations, alkaline agents for noodles, preservative preparations, tar pigment preparations, etc. For example, a health functional food in tablet form can be granulated in a usual method by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant and other additives, and then compressed and formed by adding a lubricant, etc., or the mixture can be directly compressed and formed. Further, the health functional food in tablet form can also contain a flavoring agent, etc. if necessary. Among the capsule-type health functional foods, hard capsule agents can be manufactured by filling a usual hard capsule with a mixture obtained by mixing the active ingredient of the present invention with additives such as an excipient, and soft capsule agents can be manufactured by filling a capsule base such as gelatin with a mixture obtained by mixing the active ingredient of the present invention with additives such as an excipient. The soft capsule agents can contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc. if necessary. The pill-type health functional food can be prepared by molding a mixture obtained by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant, etc. by a conventionally known method, and can be coated with sucrose or other coating agents if necessary, or the surface can also be coated with substances such as starch and talc.The granular health functional food can be produced in a granular form by a conventionally known method by mixing a mixture of an excipient, a binder, a disintegrant, etc. of the active ingredient of the present invention, and can contain a flavoring agent, a taste-correcting agent, etc. as necessary.

[0041] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are merely for more specifically explaining the present invention, and it will be apparent to those with ordinary knowledge in the art that the scope of the present invention is not limited to these examples.

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

[0043]

Table 1

[0044] As a result, as shown in Fig. 1, it was confirmed that, compared with the control group, the thickness of myofibroblasts increased in a concentration-dependent manner in the group treated with CXCL14 (Fig. 1A), and it was confirmed that CXCL14 increased the mass index of myofibroblasts in the quantitative value of fluorescence expression of myofibroblasts (Fig. 1B).

[0045] <Example 2>Confirmation of the regulation of myofibroblast synthesis and differentiation factor expression by CXCL14 The regulation of the synthesis of myofibroblasts and the expression of differentiation factors by CXCL14, which is a recombinant protein of the present invention, was confirmed. Specifically, after culturing C2C12 cells in the same manner as in Example 1 above, the expression of myofibroblast 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 blot. As myofibroblast 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 phosphorylation of S6K (T389) were confirmed. In addition, the expression of FOXO1 (Forkhead box protein O1) and FOXO3 (Forkhead box protein O3), which are proteolysis regulatory factors, was confirmed, and the expression of MuRF1 (Muscle RING-finger protein-1), which is a muscle proteolytic enzyme, and atrogin-1 (also known as Atrogin-1 or F-box only protein 32, FBXO32) was analyzed respectively.

[0046] As a result, as shown in Figure 2, in the group treated with CXCL14 (100 ng / ml) compared with the control group, the activity of AKT-S6K increased in a time-dependent manner until 2 hours after CXCL14 treatment, and it was confirmed that phosphorylation of FOXO1 / 3, a proteolysis regulator, increased. Also, it was confirmed that the expression of MuRF1 and Atrogin-1, muscle proteolytic enzymes, was significantly decreased (Figure 2A and Figure 2B). Furthermore, 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 CXCL14 recombinant protein induces an increase in the mass of myofibroblasts by increasing protein synthesis and inhibiting proteolysis in myofibroblasts.

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

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

[0049] In addition, as shown in Fig. 4, as a result of confirming the expression of muscle synthesis and differentiation factors, in the group treated with LPS, the activities of AKT-S6K-related factors, FOXO1, and FOXO3 were significantly decreased compared to the control group. However, it was confirmed that CXCL14 significantly increased the activities of AKT-S6K-related factors, FOXO1, and FOXO3 that decreased with LPS. In addition, the treatment with LPS increased the expression of MuRF1 and Atrogin-1, which are muscle proteolytic enzymes. However, it was confirmed that the increased proteolytic enzymes were significantly decreased by the treatment with CXCL14, and it was confirmed that CXCL14 improved muscle atrophy induced by bacterial infection.

