Compositions exhibiting an effect of inhibiting muscle loss or promoting muscle generation by extracellular vesicles derived from skin

Abstract

The present application relates to a composition capable of increasing the level of microRNA-26a in extracellular vesicles and the use of the composition, which can increase the level of microRNA-26a (miRNA-26a) in exosomes secreted from cells, and the exosomes can not only reduce the expression of biomarkers involved in muscle reduction, i.e. muscle ring finger protein 1 (MURF 1), human atrogin-1 (atrogin-1) and myostatin (myostatin), but also increase the expression of myogenic regulatory factor (myoD) involved in muscle generation. Therefore, the composition capable of increasing the level of microRNA-26a in extracellular vesicles can be used as an inhibitor of muscle loss and a promoter of muscle generation.

Description

Technical Field

[0001] This invention relates to a composition that can increase the level of microRNA-26a (miRNA-26a) in extracellular vesicles, and a composition comprising said composition that can exhibit the effects of inhibiting muscle loss or promoting muscle growth through skin-derived extracellular bodies. Background Technology

[0002] Muscles not only function as organs for movement but also influence the entire body, including bones, blood vessels, nerves, liver, heart, and pancreas. Bones rely on muscle strength to maintain density during movement and propulsion; therefore, weakened bones increase the risk of osteoporosis when muscles lose strength. Furthermore, muscle atrophy can hinder the formation of new blood vessels and nerves due to the influence of various substances produced in the muscles, potentially leading to cognitive decline.

[0003] Muscle loss, or muscle atrophy, is a lifelong process that begins around age 30, during which the amount of muscle tissue, the number of muscle fibers, and their size gradually decrease. This results in a gradual loss of muscle mass and strength. Mild muscle weakness can lead to increased stress on joints such as the knee, and also increases susceptibility to arthritis or falls. Furthermore, rapidly contracting muscle fibers are more susceptible to the effects of aging than slowly contracting ones. Therefore, as we age, muscles become less able to contract quickly, leading to various inconveniences in daily life. Summary of the Invention

[0004] Technical issues

[0005] In the case described above, the inventors have confirmed that when fibroblasts are treated with a specific substance, the level of microRNA-26a (miRNA-26a) can be increased in extracellular bodies secreted from cells, and the extracellular bodies can reduce the expression of genes involved in muscle loss while increasing the expression of genes involved in muscle formation.

[0006] Therefore, the object of the present invention is to provide a composition comprising a substance selected from or a combination thereof selected from the group consisting of betaine, camellia flower extract, camelliaside A, myricetin, naringenin, nobiletin, kojyl carboxy dipeptide-23, L-carnosine, and copper tripeptide, which can increase the level of microRNA-26a (miRNA-26a) in extracellular vesicles.

[0007] Another object of the present invention is to provide a composition comprising the composition as an active ingredient and exhibiting the effect of inhibiting muscle loss or promoting muscle formation through skin-derived extracellular bodies.

[0008] Problem-solving methods

[0009] To achieve the objectives described above, one aspect of the present invention provides a composition, as an active ingredient, comprising a substance selected from or a combination thereof from the group consisting of betaine, camellia extract, camelliaside A, myricetin, naringenin, naringin, kojic acid carboxylated dipeptide-23, L-carnosine, and copper tripeptide, which can increase the level of microRNA-26a in extracellular vesicles (hereinafter referred to as a composition for increasing microRNA-26a levels).

[0010] Betaine, also known as trimethylglycine (TMG), is abundant in plants such as sugarcane, sugar beets, and goji berries. It is used as a cosmetic ingredient due to its excellent skin-moisturizing properties. Myricetin is used as an antioxidant and skin care agent, while naringenin is also a cosmetic ingredient used as a skin care agent.

[0011] Kojic acid carboxylated dipeptide-23 is a compound with the following structure that can function as an antioxidant, metal ion blocker, and skin protectant:

[0012]

Chemical Formula 1

[0013]

[0014] In the chemical formula 1, R is dipeptide-23.

