KR102053208B1 - Compositions for muscle regeneration containing Ginsenoside Rb1 and Rb2 as active ingedients - Google Patents

Abstract

A composition for muscle regeneration comprising ginsenosides Rb1 and Rb2, wherein the composition is a pharmaceutical composition for treating or preventing muscle atrophy or muscle cancer, or a food for improving or alleviating muscle atrophy or muscle cancer It may be a composition. The composition modulates the activity of MyoD, a muscle transcription factor, to induce differentiation of myoblasts into myotubes, induce fibroblasts to myoblasts, or re-differentiate rhabdomyosarcoma cells to myoblasts.

Description

Composition for muscle regeneration containing Ginsenoside Rb1 and Rb2 as active ingedients}

The present invention relates to a composition for muscle regeneration, in particular to a pharmaceutical composition for the treatment or prevention of muscle atrophy (Muscle Atrophy) or muscle cancer, and a composition for health food for improving or alleviating muscle atrophy or muscle cancer It is about. The present invention also relates to the use of ginsenosides Rb1 and Rb2 for muscle regeneration, and to a method for promoting muscle regeneration comprising administering to a subject in need thereof an effective dose of ginsenosides Rb1 and Rb2.

Diseases associated with muscle abnormalities include muscle loss and related diseases such as sarcopenia, cachexia, muscle damage, muscular dystrophy and muscle fatigue. Treatment of muscle disease includes maintenance of muscle performance, strength and muscle function.

Muscle loss is characterized by a gradual loss of muscle mass, especially weakness and degeneration of skeletal or veterinary and cardiac muscle. Atrophy and hypertrophy occur in all mammalian species. Various studies have demonstrated that the same molecular mechanisms, cellular and physiological processes are identical in atrophy in both rodents and humans.

Muscle loss is due to a variety of causes and is associated with a variety of conditions, diseases and disorders. These include muscular dystrophy caused by genetic abnormalities, such as Duchenne Muscular Dystrophy (DMD), progressive muscular dystrophy, Becker-type muscular dystrophy, Dejerine-Landouzy Muscular dystrophy, Erb's muscular dystrophy, spinal muscular atrophy, and infantile axonal decompensation include, but are not limited to.

Muscle loss can also be caused by various chronic diseases and aging processes. As the body ages, some of the newly created skeletal muscle is replaced by fibrous tissue. Therefore, normal aging in humans is associated with a progressive decrease in skeletal muscle mass and strength, a condition called sarcopenia, which causes weakness and degeneration.

In addition, age-related diseases such as hypertension, impaired glucose tolerance and diabetes, obesity, dyslipidemia, atherosclerosis and cardiovascular diseases are also associated with loss of muscle mass.

In addition, cancer, autoimmune diseases, infectious diseases, HIV infection, AIDS, chronic inflammation, arthritis, malnutrition, kidney disease, chronic obstructive pulmonary disease (COPD), emphysema, rickets, chronic lower spine pain, peripheral nerve damage, spinal cord injury Other diseases, such as chemical damage, central nervous system (CNS) damage, can also be associated with or cause muscle loss. Finally, the conditions that cause muscle loss can result from lung conditions such as organ fixation due to disease, or powerlessness such as long term wheelchair use, long bed rest, fractures or trauma. Postoperative bedside care is estimated to cause loss of skeletal muscle mass of about 10% per week.

Cancers that cause muscle loss include soft-tissue sarcoma that occurs in muscle, and specifically, leiomyosarcoma and rhabdomyosarcoma.

Leiomyosarcoma and rhabdomyosarcoma are malignant tumors with symptoms such as lumps, pains, headaches, nausea and bleeding. Surgical surgery, chemotherapy, radiation therapy, etc. are mobilized for the treatment of such diseases. Drugs such as halaven (Halaven, erybulin mesylate) have been developed for drug treatment, but various side effects such as fatigue, neutropenia, nausea, hair loss, constipation, abdominal pain and fever have been reported.

Untreated muscle loss disease can lead to serious health problems, and changes that occur during muscle loss can lead to a weakened physical condition resulting in incompetent physical performance and detrimental health conditions.

Muscle loss due to chronic disease can cause premature loss of motility and increase the risk of disease-related mortality. Muscle loss due to lung use is a particularly serious problem in older people who may already have age-related deficits in muscle function and muscle mass, leading to an increase in fracture rates as well as permanent helplessness and premature death. Despite the clinical significance of the condition, there are few treatments for preventing or reversing the condition and a solution is required.

