Composition exhibiting muscle loss suppression or muscle production promotion effect through skin-derived exosomes

Abstract

A composition that increases the level of microRNA-26a in the extracellular reticulum and its use relates to a composition that increases the level of microRNA-26a (miRNA-26a) in exosomes secreted from cells, and the exosomes can decrease the expression of biomarkers involved in muscle loss, such as MURF1, atrogin-1, and myostatin, and increase the expression of myoD, which is involved in muscle production. Therefore, a composition that increases the level of microRNA-26a in the extracellular reticulum can be used to inhibit muscle loss and promote muscle production.

Description

[Technical field]

[0001] The present invention relates to a composition that increases the level of microRNA-26a (miRNA-26a) in extracellular endoplasmic reticulum and a composition that includes the composition and exhibits an effect of inhibiting muscle loss or promoting muscle production through skin-derived exosomes. [Background technology]

[0002] Muscles not only play a role as organs of human motor ability, but also affect the entire body, including bones, blood vessels, nerves, liver, heart, and pancreas. Bones maintain their density by pulling and pushing with the strength of muscles. Therefore, when muscle strength decreases, bones also become weaker, making osteoporosis more likely to occur. In addition, when muscle is reduced, various substances produced by muscles can prevent the generation of new blood vessels and nerves, which may ultimately cause a decline in cognitive function.

[0003] Muscle loss or sarcopenia begins at about age 30 and is a lifelong process. During this process, muscle tissue mass, number and size of muscle fibers are gradually reduced. The result of muscle loss is a gradual loss of muscle mass and strength. Mild muscle strength loss can increase stress on some joints (e.g., knees) and can make them more vulnerable to arthritis or falls. Furthermore, rapidly contracting muscle fibers are more affected by aging than slowly contracting muscle fibers. Thus, as aging progresses, it becomes more difficult for muscles to contract rapidly, which can cause inconvenience in daily life. Summary of the Invention [Problem to be solved by the invention]

[0004] Under the above circumstances, the inventors have confirmed that when fibroblasts are treated with a specific substance, the level of microRNA-26a (miRNA-26a) increases in exosomes secreted from the cells, and that the exosomes reduce the expression of genes involved in muscle loss while increasing the expression of genes involved in muscle production.

[0005] Therefore, an object of the present invention is to provide a composition for increasing the level of microRNA-26a (miRNA)-26a in the extracellular endoplasmic reticulum, comprising a substance selected from the group consisting of betaine, camellia flower extract, camelliaside A, myricetin, naringenin, nobiletin, kojyl carboxy dipeptide-23, L-carnosine, and copper tripeptide, or a combination thereof.

[0006] Another object of the present invention is to provide a composition comprising the above-mentioned composition as an active ingredient, which exhibits an effect of inhibiting muscle loss or promoting muscle production through skin-derived exosomes. [Means for solving the problem]

[0007] In order to achieve the above-mentioned object, one aspect of the present invention provides a composition for increasing the level of microRNA-26a in the extracellular endoplasmic reticulum (hereinafter referred to as a composition for increasing microRNA-26a level), comprising as an active ingredient a substance selected from the group consisting of betaine, camellia flower extract, camelliaside A, myricetin, naringenin, nobiletin, codylcarboxydipeptide-23, L-carnosine and copper tripeptide, or a combination thereof.

[0008] Betaine, also known as trimethylglycine (TMG), is found in large quantities in plants such as sugarcane, beets, and goji berries, and is known to have excellent skin moisturizing properties, so it is used as a cosmetic ingredient. Myricetin is used as an antioxidant and skin conditioning agent, and naringenin is also a cosmetic ingredient used as a skin conditioning agent. Cosylcarboxydipeptide-23 has the functions of an antioxidant, a sequestering agent, and a skin protective agent, and is a compound having the structure of the following chemical formula 1.

[0009] [ka]

[0010] In the above Chemical Formula 1, R is dipeptide-23.