[0050] <Example 4>Confirmation of the effect of CXCL14 on suppressing dexamethasone-induced muscle atrophy To confirm the effect of CXCL14 of the present invention on improving steroid-induced muscle atrophy, the effect of suppressing dexamethasone (DEX)-induced muscle atrophy was confirmed. Specifically, after culturing C2C12 cells in the same manner as in Example 1, they were treated with dexamethasone at a concentration of 10 μM and CXCL14 recombinant protein at 100 ng / ml, and then cultured for 48 hours. After the culture was completed, the expression of myosin heavy chain protein was analyzed by immunofluorescence staining, and the expression of muscle synthesis and differentiation factors and Total mTOR in Example 2 was analyzed by Western blot. As control groups, an untreated control group (Control), a DEX group in which muscle atrophy was induced by dexamethasone, and a group treated with CXCL14 alone were used.

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

[0052] Furthermore, as shown in Fig. 6, as a result of examining the expression of muscle synthesis and differentiation factors, in the group treated with DEX, the activities of AKT-S6K-related factors, FOXO1 and FOXO3 were significantly decreased compared with the control group. However, it was confirmed that CXCL14 significantly increased the activities of AKT-S6K-related factors, FOXO1 and FOXO3 that were decreased by DEX. In addition, the treatment with DEX increased the expression of MuRF1 and Atrogin-1, which are muscle proteolytic enzymes. However, it was confirmed that the treatment with CXCL14 significantly decreased the increased proteolytic enzymes, and it was confirmed that CXCL14 improved steroid-induced muscle atrophy.

[0053] <Example 5>Confirmation of in vivo muscle increase by local expression of CXCL14 It was confirmed whether the local expression of CXCL14 of the present invention increases muscle mass. Specifically, two types of human CXCL14 gene expression plasmid DNAs (CXCL14-Myc; SEQ ID NO: 2 or HA-CXCL14; SEQ ID NO: 3) were injected into the tibialis anterior muscle (or tibialis anterior muscle, TA) of C57BL / 6 mice by electroporation and stabilized for 3 weeks. Thereafter, the mice were humanely sacrificed, the tibialis anterior muscles were excised, and muscle transverse axis paraffin sections were prepared. As the control group, a Control group injected with a Mock plasmid, which is an empty vector, was used, and the entire process of the experiment is shown in Fig. 7. Thereafter, the CXCL14 protein expression was confirmed by immunofluorescence staining together with the laminin protein expression using an anti-Myc antibody for CXCL14-Myc having a Myc-epitope tag at the C-terminus and an anti-HA antibody for HA-CXCL14 having an HA (Hemagglutinin)-epitope tag at the N-terminus. In addition, using the ImageJ program, the cross-sectional area (CSA) of muscle fiber cells was measured and used as a measure of the mass of muscle fiber cells.

[0054] In addition, using Western blot analysis, the expression and activation levels of the main factors in the protein synthesis and degradation processes in muscle tissue lysates were measured.

[0055] As a result, as shown in Fig. 8, it was confirmed that CXCL14 was located in the interstitial space of myofibroblasts, and the Laminin protein was expressed in the basal lamina around the myofibroblasts. Also, in the group injected with CXCL14 plasmid DNA, it was confirmed that the CSA value increased and the CSA median value significantly increased compared with the Control group (Figs. 8B and 8C).

[0056] Also, as shown in Fig. 9, it was confirmed that the expression of CXCL14 increased the activity of the AKT-S6K signal transduction. Furthermore, it was confirmed that the expression of the muscle proteolytic enzymes Atrogin-1 and MuRF1 was decreased.

[0057] <Example 6>Confirmation of Inhibition of LPS-Induced Muscle Atrophy by Local Expression of CXCL14 It was confirmed whether the local expression of CXCL14 of the present invention suppresses LPS-induced muscle atrophy in vivo. Specifically, after injecting the CXCL14 gene expression plasmid (20 μg) into the TA muscle of C57BL / 6 mice by electroporation, it was stabilized for 3 weeks. Then, LPS (1 mg / kg of body weight) was intraperitoneally injected once to induce muscle atrophy. Two days after the LPS injection, the mice were humanely sacrificed, the TA muscles were excised, and muscle transverse axis paraffin sections were prepared. The whole process of the experiment is shown in Fig. 10. Then, the expressions of CXCL14 protein and laminin protein were confirmed by immunofluorescence staining. Also, using the ImageJ program, the cross-sectional area (CSA) of myofibroblasts was measured and used as a measure of the mass of myofibroblasts.