Panax Ginseng Meyer, meanwhile, has been used as a traditional medicinal plant for thousands of years in Japan, China and Korea. In Chinese medicine, ginseng has traditionally been used to energize the body and mind, prevent aging, and increase stamina and vitality. Ginsenosides were p. It is the main pharmacologically active compound of Ginseng and is classified into three types based on aglyconic residues (protopanaxadiol-, protopanaxatriol-, and oleanolic acid-type ginsenosides). The effects of ginsenosides on the differentiation of myogenic cells and on the growth of muscle in relation to C2C12 myoblasts have not been studied, and the effects of ginsenosides Rb1 and Rb2 on muscle differentiation and the formation of myotubes have not been identified It has never been.

Accordingly, the present invention relates to a pharmaceutical composition for preventing or treating muscle regeneration, specifically, muscle atrophy or muscle cancer, and more specifically, ginsenosides Rb1 and Rb2 are activated through activation of the signaling pathway of Akt. Promotes the differentiation of MyoD-mediated myoblasts, promotes the differentiation of fibroblasts into myoblasts, enhances muscle regenerative capacity, prevents fibrosis of skeletal muscle, and re-differentiates Rhabdomyosarcoma (RD) cells into myoblasts The present invention was completed by identifying the phenomenon.

Korean Patent Publication No. 10-2010-0083598

One object of the present invention to provide a composition for muscle regeneration, and more particularly to provide a pharmaceutical composition containing ginsenosides Rb1 and Rb2 as an active ingredient for treating or preventing muscle atrophy. In addition, the present invention is to provide a composition for food containing ginsenosides Rb1 and Rb2 as an active ingredient to improve or alleviate muscle atrophy.

Another object of the present invention is to provide a pharmaceutical composition containing ginsenosides Rb1 and Rb2 as active ingredients for treating or preventing muscle cancer, in particular soft tissue sarcoma, more specifically leiomyosarcoma or rhabdomyosarcoma. In addition, the present invention is to provide a food composition containing ginsenosides Rb1 and Rb2 as an active ingredient to improve or alleviate muscle cancer.

The present invention also relates to the use of ginsenoside Rg1 for muscle regeneration, and to provide a method for promoting muscle regeneration, comprising administering an effective dose of ginsenosides Rb1 and Rb2 to a subject to be administered.

In addition, the present invention provides an optimized mixing ratio of ginsenosides Rb1 and Rb2, the active ingredient of the composition, to maximize the effect of treating, preventing or improving muscle regeneration and muscular atrophy, sarcopenia, or muscle cancer do.

The present invention provides a composition for muscle regeneration comprising ginsenosides Rb1 and Rb2 as an active ingredient.

In the composition, the ratio of ginsenosides Rb1 and Rb2 may be contained in a molar ratio of Rb1: Rb2 = 1: 2 to 1: 4. Preferably, the ratio of ginsenosides Rb1 and Rb2 may be contained in a molar ratio of Rb1: Rb2 = 1: 2.5 to 1: 3.5.

The composition may induce differentiation of myoblasts into myotubes or differentiation of fibroblasts into myoblasts by regulating the activity of MyoD, a muscle transcription factor.

The composition may be a pharmaceutical composition for preventing or treating muscular atrophy.

The composition may be a composition for health food for improving or alleviating muscular atrophy.

The muscular dystrophy may include aging muscular dystrophy, degenerative muscular dystrophy, diabetic muscular dystrophy, rented muscle atrophy, Becker Muscular Dystrophy (BMD), Duchenne Muscular Dystrophy (DMD), myotonic muscular dystrophy (Myotonic Muscular Dystrophy) ), Emery-Dreifuss Muscular Dystrophy (EDMD), Distal Muscular Dystrophy (DD), Facioscapulohumeral Muscular Dystrophy (FSHD), or Congenphysio Muscular Dy can do.

The composition may be a pharmaceutical composition for preventing or treating muscle cancer.

The composition may be a composition for health food for improving or alleviating muscle cancer.

The muscle cancer may be soft tissue sarcoma, preferably, the soft tissue sarcoma may be smooth muscle sarcoma (Leiomyosarcoma) or rhabdomyosarcoma (Rhabdomyosarcoma).