[0011] Copper tripeptide is a copper complex of tripeptide (GHK-Cu) and is used as a skin conditioning agent, camelliaside A is an ingredient contained in green tea (Camellia sinensis) and is known to have wrinkle improving effects (Korean Patent Registration No. 10-0757175), and nobiletin is a polymethoxyflavone found in large amounts in citrus peels and is known to have excellent anti-inflammatory effects (Korean Patent Publication No. 2018-0046245).

[0012] L-carnosine is a dipeptide composed of two amino acids, histidine and alanine, and is known to have antioxidant and antidiabetic activities, and is therefore used as a nutritional agent and supplement. As mentioned above, the substances used in the composition of the present invention are either raw materials for cosmetics or nutrients or are derived from natural products, and therefore are safe for use on the human body.

[0013] The term "extract" as used herein includes the extract itself and all forms of extract that can be formed using the extract, such as the extract obtained by extraction treatment, a dilution or concentrate of the extract, a dried product obtained by drying the extract, a crude product or purified product of the extract, or a mixture thereof, etc. The extract of the present invention can be extracted from natural, hybrid or variant plants of the respective plants, and can also be extracted from plant tissue cultures.

[0014] The method for extracting the camellia flower extract is not particularly limited, and can be extracted by a method commonly used in the art. Non-limiting examples of the extraction method include solvent extraction, hot water extraction, ultrasonic extraction, filtration, reflux extraction, etc., and these methods can be carried out alone or in combination of two or more methods.

[0015] The extraction solvent used for the camellia flower extract is not particularly limited, and any solvent known in the art can be used. Specifically, the camellia flower extract can be extracted with any solvent selected from the group consisting of water, ethyl acetate, dichloromethane, alcohols having 1 to 4 carbon atoms, and combinations thereof, and preferably, ethanol can be used as the solvent.

[0016] The liquid camellia flower extract can be separated from the dried crushed plant material by a method such as vacuum filtration, and then can be concentrated or dried. For example, the liquid extract can be concentrated under reduced pressure using a vacuum rotary evaporator, and the liquid extract can be dried to obtain a powdered extract. The concentrated or powdered extract can be dissolved in water, alcohol, dimethyl sulfoxide (DMSO), or a mixture thereof, as necessary, for use.

[0017] The present inventors confirmed that when dermal fibroblasts were treated with each of the above substances, cultured, and then exosomes were isolated, the level of microRNA-26a (miRNA-26a) in the exosomes increased compared to when the substances were not treated (Table 1). Therefore, the extracellular vesicles may be, but are not limited to, those secreted from fibroblasts, specifically, dermal fibroblasts.

[0018] The term "extracellular vesicle" as used herein refers to an extracellular vesicle, which is a very small-sized exocytic vesicle that allows the exchange of substances such as proteins, lipids, and nucleic acids between cells and serves as a medium for physiological signal transmission, and almost all cells secrete extracellular vesicles. These are broadly classified into exosomes and microvesicles based on their size and production process. In terms of the production process, exosomes are vesicles produced within cells and secreted while folding the cell membrane inward, and have a size of about 30 to 200 nm. Microvesicles are secreted outside the cell after the cell membrane is separated from the outside, and have a size of about 50 to 1000 nm. According to one embodiment of the present invention, the extracellular vesicles may be exosomes.

[0019] According to an embodiment of the present invention, the composition for increasing microRNA-26a level may contain an active ingredient (skin-derived exosome secretion promoter) at 0.00001% by weight or more based on the total weight of the composition. More specifically, the active ingredient may be 0.00001% by weight or more, 0.0001% by weight or more, 0.0005% by weight or more, 0.001% by weight or more, 0.005% by weight or more, 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, 0.5% by weight or more, 1.0% by weight or more, 5.0% by weight or more, 10% by weight or more, or 50% by weight or more, but not more than 70% by weight. Preferably, the composition may contain 0.00001% by weight to 10% by weight or 0.005% by weight to 10% by weight of the active ingredient. Another aspect of the present invention provides a composition for inhibiting muscle loss or promoting muscle formation, comprising the composition for increasing microRNA-26a levels as an active ingredient.