[0015] Copper tripeptide is a copper complex (GHK-Cu) of tripeptides used as a skin care agent, while camelliaside A is a component contained in green tea (Camellia sinensis) and is known to have wrinkle-improving effects (Korean Patent No. 10-0757175). Furthermore, quercetin is a polymethoxyflavonoid abundant in citrus peel and is known to have excellent anti-inflammatory effects (Domestic Patent No. 2018-0046245).

[0016] L-carnosine is a dipeptide composed of two amino acids, histidine and alanine. It is known to have antioxidant and anti-diabetic activities, and is therefore used as a nutrient and supplement.

[0017] As described above, the substances used in the compositions of the present invention are raw materials for cosmetics or nutritional supplements, or are derived from natural substances, and therefore can be safely used on the human body.

[0018] As used in this invention, the term "extract" includes extracts obtained through extraction processing, diluted or concentrated extracts, dried extracts obtained by drying the extract, crude or purified extracts, or mixtures thereof, as well as extracts of the extract itself or extracts of all dosage forms supported by the extract. The extracts of this invention can be extracted from natural, hybrid, or variant plants of the respective corresponding plants, or from plant tissue cultures.

[0019] The extraction method for the camellia extract is not particularly limited and can be performed according to methods commonly used in the relevant technical field. As non-limiting examples of the extraction method, solvent extraction, hot water extraction, ultrasonic extraction, filtration, and reflux extraction can be used, and these methods can be performed individually or in combination.

[0020] The extraction solvent used in the camellia extract is not particularly limited, and any solvent known in the relevant technical field can be used. Specifically, the camellia extract can be extracted using a solvent selected from the group consisting of water, ethyl acetate, dichloromethane, alcohols with 1-4 carbon atoms, and combinations thereof, preferably ethanol.

[0021] Liquid camellia extract can be separated from dried plant powder by methods such as vacuum filtration before concentration or drying. For example, the liquid extract can be a concentrate obtained by vacuum concentration using a rotary vacuum concentrator, or it can be dried to obtain a powdered extract. The concentrated or powdered extract described above can be dissolved in water, ethanol, dimethyl sulfoxide (DMSO), or a mixture thereof as needed for use.

[0022] The inventors confirmed that when skin fibroblasts were treated with the aforementioned substance and extracellular bodies were isolated after culture, the level of microRNA-26a (miRNA-26a) in the extracellular bodies increased compared to the case without treatment with the aforementioned substance (Table 1). Therefore, the extracellular vesicles may be secreted from fibroblasts, specifically from skin fibroblasts, but are not limited thereto.

[0023] The term "extracellular vesicle" as used in this specification refers to a very small vesicle released outside the cell that facilitates the exchange of substances such as proteins, lipids, and nucleic acids between cells, thereby acting as a medium for physiological signal transmission. Almost all cells secrete extracellular vesicles. Based on size and formation process, they are broadly classified into exosomes and microvesicles. In terms of formation process, after vesicles are formed inside the cell, exosomes are secreted while the cell membrane folds inward, and their size is approximately 30 to 200 nm. Microvesicles, on the other hand, are secreted outside the cell while the cell membrane bulges outward, and their size is approximately 50 to 1000 nm.

[0024] According to a specific embodiment of the present invention, the extracellular vesicle may be an extracellular body.

[0025] In a specific embodiment of the present invention, the composition for increasing microRNA-26a levels may contain at least 0.00001 wt% of an active ingredient (an extracellular secretion-promoting substance derived from the skin) relative to the total weight of the composition. More specifically, it may contain at least 0.00001 wt%, at least 0.0001 wt%, at least 0.0005 wt%, at least 0.001 wt%, at least 0.005 wt%, at least 0.01 wt%, at least 0.05 wt%, at least 0.1 wt%, at least 0.5 wt%, at least 1.0 wt%, at least 5.0 wt%, at least 10 wt%, or at least 50 wt% but less than 70 wt% relative to the total weight of the composition. Preferably, the composition may contain from 0.00001 wt% to 10 wt% or from 0.005 wt% to 10 wt% of the active ingredient.

[0026] Another aspect of the present invention provides a composition for inhibiting muscle loss or promoting muscle growth, comprising, as an active ingredient, the composition for increasing microRNA-26a levels.