The composition for muscle regeneration according to the present invention promotes the differentiation of MyoD-mediated myoblasts through activation of the signaling pathway of Akt, promotes the differentiation of fibroblasts into myoblasts, and rhabdomyosarcoma (RD) cells. May promote re-differentiation into myoblasts.

In addition, the composition for muscle regeneration according to the present invention promotes the expression of myogenic specific proteins, can regulate the differentiation activity to promote the differentiation of myoblasts into muscle cells, and enhance the regeneration ability of muscle cells It can promote multinucleated myofibroblast formation without cell death, thereby treating or preventing, ameliorating or alleviating muscle atrophy, sarcopenia, or muscle cancer.

In addition, the present invention provides an optimized specific mixing ratio of ginsenosides Rb1 and Rb2, which are the active ingredients of the composition, when the composition satisfies the specific mixing ratio can maximize the effect of the source cell differentiation.

1 shows immunostaining results (FIG. 1A), mean myotube diameter (FIG. 1B), and immunoblotting to determine whether Rb1 and Rb2 enhance hypertrophy of Akt / mTOR signaling-mediated myotubes. Immunoblotting results (FIG. 1C, 1D).
Figure 2 shows the results of immunostaining (immunostaining) to confirm whether Rb1 or Rb2 promotes myogenic differentiation (promyogenic kinase), Akt to promote myogenic differentiation (Fig. 2A, 2F), quantitative analysis results (Fig. 2B, 2E) and immunoblotting results (Figures 2C, 2D).
3 is an immunoblotting result (FIG. 3A) and a quantitative analysis result (FIG. 3B) to examine whether synergistic effects on myogenic differentiation when Rb1 and Rb2 are combined.
4 is an immunoblotting result (FIGS. 4A and 4B) to confirm whether Rb1 and Rb2 promote heterodimerization of MyoD and E proteins.
5 is an immunoblotting result (FIG. 5A) and an immunostaining result (FIG. 5B) to confirm whether Rb1 and Rb2 enhance MyoD-mediated myogenic conversion of RD cells.

Myoblast differentiation is a finely coordinated multistage process that involves proliferation of myoblasts, cell cycle shrinkage, expression of muscle-specific transcriptional activity, cell extension and fusion to myotubes. This process is controlled by myogenic regulatory factors (MRF) such as Myf5, MRF4, MyoD and myogenin, and these transcription factors are strictly regulated to ensure efficient cell differentiation.

Akt plays an important role as a promyogenic kinase, and Akt signal transduction contributes to heterodimerization of MyoD and E proteins and changes in chromatin modification at muscle-specific loci. The Akt / mTOR pathway is an important regulator of skeletal muscle hypertrophy associated with increased protein synthesis. However, the phosphatidylinositol-3-kinase (PI3K) / Akt pathway prevents muscle atrophy by inhibiting atrophy-associated ubiquitin ligase and FoxO transcription factors.

Ginsenosides Rb1 and Rb2, classified under the Protopanaxadiol type, are the most abundant ginsenosides in Panax ginseng (CA Meyer). Rb1 and Rb2 act as AMP-activated protein kinase (AMPK) activators and can regulate glucose homeostasis. In particular, Rb1 activates the PI3K / Akt / Nrf2 pathway to reduce oxidative stress in older skeletal muscles. However, the effects of Rb1 and Rb2 on myoblast differentiation and root canal cell growth have not been reported previously.

We understand the mechanisms of molecular regulation of muscle stem cell differentiation and muscle growth, and to identify potential activators for myoblast differentiation, MyoD-reactive reporter activity and myosin heavy in myoblasts. As a regulator of MyoD-mediated myoblast differentiation for expression of chain, MHC), protopanaxatriol-type ginsenosides Rb1 and Rb2 were selected through screening.

Treatment of ginsenosides Rb1 and Rb2 enhances myocyte differentiation through upregulation of key promyogenic signals, Akt signaling, increases heterodimerization to MyoD and E proteins, and MyoD-transformation. Through this, it is possible to induce muscle differentiation of embryonic fibroblasts (10T1 / 2) and re-differentiation of rhabdomyosarcoma (RD). Muscle differentiation provides an excellent model for understanding cell fate specific and regulatory mechanisms of differentiation to enhance muscle regeneration.

In addition, the present inventors studied the effects and molecular mechanisms of ginsenosides Rb1 and Rb2 on myoblast differentiation through experiments, and as a result, it was confirmed that ginsenosides Rb1 and Rb2 promote differentiation of myoblasts.