[0020] As already mentioned, when the composition for increasing microRNA-26a levels is treated with cells, specifically skin cells, extracellular vesicles, specifically exosomes, having increased levels of microRNA-26a can be obtained.

[0021] Meanwhile, exosomes are well known to be used as a means of intercellular communication. For example, exosomes secreted from stem cells in bones reach the heart and transmit signals. In the past, it was known that hormones were mainly responsible for signal transmission between organs in the body, but it has recently been discovered that exosomes are also used for signal transmission between organs. As the skin is the largest organ of the body, it is expected that exosomes are secreted very actively. However, there has been little research on the role of exosomes secreted from the skin.

[0022] Such communication function of exosomes can also be confirmed in the present invention. Specifically, when myofiber cells were directly treated with a substance such as betaine, there was no significant change in the expression of genes related to muscle loss and myogenesis. On the other hand, when exosomes with increased levels of miRNA-26a obtained by treating with the substance were treated, the expression of genes related to muscle loss was significantly decreased and the expression of genes related to myogenesis was significantly increased (FIGS. 4-7, 9 and 10). These experimental results indicate that exosomes derived from fibroblasts and with increased levels of miRNA-26a are involved in the transmission of signals related to promoting myogenesis and suppressing muscle loss in myofiber cells.

[0023] The muscle loss-related gene can be selected from the group consisting of MURF1, atrogin-1, and myostatin, and the myogenesis-related gene can be myoD.

[0024] The composition for inhibiting muscle loss or promoting muscle production of the present invention can be prepared in any dosage form commonly prepared in the art. For example, the composition can be formulated into a solution, suspension, emulsion, paste, gel, cream, lotion, powder, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, since the composition of the present invention acts on fibroblasts, it can have a dosage form of cream, lotion, ointment or gel, and can be used in the form of a skin topical agent. The composition in such a dosage form can be prepared by a method commonly used in the art.

[0025] In addition to the active ingredient, the composition for inhibiting muscle loss or promoting muscle production of the present invention may further contain ingredients contained in general cosmetic compositions, such as moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohol, colorants, fragrances, blood circulation enhancers, cooling agents, antiperspirants, purified water, etc.

[0026] When the dosage form of the present invention is a cream or gel, animal fibers, plant fibers, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. can be used as the carrier component.

[0027] When the dosage form of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier component. In particular, when it is a spray, it can further contain an accelerator such as chlorofluorohydrocarbon, propane / butane or dimethyl ether.

[0028] When the dosage form of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, aliphatic esters of glycerol, polyethylene glycol or fatty acid esters of sorbitan.

[0029] When the dosage form of the present invention is a suspension, the carrier component can be a liquid diluent such as water, ethanol, or propylene glycol; a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester; microcrystalline cellulose, aluminum metahydroxide, bentonite, agarose, or tracant.

[0030] Yet another aspect of the present invention provides a pharmaceutical composition for preventing or treating muscle loss-related muscle diseases, comprising the composition for increasing microRNA-26a levels as an active ingredient.

[0031] The muscle loss-related muscle disease may be selected from the group consisting of sacopenia, muscular atrophy, muscular dystrophy and myasthenia.

[0032] Sarcopenia is a disease in which normal muscle mass, strength, and function are reduced due to malnutrition, decreased physical activity, aging, etc., while muscular atrophy refers to a group of various clinical and genetic disorders in which symmetrical muscle weakening or loss occurs due to genetic and other factors.

[0033] Muscular dystrophy (MD) is a muscle disease that causes the weakening of the locomotor system and impairs athletic ability, and is characterized by the gradual weakening of skeletal muscles, muscle protein deficiency, and necrosis of muscle cells and tissues. Myasthenia gravis is a disease in which muscles become abnormally weak and fatigued, and if not treated appropriately, muscle weakness may suddenly worsen, and in severe cases, even the respiratory muscles may become weak, leading to respiratory paralysis.