[0027] As described above, when cells, specifically skin cells, are treated with the composition that increases the level of microRNA-26a, extracellular vesicles, specifically extracellular bodies, with increased levels of microRNA-26a can be obtained.

[0028] Furthermore, extracellular bodies are known to be used as a means of intercellular communication. For example, extracellular bodies secreted from stem cells in bone can transmit signals after reaching the heart. While it was previously believed that signal transduction between organs was primarily handled by hormones, recent findings suggest that extracellular bodies also play a role in this process. Because the skin is the largest organ in the human body, its extracellular body secretion is expected to be highly active; however, research on the functions of extracellular bodies secreted from the skin is limited.

[0029] The communication function of extracellular bodies as described above can also be confirmed through this invention. Specifically, when myofibroblasts are treated with substances such as betaine, no significant changes were observed in the expression of muscle loss and myogenesis-related genes. However, when treated with extracellular bodies whose miRNA-26a levels were increased by treatment with the aforementioned substances, a significant decrease in the expression of muscle loss-related genes and a significant increase in the expression of myogenesis-related genes were observed. Figures 4 to 7 , Figure 9 as well as Figure 10 The experimental results described above indicate that extracellular bodies derived from fibroblasts with elevated levels of miRNA-26a participate in promoting muscle growth and inhibiting muscle loss-related signaling in muscle fibroblasts.

[0030] The muscle loss-related genes can be selected from the group consisting of muscle ring finger protein 1 (MURF 1), human dystrophin-1 (atrogin-1), and myostatin, while the muscle generation-related genes can be myogenic regulatory factors (myoD).

[0031] The compositions of the present invention for inhibiting muscle loss or promoting muscle regeneration can be manufactured in any dosage form commonly manufactured in the relevant industry, such as solutions, suspensions, emulsions, pastes, gels, creams, emulsions, powders, foundations, creamy foundations, waxy foundations, and sprays, but are not limited thereto. Specifically, because the compositions of the present invention act on fibroblasts, they can be used in dosage forms such as creams, emulsions, ointments, or gels, and can be used as topical skin preparations. The compositions in the dosage forms described above can be manufactured according to methods commonly used in the relevant art.

[0032] The composition of the present invention for inhibiting muscle loss or promoting muscle growth may, in addition to the active ingredient, include ingredients commonly found in cosmetic compositions. These optional ingredients may be microparticles 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 promoters, cooling agents, antiperspirants, and purified water.

[0033] When the dosage form of the present invention is a cream or gel, the carrier component may include animal fiber, plant fiber, wax, paraffin, starch, astragalus gum, cellulose derivatives, polyethylene glycol, silicone resin, bentonite, silica, talc, or zinc oxide.

[0034] When the dosage form of the present invention is a powder or a spray, the carrier component may include lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder, especially in the case of a spray, it may contain accelerators such as chlorofluorocarbons, propane / butane or dimethyl ether.

[0035] In the case of the present invention and the behavior of the solution or emulsion, the carrier component may be a solvent, solvator or emulsifier, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylethylene glycol oil, aliphatic glycerides, polyethylene glycol or fatty acid esters of sorbitol.

[0036] When the dosage form of the present invention is a suspension, the carrier component may be liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol or polyoxyethylene sorbitol ester and polyoxyethylene dehydrated sorbitol ester, microcrystalline cellulose, aluminum hydroxide, bentonite, agar or astragalus gum, etc.

[0037] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases associated with muscle loss, comprising, as an active ingredient, a composition for increasing microRNA-26a levels.

[0038] The muscle diseases associated with sarcopenia may be selected from the group consisting of sarcopenia, muscular atrophy, muscular dystrophy, and myasthenia gravis.

[0039] Sarcopenia is a disease that causes a decline in normal muscle mass, strength, and function due to malnutrition, reduced physical activity, and aging. Muscular dystrophy, on the other hand, is a highly diverse clinical and genetic disease characterized by symmetrical muscle weakness or loss due to genetic or other causes.