In the present invention, the molecular framework of ginsenosides Rb1 and Rb2 which promotes hypertrophy of myotube cells by upregulating Akt / mTOR signaling and enhancing Akt-mediated myogenic differentiation with active heterodimerization of MyoD Furthermore, it has been confirmed that ginsenosides Rb1 and Rb2 can prevent and treat age-related muscular dystrophy by increasing the differentiation of RD cells into myogenic cells.

In conclusion, ginsenosides Rb1 and Rb2 of the present invention are myogenic cell differentiation and root canal cells through activation of Akt-mediated gene expression accompanied by major muscle differentiation signals, particularly activated Akt / mTOR signal transduction and active heterodimerization of MyoD. May promote hypertrophy and positively regulate MyoD-mediated myoblast differentiation to regenerate damaged muscle or treat atrophy and muscle cancer.

According to one embodiment of the present invention, ginsenosides Rb1 and Rb2 may be a composition for muscle regeneration containing an active ingredient.

The muscle regeneration may be regeneration of damaged muscle cells. The damage may be due to genetic factors and environmental factors such as surgery, cancer, inflammation, infection. Muscle regeneration may be achieved through muscle cell differentiation, and the muscle cell differentiation may be differentiation into myocytes and muscle cells such as myocytes or fibroblasts capable of differentiation into muscle cells, and the differentiation into the muscle cells may be MyoD. It may be due to the activity regulation of transcriptional regulators.

When the ginsenosides Rb1 and Rb2 are treated in combination, a much better effect can be obtained compared with the case where the ginsenosides Rb1 or Rb2 are treated respectively. Specifically, expression of MHC may be more effective when co-treated with Rb1 and Rb2, and C2C12 cells co-treated with Rb1 and Rb2 may show a gradual increase in phosphorylation of Akt and also contain six or more nuclei. The proportion of MHC-positive myotubes can be significantly increased.

 In addition, in the present invention, the ratio of ginsenosides Rb1 and Rb2 may be preferably contained in a molar ratio of Rb1: Rb2 = 1: 2 to 1: 4, and more preferably in a molar ratio of Rb1: Rb2 = 1: 2.5 to 1: 3.5 may be contained. When the mixing ratio of the ginsenosides Rb1 and Rb2 satisfies the above range, MHC expression may be more effective than when the range of the combination does not satisfy the above range, and MHC-positive root canal containing 6 or more nuclei. The advantage is that cells can be obtained at a higher rate.

According to one embodiment of the invention, the composition of the present invention may be prepared as a pharmaceutical composition for the treatment or prevention of muscular atrophy.

In addition, the composition of the present invention may be prepared as a composition for health food for the prevention or improvement of muscular atrophy.

Muscular atrophy may include aging sarcopenia, degenerative sarcopenia, diabetic sarcopenia.

The muscular dystrophy includes: atrophic atrophy, Becker Muscular Dystrophy (BMD), Duchenne Muscular Dystrophy (DMD), Myotonic Muscular Dystrophy, Emery-Dreisfufu (Emery-Dreisfu) Muscular Dystrophy (EDMD), Distal Muscular Dystrophy (DD), Facioscapulohumeral Muscular Dystrophy (FSHD), and Congenital Muscular Dystrophy (CMD). no.

Although it is possible to administer Rb1 and Rb2 of the present invention for use in the treatment and prevention of muscular dystrophy, it is preferred that Rb1 and Rb2 are included as active ingredients of the pharmaceutical composition.

According to one embodiment of the present invention, the composition of the present invention may be prepared as a pharmaceutical composition for the treatment or prevention of muscle cancer.

In addition, the composition of the present invention may be prepared as a composition for health food for the prevention or improvement of muscle cancer.

The muscle cancer may be soft tissue sarcoma, and the soft tissue sarcoma may include leiomyosarcoma or rhabdomyosarcoma, but is not limited thereto.

Although it is possible to administer Rb1 and Rb2 of the present invention for use in the treatment and prevention of muscle cancer, it is preferred that Rb1 and Rb2 are included as active ingredients of the pharmaceutical composition.

The pharmaceutical composition according to the present invention may contain the ginsenosides Rb1 and Rb2 as an active ingredient and may include a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included.

In addition, diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Suitable pharmaceutically acceptable carriers and formulations may be preferably formulated according to each component using the methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, or external skin preparation.