[0034] It will be understood that the terms or elements referred to in the pharmaceutical composition and in the description of the composition for increasing microRNA-26a levels are similar to those referred to in the description of the claimed composition for increasing microRNA-26a levels.

[0035] The pharmaceutical composition of the present invention may contain a pharma- ceutically acceptable carrier in addition to the active ingredient. The pharma- ceutically acceptable carrier is one that is commonly used in formulations, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the composition may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0036] The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, by application to the skin, intravenous, subcutaneous, or intraperitoneal injection or local application) depending on the intended method, but parenteral administration is preferred.

[0037] When the active ingredient of the present invention is formulated into preparations such as tablets, capsules, chewable tablets, powders, liquids, suspensions and the like for oral administration, the preparations may contain binders such as gum arabic, corn starch, microcrystalline cellulose or gelatin, excipients such as dicalcium phosphate or lactose, disintegrating agents such as alginic acid, corn starch or potato starch, lubricants such as magnesium stearate, sweeteners such as sucrose or saccharin, and flavoring agents such as peppermint, methyl salicylate or fruit flavors.

[0038] The parenteral administration form may be a transdermal administration form, such as, but not limited to, an injection, adhesive, ointment, lotion, gel, cream, spray, suspension, emulsion, suppository, patch, etc.

[0039] Furthermore, the pharmaceutical composition may be in the form of an external skin preparation, and the external skin preparation is a general term that can include anything that is applied outside the skin, and includes pharmaceuticals in various dosage forms.

[0040] The pharmaceutical composition of the present invention is administered in a pharma- ceutical effective amount. In the present invention, the term "pharma-ceutical effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined by factors including the type of disease of the patient, the severity of symptoms, the activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, duration of treatment, drugs used concomitantly, and other factors well known in the medical field. For example, the pharmaceutical composition of the present invention can be administered at a dose of 1 μg / kg to 200 mg / kg, preferably 50 μg / kg to 50 mg / kg, once a day or in divided doses three times a day. The above dosages are not intended to limit the scope of the present invention in any way.

[0041] The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in a single or multiple doses. It is important to administer an amount that provides maximum effect at the minimum amount without side effects, taking into consideration all of the above factors, and this can be easily determined by a person skilled in the art.

[0042] Yet another aspect of the present invention provides a composition (hereinafter referred to as an extracellular vesicle composition) containing as an active ingredient extracellular vesicles obtained by treating cells with a substance selected from the group consisting of betaine, camellia flower extract, camelliaside A, myricetin, naringenin, nobiletin, codylcarboxydipeptide-23, L-carnosine, and copper tripeptide, or a combination thereof, and a composition for inhibiting muscle loss or promoting muscle formation, containing the extracellular vesicle composition as an active ingredient.

[0043] The present inventors confirmed that when skin-derived fibroblasts were treated with the above-mentioned substances, the level of microRNA-26a increased in exosomes secreted from the fibroblasts, and that the exosomes have the effect of suppressing muscle loss or promoting muscle production (Figures 4 to 7, 9 and 10).

[0044] It will be understood that the terms or elements referred to in the extracellular vesicle composition and in the description of the composition for increasing microRNA-26a levels are similar to those referred to in the description of the claimed composition for increasing microRNA-26a levels.

[0045] Yet another aspect of the present invention provides a method for producing extracellular vesicles having increased levels of microRNA-26a, comprising the steps of:

[0046] treating the cells with a substance selected from the group consisting of betaine, camellia flower extract, camelliaside A, myricetin, naringenin, nobiletin, codylcarboxydipeptide-23, L-carnosine, and copper tripeptide, or a combination thereof; and Recovering the extracellular vesicles from the cell culture medium.

[0047] According to one embodiment of the present invention, the cells are skin-derived fibroblasts, and the extracellular vesicles can be, but are not limited to, exosomes.