[0040] Muscular dystrophy (MD) is a muscle disorder that weakens muscles and impairs motor function. It is characterized by progressive skeletal muscle aging, muscle protein deficiency, and necrosis of muscle cells and tissues. Myasthenia gravis is a condition characterized by abnormal weakness or fatigue of muscles. If left untreated, it can lead to sudden muscle weakness and, in severe cases, even respiratory paralysis due to weakened respiratory muscles.

[0041] In the pharmaceutical composition, any part of the term or element that is identical to the description in the composition for increasing microRNA-26a levels may be understood to have the same meaning as the description in the requested composition for increasing microRNA-26a levels.

[0042] In addition to the active ingredient, the pharmaceutical composition of the present invention may also contain a pharmaceutically permissible carrier. In this case, the pharmaceutically permissible carrier may be a substance commonly used in formulation, including, but not limited to, substances such as lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyridinone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. Furthermore, in addition to the aforementioned components, additional components such as lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives may be added.

[0043] The pharmaceutical compositions of the present invention can be administered orally or non-orally according to the desired method (e.g., skin application, intravenous injection, subcutaneous injection, intraperitoneal injection, or local administration), but non-oral administration is preferred.

[0044] When the active ingredients of the present invention are formulated into preparations such as tablets, capsules, chewable tablets, powders, liquids, and suspensions for oral administration, they may contain binders such as gum arabic, corn starch, microcrystalline cellulose, or gelatin; excipients such as dicalcium phosphate or lactose; disintegrants such as alginic acid, corn starch, or potato starch; lubricants such as magnesium stearate; sweeteners such as sucrose or gentian; and flavorings such as peppermint, methyl salicylate, or fruit flavorings.

[0045] In addition, the non-oral administration form can be a transdermal formulation, such as an injection, adhesive, ointment, lotion, gel, cream, spray, suspension, emulsion, suppository and patch, but is not limited to these.

[0046] Furthermore, the pharmaceutical composition may be in the form of a topical skin preparation, which is a general term encompassing any dosage form that can be applied to the external skin, including pharmaceuticals in multiple dosage forms.

[0047] The pharmaceutical compositions of the present invention are administered in pharmaceutically effective amounts. In this invention, a "pharmaceutically effective amount" means an amount sufficient to treat a disease in a reasonable benefit / risk ratio applicable to medical treatment. The level of an effective amount can be determined based on the patient's disease type, severity, drug activity, drug sensitivity, timing of administration, route of administration, excretion rate, treatment duration, factors including concurrently used drugs, and other factors known in the medical field. For example, the pharmaceutical compositions of the present invention can be administered once daily or three times daily at a volume of 1 μg / kg to 200 mg / kg, preferably 50 μg / kg to 50 mg / kg. The dosages described are not intended to limit the scope of the invention in any way.

[0048] The pharmaceutical compositions according to the invention can be administered as a standalone therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, or alone or in multiple doses. It is important that the dosage be administered in a minimum amount that yields the maximum effect without any side effects, taking all of the aforementioned factors into account; this can be easily determined by those skilled in the art.

[0049] Another aspect of the present invention provides a composition comprising, as an active ingredient, extracellular vesicles obtained by treating cells with a substance selected from the group consisting of betaine, camellia extract, camelliaside A, myricetin, naringenin, naringin, kojic acid carboxylated dipeptide-23, L-carnosine, and copper tripeptide, or a combination thereof (hereinafter referred to as extracellular vesicle composition), and a composition comprising the extracellular vesicle composition as an active ingredient for inhibiting muscle loss or promoting muscle growth.

[0050] The inventors confirmed that when fibroblasts derived from the skin were treated with the aforementioned substance, the level of microRNA-26a in the extracellular bodies secreted by the fibroblasts increased, and that the extracellular bodies had the effect of inhibiting muscle loss or promoting muscle regeneration. Figures 4 to 7 , Figure 9 as well as Figure 10 ).

[0051] In the context of the extracellular vesicle composition, any part of the term or element that is identical to the description in the composition for increasing microRNA-26a levels may be understood to have the same meaning as the description in the requested composition for increasing microRNA-26a levels.