The method of administering the pharmaceutical composition of the present invention is not particularly limited, but may be parenterally or orally administered, such as intravenous, subcutaneous, intraperitoneal, inhalation, dermal application or topical application, depending on the desired method.

Dosage varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient.

Daily dosage refers to the amount of therapeutic substance of the invention sufficient for treatment for a disease state alleviated by administration to a subject in need thereof. Effective amounts of therapeutic agents depend on the particular compound, disease state and severity thereof, and on the individual in need thereof, and can be routinely determined by one skilled in the art. By way of non-limiting example, the dosage of the composition according to the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient and may be based on an adult patient weighing 70 kg. At this time, it is generally 0.01 to 1000 mg / day, preferably 1 to 500 mg / day, and may be dividedly administered once to several times a day at regular time intervals.

When the composition of the present invention is prepared as a health food composition, not only contains the ginsenosides Rb1 and Rb2 as active ingredients, but also includes components commonly added in food production, for example, proteins, carbohydrates. And fats, nutrients, seasonings and flavoring agents. For example, when the food composition of the present invention is prepared with a drink, in addition to ginsenosides Rb1 and Rb2 of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, and fruit juice may be further included.

Ginsenosides Rb1 and Rb2 may provide muscle regeneration applications, and more specifically, may be used for preventing or treating muscle atrophy or muscle cancer, or for improving or alleviating muscle atrophy or muscle cancer.

The present invention may provide a method for promoting muscle regeneration, comprising administering to a subject in need thereof an effective dose of ginsenosides Rb1 and Rb2.

Specifically, it may be a method of preventing or treating muscular atrophy or muscle cancer, including administering ginsenosides Rb1 and Rb2 according to the present invention, and may also be a method of improving or alleviating muscular atrophy or muscle cancer. .

The effective dosage means the amount of ginsenosides Rb1 and Rb2 effective for promoting muscle regeneration by the present invention. Ginsenosides Rb1 and Rb2 of the present invention can be used as they are or in a form that can be administered to an animal. In addition, ginsenosides Rb1 and Rb2 of the present invention may have a powder, suspension, or extract form, but is not limited thereto.

The administration subject is an animal, preferably a mammal (a mammal including a human), and includes cells and tissue organs of the animal. Such subjects can include patients in need of treatment.

Hereinafter, examples are provided to help understand the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example

 (1) Experimental Materials

Ginsenosides Rb1 and Rb2 were provided by Dr. Soo-Nam Kim (Korea Institute of Science and Technology, Natural Products Research Institute, Gangneung, Korea).

(2) cell culture

C2C12 myoblasts, rhabdomyosarcoma (RD) cells and embryonic kidney 293T cells were cultured (Bae et al., 2009; Bae et al., 2010). C2C12 cells were maintained in DMEM containing 15% FBS (fetal bovine serum; Life Technologies, Carlsbad, Calif.). To induce differentiation of C2C12 myoblasts, C2C12 cells were migrated to differentiation medium (DM) containing 2% horse serum (Life Technologies) and myotube formation was observed for about 48-72 hours. . The efficiency of myotube cell formation was quantified using a transient differentiation assay (Bae et al., 2010). To induce hypertrophic myoblast differentiation, C2C12 cells were differentiated for 2 days and then treated with Rb1 and RB2 for an additional 2 days in DM. Additional cell lines, RD and 293T were incubated in DMEM containing 15% FBS.

(3) Immunblotting and immunoprecipitation analyses

Cell lysis buffer containing complete protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) [pH 7.2, 10 mM Tris-HCl, 150 mM sodium chloride (NaCl), 1 mM ethylene diamine tetraacetic acid (EDTA), 1% Triton X-100 Cells were lysed. Cell lysates were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The primary antibodies used are shown in Table 1 below.

For immunoprecipitation assay (IP), C2C12 or MyoD-type infected 293T cells were treated with Rb1 and Rb2 for 36 hours, incubated in cell lysis buffer and then immunoprecipitated with anti-E2A antibody at 4 ° C. overnight. I was. Immunoprecipitation agents were mixed with Protein G agarose beads (Roche Diagnostics) at 4 ° C. for 2 hours and analyzed by immunoblotting with antibodies against MyoD and E2A.

(4) immune cell chemistry

For immunostaining for MHC expression, C2C12 cell line was fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.2% Triton X-100 in phosphate buffered saline and sealed to prevent anti-MHC Stained with. It was then treated with Alexa Fluor 568-conjugated secondary antibody (Molecular Probes, Eugene, OR). Images were obtained with a Zeiss LSM-510 meta confocal microscope and processed with ZEN-2 software (Carl Zeiss AG, Oberkochen, Germany).