[0048] Meanwhile, the method for separating extracellular vesicles and / or exosomes from a cell culture solution is as disclosed in Examples 1 and 2. However, in addition to the above-mentioned separation methods, various methods known in the art can be used to separate extracellular vesicles and / or exosomes from a cell culture solution.

[0049] For example, for the separation of extracellular vesicles and / or exosomes, known separation methods such as ultrafiltration, density gradient centrifugation, tangential flow filtration, size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, or polymer based precipitation can be used. However, the exosome separation method is not limited to the above-mentioned methods, and it goes without saying that various separation methods that are used in the art or that can be used in the future can be adopted. Effect of the Invention

[0050] According to an embodiment of the present invention, a composition for increasing the level of microRNA-26a in extracellular vesicles can be used to produce exosomes with increased levels of microRNA-26a. The exosomes can reduce the expression of biomarkers involved in muscle loss, such as MURF1, atrogin-1, and myostatin, while increasing the expression of myoD, which is involved in muscle production. Therefore, the composition for increasing the level of microRNA-26a in extracellular vesicles can be useful for inhibiting myolytic muscle loss or promoting muscle production. [Brief description of the drawings]

[0051] [Figure 1] The figures show the results of examining cell viability after treating fibroblasts (A) and myofibrocytes (B) with different concentrations of betaine. [Diagram 2] The figures show the results of examining whether or not fluorescently labeled fibroblast-derived exosomes are taken up into muscle fibrocytes after treatment with the exosomes. [Diagram 3] 1 shows the results of examining the levels of miRNA-26a in exosomes derived from fibroblasts isolated after treatment with different concentrations of betaine. [Figure 4] The figure shows the results of examining the expression levels of muscle loss markers, maf1, atrogin1, and myostatin, after treating muscle fibrocytes with betaine or fibroblast-derived exosomes. [Diagram 5] The figure shows the results of examining the expression level of MyoD, a myogenic marker, after treating muscle fibrocytes with betaine or fibroblast-derived exosomes. [Figure 6] The figure shows the results of examining the protein levels of muscle loss markers, maf1, atrogin1, and myostatin, after treating muscle fibrocytes with betaine or fibroblast-derived exosomes. [Figure 7] The figure shows the results of examining the protein level of MyoD, a myogenic marker, after treating muscle fibrocytes with betaine or fibroblast-derived exosomes. [Figure 8] The results are shown in which muscle fibroblasts were treated with dexamethasone, and then the expression levels of Maf1 and MyoD were examined according to the treatment concentration of the miRNA-26a analog. [Figure 9] The results show that after treating fibroblasts with camellia flower extract, the levels of miRNA-26a were examined in isolated exosomes. [Figure 10] The figures show the results of examining the expression levels of muscle loss markers, Maf1 (A) and myostatin (B), after muscle fibrocytes were treated with camellia flower extract or exosomes isolated from fibroblasts treated with camellia flower extract. BEST MODE FOR CARRYING OUT THEINVENTION

[0052] Hereinafter, one or more embodiments will be described in more detail with reference to examples. However, these examples are intended to illustratively explain one or more embodiments, and the scope of the present invention is not limited to these examples.

[0053] Example 1: Exosome isolation and analysis 1-1. Confirmation of cytotoxicity of betaine

[0054] Human skin dermal cells were normal human dermal fibroblasts (NHDFs) isolated from adult specimens and purchased from LONZA (Cat. CC-2511).Myofibrocytes were C2C12 cells, myoblasts isolated and cultured from C3H mice, and purchased from ATCC (CRL-1772).

[0055] Human dermal cells (human fibroblasts) HS68 (hereafter referred to as FB) or C2C12 (passage 27) were cultured in a 96-well plate at 1x10 4The cells were seeded at a concentration of 100 cells / well and cultured in a 5% CO2, 37°C incubator for 24 hours. After culture, the cells were treated with different concentrations of betaine and further cultured for 24 hours, and an untreated group was used as a control. After washing the cells with PBS, they were further cultured in CCK-8 (DONGJIN, CK04-11) for 2 hours and the absorbance was measured at 450 nm using a microplate reader. From the measurement results, the value of the control group was set to 100, and the relative cell survival rate according to the betaine treatment concentration was calculated. As a result, it was confirmed that betaine treatment did not affect the viability of FB and C2C12 (Figures 1A and B).