[0052] Another aspect of the present invention provides a method for producing extracellular vesicles in which the level of microRNA-26a is increased, comprising the following steps:

[0053] The steps include treating cells with a substance selected from or a combination thereof consisting of betaine, camellia extract, camelliaside A, myricetin, naringenin, kojic acid carboxylated dipeptide-23, L-carnosine, and copper tripeptide; and recovering extracellular vesicles from the cell culture medium.

[0054] In a specific embodiment of the present invention, the cells may be fibroblasts derived from the skin, and the extracellular vesicles may be extracellular bodies, but are not limited thereto.

[0055] Furthermore, the method for isolating extracellular vesicles and / or extracellular bodies from the cell culture medium is as described in Examples 1-2. However, in addition to the isolation method described above, various methods known in the art can also be used as methods for isolating extracellular vesicles and / or extracellular bodies from the cell culture medium.

[0056] For example, to separate extracellular vesicles and / or extracellular bodies, 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, extracellular body separation methods are not limited to those described above, but can utilize various separation methods currently in use or likely to be used in the relevant industry.

[0057] Invention Effects

[0058] Using a composition according to one embodiment of the present invention that can enhance the level of microRNA-26a in extracellular vesicles, extracellular bodies with enhanced microRNA-26a levels can be manufactured. Because these extracellular bodies can not only reduce the expression of biomarkers involved in muscle loss, namely muscle ring finger protein 1 (MURF 1), atrogin-1, and myostatin, but also increase the expression of myogenic regulatory factor (myoD), which is involved in muscle formation, the composition that can enhance the level of microRNA-26a in extracellular vesicles can be effectively used for inhibiting muscle loss or promoting muscle formation. Attached Figure Description

[0059] Figure 1 This is a result confirming the cell survival rate after treating long fibroblasts (A) and muscle fibroblasts (B) with different concentrations of betaine.

[0060] Figure 2This is a result confirming whether muscle fiber cells are absorbed into the cells after treatment with fluorescently labeled extracellular bodies derived from fibroblasts.

[0061] Figure 3 This is a confirmation of the level of miRNA-26a in extracellular bodies derived from fibroblasts isolated by treatment with different concentrations of betaine.

[0062] Figure 4 This is a result confirming the expression levels of muscle loss markers, namely muscle ring finger protein 1, human dystrophin-1, and myostatin, after treatment of muscle fibroblasts with betaine or extracellular bodies derived from fibroblasts.

[0063] Figure 5 This is a result confirming the expression level of myogenic regulatory factor (MyoD), a marker of muscle formation, after treatment of muscle fibroblasts with betaine or extracellular bodies derived from fibroblasts.

[0064] Figure 6 This is the result of confirming the protein levels of muscle loss markers, namely muscle ring finger protein 1, human dystrophin-1, and myostatin, after treatment of muscle fibroblasts with betaine or extracellular bodies derived from fibroblasts.

[0065] Figure 7 This is a result confirming the protein levels of myogenic regulatory factor (MyoD), a marker of muscle formation, after treatment of muscle fibroblasts with betaine or extracellular bodies derived from fibroblasts.

[0066] Figure 8 This is a result confirming the expression levels of muscle ring finger protein 1 and myogenic regulatory factor (MyoD) at different treatment concentrations of miRNA-26a derivatives after treatment of muscle fibroblasts with dexamethasone.

[0067] Figure 9 This is a confirmation of the level of miRNA-26a in the extracellular bodies isolated after fibroblasts were treated with camellia extract.

[0068] Figure 10 This is a result confirming the expression levels of muscle wetting markers, namely muscle ring finger protein 1 (A) and myostatin (B), after treating muscle fibroblasts with camellia extract or extracellular bodies isolated from fibroblasts treated with camellia extract. Detailed Implementation

[0069] The following will provide a detailed description of one or more specific examples through embodiments. However, these embodiments are merely illustrative examples, and the scope of the present invention is not limited by these embodiments.

[0070] Example 1: Isolation and Analysis of Extracellular Bodies

[0071] 1-1. Confirmation of the cytotoxicity of betaine

[0072] As human dermal cells, Normal Human Dermal Fibroblasts (NHDF, fibroblasts) isolated from adult samples were purchased from LONZA (Cat. CC-2511). As muscle fibroblasts, myoblasts (C2C12 cells) isolated and cultured from C3H mice were purchased from ATCC (CRL-1772).