(5) Statistical analysis

The experiment was performed three times independently. T-tests in each experiment were used to access the significance of the difference between the two mean values, with * p <0.05 and ** p <0.01 considered statistically significant.

2. Experimental Example

(1) Rb1 and Rb2, which promote myocardial cell hypertrophy through activation of the Akt / mTOR signaling pathway

Skeletal muscle mass is tightly regulated by Akt / mTOR signals, which act as promoters of muscle protein synthesis and hypertrophy. To determine whether ginsenosides Rb1 and Rb2 can promote the enlargement of myotubes, C2C12 myoblasts were induced to differentiate for 2 days and then treated with Rb1 or Rb2 for an additional 2 days. The cells were then subjected to immunostaining for MHC to assess the formation of myotubes. More specific information related to this can be confirmed through FIG. 1.

C2C12 cells were induced to differentiate for 2 days in DM (differentiation medium) and then treated with Rb1 or Rb2 for an additional 2 days. Myotube cell formation was analyzed by MHC (myosin heavy chain) immunostaining. DAPI (4 ', 6-diamidine-2'-phenylindole dihydrochloride) was used to visualize the nucleus. In immunostaining results, MHC expression was red and nucleus was blue (FIG. 1A).

The average diameter of the root canal cells was measured by NIS-Elements F software (Nikon, Tokyo, Japan). Data were presented as mean determination values of 6 fields ± 1SD (standard deviation) (** p <0.01 compared to control) (FIG. 1B).

In order to confirm whether Rb1 and Rb2 enhance the expression of muscle-specific proteins and include signal transduction pathways, immunoblotting was performed on muscle-specific proteins MHC, MyoD and myogenin. Pan-Cadherin was used as loading control (FIG. 1C).

In addition, immunoblotting was performed on the whole and phosphorylated forms of Akt, mTOR, p70S6K and 4E-BP1. α-Tubulin was used as a loading control (FIG. 1D).

As a result, Rb1- or Rb2-treated C2C12 cells formed larger, thicker multinucleated myotubes, dose-dependently, as compared to control cells (FIG. 1A). Measurement of the diameter of the root canal cells confirmed that Rb1 or Rb2 treatment increased the thickness of the root canal cells, in particular about 3.31 and 2.08 fold increase at 100 nM Rb1 and Rb2 (FIG. 1B).

In addition, to further investigate whether these ginsenosides regulate muscle specific proteins and muscle hypertrophy related signaling pathways, C2C12 cells were differentiated under the same experimental conditions as described above, and Rb1 treatment was performed by immunoblotting analysis. Compared with the control cells, the expression of MHC was sharply increased, and the expression of MyoD and myogenin was slightly improved at 4 days of differentiation (FIG. 1C).

Similar to Rb1, Rb2 treatment promoted expression of MHC and myogenin at the same point, while MyoD expression was constant in Rb2-treated myotubes. Treatment with Rb1 or Rb2 induced phosphorylation of Akt and increased phosphorylation of Akt downstream targets such as mTOR, p70S6K and 4E-BP1 compared to control cells (FIG. 1D).

These results indicate that Rb1 and Rb2 may induce myocardial cell hypertrophy and regain muscle regeneration capacity depending on Akt / mTOR signal.

(2) Activates myogenic kinase, Akt, Rb1 and Rb2 Enhance Akt-mediated Myoblast Differentiation

The effects of Rb1 and Rb2 on myoblast differentiation were examined. More detailed information related to this can be confirmed through FIG. 2.

Rb1-treated C2C12 cells were differentiated for 2 days in vehicle DMSO or various concentration conditions, followed by MHC immunostaining. (FIG. 2A). MHC-positive myocytes were quantified by the number of nuclei per myotube. Values are expressed as mean ± SD of three independent experiments (** P <0.01 compared to control) (FIG. 2B). Cell lysates were subjected to immunoblotting with antibodies to MHC, MyoD, myogenin, phosphorylated Akt and Akt. Pan-Cadherin was used as loading control (FIG. 2C).