[0058] Also, using Western blot analysis, the expression and activation levels of the main factors in the protein synthesis and degradation processes in muscle tissue lysates were measured.

[0059] As a result, as shown in Fig. 11, in the group injected with LPS, the CSA value and the CSA median value were significantly decreased compared with the control group, while in the group injected with the CXCL14 plasmid, the CSA value and the CSA median value were significantly increased, confirming that the overexpression of CXCL14 suppresses LPS-induced muscle atrophy.

[0060] Also, as shown in Fig. 12, it was confirmed that the expression of CXCL14 increases the activities of FOXO1 and FOXO3, and it was confirmed that the expression of Atrogin-1 and MuRF1, which are muscle proteolytic enzymes, was decreased.

[0061] <Example 7>Confirmation of suppression of dexamethasone-induced muscular atrophy by local expression of CXCL14 It was confirmed whether the local expression of CXCL14 of the present invention suppresses dexamethasone (DEX)-induced muscle atrophy in vivo. Specifically, after injecting the CXCL14 gene expression plasmid (20 μg) into the TA muscle of C57BL / 6 mice by electroporation, it was stabilized for 3 weeks. Thereafter, DEX (20 mg / kg of body weight) was intraperitoneally injected once a day for 6 days to induce muscular atrophy. The mice were humanely sacrificed at 1 week after DEX injection, the TA muscle was excised, and muscle transverse axis paraffin sections were prepared. The whole process of the experiment is shown in Fig. 13. Thereafter, the expression of CXCL14 protein and laminin protein was confirmed by immunofluorescence staining. Also, using the ImageJ program, the cross sectional area (CSA) of muscle fiber cells was measured and used as a measure of the mass of muscle fiber cells.

[0062] Also, using Western blot analysis, the degree of expression and activation of the main factors in the protein synthesis and degradation processes in muscle tissue lysates was measured.

[0063] As a result, as shown in Fig. 14, in the group injected with DEX, the CSA value and the CSA median value were significantly decreased compared with the control group. However, in the group injected with the CXCL14 plasmid, the CSA value and the CSA median value were significantly increased, confirming that the overexpression of CXCL14 inhibits dexamethasone-induced muscle atrophy.

[0064] In addition, as shown in Fig. 15, it was confirmed that the expression of CXCL14 increases the activities of FOXO1 and FOXO3, and it was also confirmed that the expression of Atrogin-1 and MuRF1, which are muscle proteolytic enzymes, was decreased.

[0065] Therefore, it was confirmed in the present invention that CXCL14 increases muscle differentiation and synthesis and increases muscle mass. In addition, in LPS-induced muscle atrophy or DEX-induced muscle atrophy, it was confirmed that muscle loss was suppressed, the expression of muscle synthesis and differentiation factors was increased, and the expression of muscle-degrading proteins was suppressed. It was also confirmed that CXCL14 promotes muscle synthesis and differentiation and inhibits muscle breakdown in vivo.

Claims

1. A pharmaceutical composition for preventing or treating a muscle disease, comprising CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient.

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

1.

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

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

3.

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

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

7. The composition of 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 of 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 of claim 7, wherein the muscle synthesis and differentiation factor is (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 a disease selected from the group consisting of muscular atrophy, myopathy, muscle degeneration, myasthenia, muscle injury, dystrophinopathy, myopathy, muscular dystrophy, cachexia, and sarcopenia.

14. The composition of claim 1 , wherein the muscle disease is bacterial infection or steroid induced.

15. A food composition for preventing or improving a muscle disease, comprising CXCL14 (CXC motif chemokine ligand 14) protein as an active ingredient.

Images