[0073] The passage 27 cells, namely human fibroblast HS68 (hereinafter referred to as FB) or C2C12, were cultured in 96-well plates at a concentration of 1×10⁻⁶. 4 Cells were seeded at a concentration of [number] cells / well and cultured for 24 hours in a 5% CO2 incubator at 37°C. Following culture, cells were treated with different concentrations of betaine and cultured for an additional 24 hours, while an untreated control group was used. After washing with phosphate-buffered saline (PBS), cells were cultured for 2 hours on CCK-8 (DONGJIN, CK04-11), and absorbance was measured at 450 nm using a microplate reader. The relative cell viability at different betaine treatment concentrations was calculated using the control group as 100.

[0074] The results confirm that even with treatment using betaine, the survival rates of FB and C2C12 did not change. Figure 1 (A and B in the text).

[0075] 1-2. Isolation of extracellular bodies

[0076] Culture medium from passage 27 (FB) cells treated with betaine for 48 hours was collected and centrifuged at 3000 x g for 30 minutes. Following centrifugation, only the supernatant was recovered and transferred to Ultra-15 Centrifugal Filter Units (Amicon®, MERCK, C7715), followed by centrifugation at 4000 x g for 40 minutes. Only the supernatant was recovered, and half the volume of the supernatant was added to Total Exosome Isolation Reagent (Invitrogen, Cat No. 4478539), and the reaction was carried out overnight at 4°C. The following day, centrifugation was performed at 10000 x g for 1 hour, and the final extracellular particles were resuspended in island phosphate-buffered saline (PBS) after the supernatant was removed by suction. The extracellular particles obtained in this embodiment will be referred to as "fibroblast-derived extracellular particles".

[0077] To confirm the size of extracellular bodies derived from fibroblasts (FB), dynamic light scattering was measured using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK), and the results were analyzed using Dynamic V6 software.

[0078] The analysis results showed that the size of the extracellular bodies derived from fibroblasts ranged from 50 to 150 nm.

[0079] In addition, to quantify extracellular bodies derived from fibroblasts (FB), the absorbance at 405 nm was measured using the EXOCET Exosome Quantitation Kit (System Biosciences, USA).

[0080] Based on the analysis results, all experimental groups were treated with fibroblast-derived extracellular bodies at a concentration of 20 μg / ml.

[0081] 1-3. Confirmation of whether extracellular bodies derived from fibroblasts are absorbed by cells.

[0082] The study confirmed whether fibroblast-derived extracellular bodies could be absorbed into C2C12 cells. C2C12 cells were seeded at 1.5 × 10⁶ cells per cell line on Lab-Tek chamber slides (Nunc Penfield, NY). 4The cells were cultured, and after 20 minutes of treatment with bodipy TR ceramide staining reagent, excess staining reagent was removed using a purification kit. C2C12 cells were then observed using confocal microscopy after 30 minutes of treatment with the fibroblast-derived extracellular bodies labeled with the staining reagent.

[0083] The observation results confirmed that a signal representing extracellular bodies, shaped like red spots, was observed in C2C12 cells, indicating that extracellular bodies originating from fibroblasts were absorbed into the cells.

[0084] 1-4. Confirmation of miRAN-26a levels in extracellular bodies derived from fibroblasts treated with betaine

[0085] In 2×10 6 After seeding FB cells into 75T flasks and culturing for 24 hours, they were treated with betaine (0.1, 1, and 10 mM). After an additional 48 hours of culture, extracellular bodies were isolated according to the methods in Examples 1-2. Subsequently, microRNA (hereinafter referred to as miRNA) was extracted from the extracellular bodies isolated from the culture medium using the RNeasy plus mini kit (Qiagen, Germany) according to the manufacturer's specifications.