Likewise, Rb2-treated C2C12 cells were differentiated for 2 days in vehicle DMSO or various concentration conditions, followed by MHC immunostaining (FIG. 2F). MHC-positive myocytes were quantified by the number of nuclei per myotube. Values are expressed as mean ± SD of three independent experiments (** P <0.01 compared to control) (FIG. 2E). Cell lysates were subjected to immunoblotting with antibodies to MHC, MyoD, myogenin, phosphorylated Akt and Akt. Pan-Cadherin was used as loading control (FIG. 2C).

As a result, as confirmed by MHC immunostaining, Rb1 treatment dose-dependently increased the formation of multinuclear MHC-positive myotubes (FIG. 2A). As a result of quantification of myotube cell formation, Rb1 treatment significantly increased the proportion of large myotubes containing 6 or more nuclei compared to the control, but substantially reduced myocytes in monocytes dose-dependently (FIG. 2B). In addition, Rb1 treatment enhanced the expression of MHC, MyoD and myogenin and increased phosphorylation of Akt, as assessed by immunoblotting assay (FIG. 2C).

Similar to the results of Rb1, Rb2-treated C2C12 cells showed gradual expression of MHC and myogenin and dose-dependently increased phosphorylation of Akt (FIG. 2D). However, unlike Rb1, MyoD expression was constant in Rb2-treated C2C12 cells. Rb2 treatment increased the formation of MHC-positive root canal cells with six or more nuclei dose-dependently (FIGS. 2E and 2F).

These results indicate that the effect of Rb1 and Rb2 on promoting muscle differentiation depends on the activation of Akt at the morphological and biochemical levels.

(3) Myogenic differentiation effect is further enhanced when Rb1 and Rb2 are used in combination

The effects of the combination of Rb1 and Rb2 on myoblast differentiation were examined. More detailed information related to this can be confirmed through FIG. 3.

C2C12 cells were simultaneously treated with a combination of Rb1 and Rb2 and then induced differentiation for 2 days. The cell lysates were subjected to immunoblotting with antibodies to MHC, phosphorylated Akt and Akt. Pan-Cadherin was used as loading control (FIG. 3A). MHC-positive myocytes were quantified by the number of nuclei per myotube (** P <0.01 compared to control).

As a result, the expression of MHC was more effective when Rb1 and Rb were combined, as compared with when ginsenoside Rb1 or Rb2 was treated, respectively. In addition, treatment with a combination of 25 nM Rb1 and 75 nM Rb2 showed more effective expression than the other cases (FIG. 3A). Similar to the results of MHC expression, C2C12 cells simultaneously treated with Rb1 and Rb2 showed progressive phosphorylation of Akt.

In addition, as a result of quantifying myotube formation under the same experimental conditions, MHC-positive myotube cells containing 6 or more nuclei were treated with C2C12 cells treated with Rb1 and Rb2, respectively, compared to cells treated with Rb1 or Rb2. The ratio of was significantly increased. Among them, the combination treatment of 25 nM Rb1 and 75 nM Rb2 showed the highest ratio of MHC-positive myotubes containing 6 or more nuclei (FIG. 3B).

These results indicate that synergistic effects on Akt-mediated myogenic differentiation can be achieved through the combination of Rb1 and Rb2.

(4) Heterodimerization of MyoD and E Proteins heterodimerization Rb1 and Rb2 to promote

MyoD functions by binding to DNA as a heterodimer with a widely expressed E protein to activate muscle specific gene transcription. To determine whether Rb1 and Rb2 can induce heterodimer formation of MyoD and E proteins, C2C12 cells were treated with Rb1 or Rb2 for 2 days (2D) followed by IP analysis. More detailed information related to this can be confirmed through FIG. 4.

C2C12 cells (FIG. 4A) or MyoD-type infected 293T cells (FIG. 4B) were treated with Rb1 or Rb2 and then differentiated for 2 days. Cell lysates were then immunoprecipitated with anti-E2A antibodies, followed by immunoblotting assays using anti-MyoD antibodies. α-Tubulin was used as loading control.

As a result, Rb1 or Rb2 treatment increased the amount of MyoD in the precipitate with E2A antibody compared to DMSO treatment. Protein levels of E2A and MyoD remained constant in lysates of C2C12 cells treated with Rb1 or Rb2 compared to control cells (FIG. 4A). In addition, consistent with endogenous interactions, exogenous MyoD protein strongly bound to E2A protein in Rb1- or Rb2-treated C2C12 cells (FIG. 4B). However, protein levels of MyoD and E2A were constant in whole cell lysates regardless of treatment with Rb1 or Rb2.