[0056] 1-2.Exosome isolation

[0057] The medium from the passage 27 FBs that had been subcultured and treated with betaine for 48 hours was collected and centrifuged at 3000×g for 30 minutes. After centrifugation, only the supernatant was collected and transferred to Ultra-15 Centrifugal Filter Units (Amicon (registered trademark), MERCK, C7715), and centrifuged at 4000×g for 40 minutes. Only the supernatant was collected, and Total Exosome Isolation Reagent (Invitrogen, Cat No#4478539) was added to only 1 / 2 of the supernatant volume, and reacted at 4°C overnight. The next day, the cells were centrifuged at 10000×g for 1 hour, the supernatant was removed by suction, and the final exosome pellet was resuspended in PBS. The exosomes obtained in this example are hereinafter referred to as "fibroblast-derived exosomes".

[0058] To confirm the size of fibroblast (FB)-derived exosomes, dynamic light scattering was measured using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK), and the results were analyzed using Dynamic V6 software. Analysis showed that the size of fibroblast-derived exosomes ranged from 50 to 150 nm.

[0059] Furthermore, to quantify fibroblast (FB)-derived exosomes, absorbance was measured at 405 nm using the EXOCET Exosome Quantitation Kit (System Biosciences, USA). Based on the analysis results, all experimental groups were treated with fibroblast-derived exosomes at a concentration of 20 μg / ml.

[0060] 1-3. Confirmation of cellular uptake of fibroblast exosomes

[0061] We examined whether fibroblast-derived exosomes could be taken up by C2C12 cells. C2C12 cells were plated on Lab-Tek chamber slides (Nunc Penfield, NY) at 1.5 × 10 4 The fibroblast-derived exosomes were treated with bodipy TR ceramide staining reagent for 20 minutes, and excess staining reagent was removed using a cleanup kit. The stained fibroblast-derived exosomes were treated with C2C12 cells for 30 minutes and then observed under a confocal microscope.

[0062] As a result of the observation, red dot-like signals indicating exosomes were confirmed in C2C12 cells, indicating that fibroblast-derived exosomes were taken up into the cells (Figure 2).

[0063] 1-4. Confirmation of miRNA-26a levels in exosomes derived from betaine-treated fibroblasts

[0064] 2 x 10 FB cells in a 75T flask 6After inoculation and culturing for 24 hours, the cells were treated with betaine (0.1, 1 and 10 mM). After further culturing for 48 hours, exosomes were isolated by the method of Example 1-2, and microRNA (hereinafter referred to as miRNA) was extracted from the exosomes isolated from the culture medium using an RNeasy plus mini kit (Qiagen, Germany) according to the manufacturer's protocol.

[0065] Real-time qPCR was performed with a Taqman probe targeting mature RNA. The relative expression levels of intracellular target miRNAs were expressed as relative percentages after normalization with the expression level of RNU48, which was used as a housekeeping gene for miRNA quantification. The relative expression level of exosomal miRNA-26a was expressed as relative percentages after normalization with the expression level of microRNA-26a, which was used as a housekeeping gene for miRNA quantification in fibroblast exosomes. All real-time qPCR analyses were performed using an Applied Biosystems 7500 (Applied Biosystems) instrument.

[0066] The analysis confirmed that the level of miRNA-26a was increased in exosomes isolated from betaine-treated FB cells compared to the control group that was not treated with betaine (Figure 3).

[0067] 1-5. Additional exosome isolation

[0068] The FB was treated with the substances shown in Table 1 below, and exosomes were isolated in the same manner as in Example 1-1, and the level of miRNA-26a in the exosomes was confirmed by the method in Example 1-4.