[0086] Real-time qPCR was performed using TaqMan probes targeting mature RNA. Relative expression levels of intracellular target miRNAs were averaged using the expression levels of RNU48 (used as a housekeeping gene) for miRNA quantification and recorded as relative %. Relative expression levels of extracellular miRNA-26a were averaged using the expression levels of microRNA-26a (used as a housekeeping gene) for quantifying extracellular miRNAs derived from fibroblasts and recorded as relative %. All real-time qPCR analyses were performed using an Applied Biosystems 7500 system.

[0087] Analysis of the results confirmed that, compared with the control group not treated with betaine, the level of miRNA-26a was increased in the extracellular bodies isolated from FB cells treated with betaine. Figure 3 ).

[0088] 1-5. Isolation of additional extracellular bodies

[0089] After treating FB with the substances listed in Table 1 below, the extracellular bodies were isolated using the same method as in Example 1-1. The level of miRNA-26a in the extracellular bodies was then confirmed using the method described in Examples 1-4.

[0090] Table 1

[0091]

[0092]

[0093] Example 2: Confirmation of gene expression changes in muscle fibroblasts

[0094] 2-1. Confirmation of Muscle Loss / Changes in Myogenic Marker Expression - qPCR

[0095] C2C12 cells were cultured in 6-well plates at a density of 1.5 × 10⁻⁶ cells / well. 5 C2C12 cells were seeded at a concentration of [number] cells / well and cultured for 24 hours. Next, the medium was replaced with medium supplemented with 2% horse serum and cultured for an additional 72 hours to promote differentiation. After differentiation, the cells were treated with 0.5 mM betaine or extracellular fibroblasts obtained in Examples 1-2 (approximately 20 μg / ml) and cultured for an additional 48 hours. Total RNA was extracted using the RNeasyplus mini kit, and cDNA was synthesized using superscript™ m (Invitrogen, USA). Analysis was then performed using qPCR with the gene's TaqMan probe.

[0096] Analysis confirmed that, compared to the control group, treatment of C2C12 muscle fibroblasts with betaine increased the expression of muscle loss markers MURF1 (muscle ring finger 1) and atrogin-1, while myostatin expression remained at a similar level. However, none of the three genes showed significant changes. Conversely, treatment of C2C12 cells with extracellular bodies derived from fibroblasts significantly reduced the expression of muscle loss markers MURF1, atrogin-1, and myostatin. Figure 4 (A to C in the original text).

[0097] Furthermore, when betaine was used to directly treat myofibroblasts (C2C12 cells), the expression of the myogenic marker Myogenic differentiation 1 (MyoD) was increased compared to the control group, but not significantly. Conversely, treatment with extracellular bodies derived from fibroblasts confirmed a significant increase in the expression of MyoD. Figure 5 ).

[0098] 2-2. Confirmation of Muscle Loss / Changes in Myogenic Marker Expression - Western Blotting

[0099] C2C12 cells were cultured in 6-well plates at a rate of 5 × 10⁻⁶. 5 C2C12 cells were seeded at a concentration of [number] cells / well and cultured for 24 hours. Next, the medium was replaced with medium supplemented with 2% horse serum and cultured for an additional 72 hours to promote differentiation. After differentiation, the cells were treated with betaine (0.1, 0.5 mM) or extracellular fibroblasts and cultured for an additional 48 hours. Cells were then lysed by adding a solubilization buffer containing proteolytic enzyme inhibitors (1% NP40, 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.01 M MgCl2), and protein concentration was determined by the BCA (bovine carbonic anhydrase) method. Quantified proteins were separated by SDS-PAGE and transferred to PVDF membranes, and the protein expression levels of various muscle loss markers were confirmed using antibodies.

[0100] The results showed that the expression of muscle loss markers did not change when C2C12 cells were treated directly with 0.1 or 0.5 mM betaine. However, the expression of muscle loss markers, namely muscle ring-finger protein-1, dystrophin-1, and myostatin, was reduced when treated with extracellular bodies derived from fibroblasts. Figure 6 Furthermore, the expression of myogenic marker Myogenic regulatory factor (MyoD) did not change when C2C12 cells were treated directly with betaine, but it increased when treated with extracellular bodies derived from fibroblasts. Figure 7 ).