MyoD has recently been shown to interact directly with the Baf60c subunit of the SWI / SNF chromatin modification complex and is bound to regulatory elements before differentiation begins. AKT signaling promotes the recruitment of MyoD to the chromatin of muscle genes, which is an event MyoD requires to activate chromatin modification activity of p38-mobilized SWI / SNF complexes.

These results indicate that myoD differentiation by treatment of Rb1 and Rb2 depends on MyoD.

(5) Rb1 and Rb2 enhance MyoD-mediated myogenic conversion of rhabdomyosarcoma (RD) cells

To determine if Rb1 and Rb2 could restore impaired myogenic differentiation of RD cells, RD cells transiently transfected with MyoD were treated with Rb1 and Rb2 for 4 days (D4). More detailed information related to this can be confirmed through FIG. 5.

MyoD-expressing RD cells were induced to differentiate for 4 days by treatment with Rb1 or Rb2. Cell lysates were subjected to immunoblotting with antibodies against MHC, MyoD and myogenin. α-Tubulin was used as loading control. The experiment was repeated three times, all with similar results (FIG. 5A). Immunostaining was performed to show the formation of myotubes, and MHC expression was red and nucleus was blue (FIG. 5B).

As a result, it was confirmed that the Rb1 or Rb2 treatment restored the expression of MHC and myogenin in RD cells transfected with MyoD without any change in MyoD expression (FIG. 5A). In addition, it was confirmed that treatment with Rb1 or Rb2 increased the formation of MHC-positive myotubes containing three or more nuclei in RD cells transfected with MyoD (FIG. 5B).

These results indicate that Rb1 and Rb2 can induce MyoD-mediated transdifferentiation to induce differentiation of rhabdomyosarcoma (RD) cells into myoblasts, thereby enhancing MyoD-mediated myogenic differentiation of RD cells.

In other words, the composition for muscle regeneration containing ginsenosides Rb1 and Rb2 according to the present invention promotes heterodimerization of MyoD and E proteins, and promotes the heterodimerization of MyoD and E proteins with myotubes downstream of Akt and its downstream signal transduction. Increasing hypertrophy can improve myoblast differentiation and hypertrophy, induce MyoD-mediated transdifferentiation and induce differentiation of rhabdomyosarcoma (RD) cells into myoblasts, moreover, with ginsenoside Rb1 By simultaneously containing Rb2 and containing Rb1 and Rb2 in a specific ratio, a more synergistic effect can be obtained in terms of myoblast differentiation.

Claims (11)

A composition for muscle regeneration comprising ginsenosides Rb1 and Rb2 as an active ingredient. The composition of claim 1, wherein the ratio of ginsenosides Rb1 and Rb2 is in a molar ratio of Rb1: Rb2 = 1: 2 to 1: 4. The composition for muscle regeneration according to claim 2, wherein the ratio of ginsenosides Rb1 and Rb2 is Rb1: Rb2 = 1: 2.5 to 1: 3.5 in molar ratio. The composition according to claim 1, wherein the composition regulates the activity of MyoD, a muscle transcription factor, to induce differentiation of myoblasts into myotubes or to differentiate fibroblasts into myoblasts. Pharmaceutical composition for the prevention or treatment of muscular dystrophy containing ginsenosides Rb1 and Rb2 as an active ingredient. Health food composition for improving or alleviating muscular dystrophy containing ginsenosides Rb1 and Rb2 as an active ingredient. The muscular dystrophy according to claim 5, wherein the muscular dystrophy is aging muscular dystrophy, degenerative muscular dystrophy, diabetic muscular dystrophy, rented muscular atrophy, Becker Muscular Dystrophy (BMD), Duchenne Muscular Dystrophy (DMD), myotonia Myotonic Muscular Dystrophy, Emery-Dreifuss Muscular Dystrophy, EDMD, Distal Muscular Dystrophy, DD, Facioscapulohumeral Muscular Dystrophy, FSgen Muscular Dystrophy Dystrophy, CMD). A pharmaceutical composition for preventing or treating muscle cancer containing ginsenosides Rb1 and Rb2 as an active ingredient. Health food composition for improving or alleviating muscle cancer containing ginsenosides Rb1 and Rb2 as an active ingredient. The pharmaceutical composition of claim 8, wherein the muscle cancer is soft-tissue sarcoma. 11. The pharmaceutical composition of claim 10, wherein the soft tissue sarcoma is Leiomyosarcoma or Rhabdomyosarcoma.

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