[0069] [Table 1]

[0070] Example 2: Confirmation of changes in gene expression in muscle fiber cells 2-1. Confirmation of changes in muscle loss / muscle formation marker expression - qPCR

[0071] C2C12 cells were cultured in a 6-well plate at 1.5 × 10 5 The cells were seeded at a concentration of 1 / well and cultured for 24 hours. The medium was then replaced with one containing 2% horse serum, and further cultured for 72 hours to promote differentiation of C2C12 cells. After differentiation was completed, the cells were treated with 0.5 mM betaine or the fibroblast-derived exosomes (approximately 20 μg / ml) obtained in Examples 1 and 2, and further cultured for 48 hours. Total RNA was extracted using the RNeasy plus mini kit, and analyzed by SuperScript TM cDNA was synthesized using ELISA kit III (Invitrogen, USA), and then analyzed by qPCR using the Taqman probe of the target gene.

[0072] The analysis showed that when C2C12 cells, a muscle fibroblast cell line, were directly treated with betaine, the expression of muscle loss markers MURF1 (muscle ring finger 1) and atrogin-1 increased slightly compared to the control group, while the expression of myostatin was at a similar level. However, the changes in all three genes were not significant. On the other hand, when C2C12 cells were treated with fibroblast-derived exosomes, the expression of muscle loss markers MURF1, atrogin-1, and myostatin was significantly decreased (Figure 4A-C).

[0073] Furthermore, when C2C12 cells, a muscle fibroblast cell, were directly treated with betaine, the expression of the myogenic marker MyoD (myogenic differentiation 1) was increased compared to the control group, but not to a significant level. On the other hand, when fibroblast-derived exosomes were treated, the expression of the myogenic marker MyoD was confirmed to be increased to a significant level (Figure 5).

[0074] 2-2. Confirmation of changes in muscle loss / muscle formation marker expression - Western blotting

[0075] 5 × 10 C2C12 cells in a 6-well plate 5 The cells were seeded at a concentration of 1000 / well and cultured for 24 hours. The medium was then replaced with 2% horse serum and further cultured for 72 hours to promote the differentiation of C2C12 cells. After differentiation was completed, the cells were treated with betaine (0.1, 0.5 mM) or fibroblast-derived exosomes and further cultured for 48 hours. The C2C12 cells were then lysed in a lysis buffer (1% NP40, 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl and 0.01 M MgCl2) containing a protease inhibitor, and the protein concentration was measured by the BCA (bovine carbonic anhydrase) method. The quantified proteins were separated by SDS-PAGE and transferred to a PVDF membrane, and the protein expression level of each muscle loss marker was confirmed with antibodies.

[0076] As a result of the confirmation, it was confirmed that when C2C12 cells were directly treated with betaine at a concentration of 0.1 or 0.5 mM, there was no change in the expression of muscle loss markers, but when fibroblast-derived exosomes were treated, the expression of muscle loss markers MAFF1 (muscle Ring-finger protein-1), atrogin1, and myostatin was reduced (Figure 6). Furthermore, the expression of myogenesis marker MyoD was not changed when C2C12 cells were directly treated with betaine, but was increased by fibroblast-derived exosome treatment (Figure 7).

[0077] Example 3: Confirmation of changes in gene expression by miRNA-26a

[0078] Since it was confirmed that the level of miRNA-26a increased in fibroblast-derived exosomes by betaine treatment, the efficacy of miRNA-26a in suppressing muscle loss and promoting muscle formation was confirmed as follows. C2C12 cells were treated with 100 μM dexamethasone, which is known to cause muscle loss, and cultured with 10 and 20 nM miRNA-26a mimics. Then, changes in the expression of muscle loss and myogenesis markers were confirmed by Western blotting.

[0079] As a result of confirmation, it was found that treatment with miRNA-26a analogue reduced the expression of Maf1, a marker that suppresses muscle loss, and increased the expression of MyoD, a marker that promotes muscle production (Figure 8).