[0101] Example 3: Confirmation of gene expression changes based on miRNA-26a

[0102] Since it has been confirmed that the level of miRNA-26a in the extracellular bodies derived from fibroblasts increases upon treatment with betaine, the miRNA-26a-based inhibitory effect on muscle loss and myogenesis was investigated using the method described below. C2C12 cells were treated with 100 μM of dexamethasone, a known muscle-inducing agent, and then treated and cultured with 10 and 20 nM of miRNA-26a analogs (mimics). Subsequently, Western blotting was used to confirm changes in the expression of markers of muscle loss and myogenesis.

[0103] The results confirmed that treatment with miRNA-26a analogs reduced the expression of muscle loss inhibitory marker 1 (Ring Finger Protein 1) and increased the expression of muscle growth promoting marker Myogenic Factor (MyoD). Figure 8 ).

[0104] Example 4: Confirmation of the efficacy of extracellular bodies treated with camellia extract

[0105] 4-1. Preparation of Camellia Flower Extract and Isolation of Extracellular Bodies

[0106] Camellia japonica flowers were dried overnight at 50°C using a hot air dryer and then pulverized. The dried camellia flowers (100g) were extracted overnight at room temperature using 70% (v / v) ethanol. After filtration, the solvent was removed using a rotary vacuum evaporator, and the mixture was freeze-dried to produce the camellia flower extract.

[0107] Next, after treating fibroblasts (FB) with camellia extract for 48 hours, extracellular bodies were isolated according to the methods described in Examples 1-2. Furthermore, the level of miR-26a in the extracellular bodies isolated from the culture medium according to the methods described in Examples 1-4 was confirmed. The confirmation results showed that, compared with the untreated group (Control), the level of miRNA-26a in the extracellular bodies isolated from FB treated with 50 ppm of camellia extract was increased. Figure 9 ).

[0108] 4-2. Confirmation of Muscle Loss / Changes in Myogenic Marker Expression - qPCR

[0109] C2C12 cells were cultured in 6-well plates at a density of 1.5 × 10⁻⁶ cells / well. 5C2C12 cells were seeded at a concentration of [number] cells / well and cultured for 24 hours. Next, the medium was replaced with medium supplemented with 2% horse serum and cultured for an additional 72 hours to promote differentiation. After differentiation, the cells were treated with 50 ppm camellia extract or the extracellular matrix obtained in Example 4-1 (approximately 20 μg / ml) and cultured for an additional 48 hours. The expression changes of the target gene were then confirmed by qPCR according to the method in Example 2-1.

[0110] The results showed that, compared with the untreated group (control group) and the experimental group treated directly with camellia extract, the expression of muscle loss markers, namely muscle ring finger protein 1 and myostatin, was significantly reduced in the experimental group treated with the extracellular bodies obtained in Example 4-1. Figure 10 (A and B in the text).

Claims

1. The use of one or more of the following substances in vitro—betaine, camellia flower extract, camelliaside A, myricetin, naringenin, nobiletin, kojyl carboxy dipeptide-23, L-carnosine, and copper tripeptide—to increase the level of microRNA-26a (miRNA-26a) in the extracellular bodies secreted by fibroblasts, characterized in that, When the substance was used to treat fibroblasts derived from the skin, the level of microRNA-26a in the extracellular bodies secreted by the fibroblasts increased.

2. A composition for inhibiting muscle loss or promoting muscle growth, The active ingredient includes an extracellular substance obtained by treating fibroblasts with a substance selected from the group consisting of betaine, camellia extract, camellia glycoside A, myricetin, naringenin, naringin, kojic acid carboxylated dipeptide-23, L-carnosine, and copper tripeptide.

3. The composition for inhibiting muscle loss or promoting muscle growth according to claim 2, This increases the level of microRNA-26a in the extracellular body.

4. A method for producing extracellular bodies with enhanced levels of microRNA-26a, comprising: The step of treating fibroblasts with a substance selected from the group consisting of betaine, camellia extract, camelliaside A, myricetin, naringenin, naringin, kojic acid carboxylated dipeptide-23, L-carnosine, and copper tripeptide; and, The steps for recovering extracellular bodies from fibroblast culture medium.

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