[0080] Example 4: Efficacy of exosomes treated with camellia flower extract 4-1. Preparation of camellia flower extract and isolation of exosomes

[0081] Camellia flowers (Camellia Japonica Flowers) were dried overnight at 50°C using a hot air dryer and crushed. The dried flowers (100 g) were extracted overnight at room temperature with 70% (v / v) ethanol. After a filtration process, the solvent was removed using a rotary vacuum evaporator and the extract was freeze-dried to prepare the camellia flower extract.

[0082] Then, fibroblasts (FB) were treated with camellia flower extract for 48 hours, and exosomes were isolated by the method of Example 1-2. In addition, the miR-26a level was confirmed in exosomes isolated from the culture medium by the method of Example 1-4. As a result, it was confirmed that the miRNA-26a level was increased in exosomes isolated from FB treated with camellia flower extract at a concentration of 50 ppm, compared to the untreated control group (Control) (Figure 9).

[0083] 4-2. Confirmation of changes in muscle loss / muscle formation marker expression - qPCR

[0084] C2C12 cells were cultured in a 6-well plate at 1.5 × 105 The cells were seeded at a concentration of 1000 cells / well and cultured for 24 hours. The medium was then replaced with one containing 2% horse serum, and further cultured for 72 hours to promote the differentiation of C2C12 cells. After differentiation was complete, the cells were treated with 50 ppm of camellia flower extract or the exosomes (approximately 20 μg / ml) obtained in Example 4-1 and further cultured for 48 hours. Then, the expression changes of the target genes were confirmed by qPCR according to the method in Example 2-1.

[0085] As a result, it was found that the expression of muscle loss markers MAF-1 and myostatin was significantly reduced in the experimental group treated with the exosomes obtained in Example 4-1, compared to the untreated control group and the experimental group directly treated with camellia flower extract (Figures 10A and B).

Claims

1. A composition for inhibiting muscle loss or promoting muscle production, comprising: The present invention relates to a method for treating a fibroblast-related condition, comprising administering to a subject a substance selected from the group consisting of betaine, camellia flower extract, camelliaside A, myricetin, naringenin, nobiletin, L-carnosine, and copper tripeptide, or a combination thereof, for increasing the level of microRNA-26a (miRNA-26a) in extracellular vesicles secreted from fibroblasts. A composition for inhibiting muscle loss or promoting muscle formation.

2. The extracellular vesicles are exosomes The composition for inhibiting muscle loss or promoting muscle formation according to claim 1.

3. The composition reduces the expression of muscle loss-related genes selected from the group consisting of muscle ring-finger protein-1 (MURF1), atrogin-1, and myostatin. The composition for inhibiting muscle loss or promoting muscle formation according to claim 1.

4. The composition increases the expression of the myoD gene, which promotes muscle production. The composition for inhibiting muscle loss or promoting muscle formation according to claim 1.

5. The composition is in the form of a cream, lotion, ointment or gel. The composition for inhibiting muscle loss or promoting muscle formation according to claim 1.

6. The composition according to claim 1 is contained as an active ingredient. A pharmaceutical composition for preventing or treating muscle loss-related muscle diseases, comprising:

7. The muscle loss-related muscle disease is selected from the group consisting of sarcopenia, muscular atrophy, muscular dystrophy, and myasthenia. The pharmaceutical composition for preventing or treating muscle loss-related muscle diseases according to claim 6.

8. Treating fibroblasts with a substance selected from the group consisting of betaine, camellia flower extract, camelliaside A, myricetin, naringenin, nobiletin, L-carnosine and copper tripeptide, or a combination thereof; and and recovering extracellular vesicles from the culture medium of the fibroblasts. A method for producing extracellular vesicles having increased levels of microRNA-26a, comprising:

9. The extracellular vesicles are exosomes A method for producing extracellular vesicles having increased levels of microRNA-26a as described in claim 8.

10. The composition for inhibiting muscle loss or promoting muscle formation is applied to the skin. The composition for inhibiting muscle loss or promoting muscle formation according to claim 1.

Images