KR20240052301A - Composition for promoting muscle stem cell regeneration comprising Farnesol - Google Patents

Abstract

The present invention relates to a composition for promoting the proliferation of muscle stem cells containing farnesol. More specifically, it has the effect of promoting the growth of muscle stem cells containing farnesol and at the same time promoting differentiation by increasing the proliferation of muscle cells. It relates to a composition and a method of promoting muscle stem cell proliferation using farnesol.

Description

A composition for promoting muscle stem cell proliferation and differentiation containing farnesol. {Composition for promoting muscle stem cell regeneration comprising Farnesol}

The present invention relates to the use of farnesol for promoting muscle stem cell proliferation and differentiation and to a composition containing the same for promoting muscle stem cell proliferation and differentiation.

Skeletal muscle has a resilient regenerative capacity governed primarily by muscle stem cells called satellite cells. Normally, satellite cells remain quiescent and can self-renew and differentiate when activated by injury stimuli. Satellite cells in the quiescent state express Pax7, and when satellite cells are activated, they express myogenic transcription factors such as Myf5 and MyoD. This is followed by expansion and differentiation of progenitor cells, consistent with decreased Pax7 expression and increased Myogenin expression. The ability of satellite cells to proliferate and return to a quiescent state is critical for the maintenance of stem cell populations. Satellite cells are known to have decreased cell number and proliferation ability due to aging. Satellite cells are remarkably resistant to cellular stress, but gradually lose their ability to proliferate and differentiate with aging. Aging and dysfunction of satellite cells are particularly associated with cellular senescence, which is caused by the accumulation of DNA damage and irreversible cell cycle arrest. Senescent cells have a flat shape and are characterized by activating senescence-β galactosidase (SA-β-gal) and upregulating the cyclin-dependent kinase inhibitors p16Ink4a (p16) and p21Waf1 (p21). Have. Additionally, senescent cells develop a senescence-associated secretory phenotype (SASP), producing inflammatory cytokines and insulin-like growth factor binding proteins. Dysregulation of cell cycle inhibitors is an important mechanism underlying cellular aging, but the molecular mechanisms of satellite cell aging are still largely unknown.

Accordingly, while conducting research to improve the decrease in the number of muscle stem cells due to decline in proliferation and differentiation ability due to aging, the present inventors discovered that Farnesol is involved in the mechanism related to the proliferation of muscle stem cells. and completed the present invention.

Therefore, an object of the present invention is to provide a composition for promoting proliferation and differentiation of muscle stem cells containing farnesol.

Another object of the present invention is to provide a composition for muscle fiber regeneration containing farnesol.

In order to achieve the above object, the present invention provides a composition for promoting proliferation and differentiation of muscle stem cells containing farnesol.

In order to achieve another object of the present invention, the present invention provides a composition for muscle fiber regeneration containing farnesol.

Hereinafter, the present invention will be described in detail.

In one aspect of the present invention, the present invention relates to a composition for promoting proliferation and differentiation of muscle stem cells containing farnesol.

Farnesol is a noncyclic sesquiterpene consisting of 15 carbons, and its IUPAC name is (2E,6E)-3,7,11-trimethyldodeca-2,6,10-triene-1- It's all coming. Farnesol has the structure of Chemical Formula 1 below.

The farnesol may be commercially sold, directly synthesized, or extracted from natural products. The term “pharmaceutically acceptable salt” refers to the customary acid addition salts used in the pharmaceutical field, for example in the field of demyelinating diseases, for example from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid or nitric acid. Derivatized salts and acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, citric acid, maleic acid, malonic acid, methanesulfonic acid, tartaric acid, malic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid. , salts derived from organic acids such as oxalic acid or trifluoroacetic acid. The salts also include common metal salt forms, for example salts derived from metals such as lithium, sodium, potassium, magnesium, or calcium. The acid addition salt or metal salt can be prepared according to conventional methods.

“Solvate” means a complex or aggregate formed by one or more solute molecules, farnesol or a pharmaceutically acceptable salt thereof, and one or more solvent molecules. Solvates may be complexes or aggregates formed with, for example, water, methanol, ethanol, isopropanol or acetic acid.

The farnesol may also be in the form of its stereoisomers. The stereoisomers include all stereoisomers such as enantiomers and diastereomers. The farnesol may be in stereoisomerically pure form or a mixture of one or more stereoisomers, for example, a racemic mixture. Separation of specific stereoisomers can be performed by any of the conventional methods known in the art.

In the present invention, farnesol is used to promote proliferation and differentiation of muscle stem cells. In the early stages of regeneration, satellite cells differentiate into mononucleated myoblasts through cell proliferation and differentiation processes. The mononucleated myoblasts proliferate and differentiate and fuse into multinucleated myotubes, and finally the mature myotubes differentiate into new myofibers. Each stage of this muscle regeneration process is regulated by different myogenic regulatory factors (MRF) and cell cycle regulators. In one embodiment of the present invention, farnesol was confirmed to regulate the expression and activity of the above cell cycle regulators and myogenic regulators, and through this, it was confirmed that it promoted the proliferation and differentiation of muscle stem cells.

In one embodiment of the present invention, farnesol was confirmed to improve the expression level of cell cycle regulators Ccnb1, Ccnb2, Ccnd1, Ccne1, Ccnf, Ccna2, Mcm3, Mcm6, Mcm7 and AurkB, as well as myogenic regulators. It was confirmed that the expression levels of (muscle stem cell differentiation factors) Pax7, MyoD, Myog, and Myf5 were improved. Through this, it was confirmed through cell and animal experiments that the process of differentiation of myogenic cells into muscle cells was further activated.

In the present invention, the “muscle stem cells” may be damaged by external stimuli or muscle disease, but are not limited thereto, and may be aged cells or normal muscle stem cells. Additionally, the muscle stem cells may be satellite cells or myoblasts, but are not limited thereto, and more preferably satellite cells.

In addition, farnesol of the present invention not only promotes the proliferation and differentiation of muscle cells, but also increases the actual muscle fiber thickness, which can have a regenerative effect on damaged or aged muscles and can further increase muscle mass.

The composition of the present invention can be used for various purposes such as pharmaceuticals, health functional foods, functional foods, animal feed, and cell culture compositions, and has the effect of promoting the proliferation and differentiation of muscle stem cells.

In addition, the composition for promoting muscle stem cell proliferation and differentiation may further have a preventive or therapeutic effect on various muscle diseases related to a decrease in muscle mass and strength. The muscle disease is not limited thereto, but is a disease in which symptoms can be alleviated by promoting the proliferation and differentiation of muscle stem cells and the resulting regeneration of muscle fibers, and more specifically, DMD (muscular dystrophy), Lou Gehrig's disease (ALS), This may include diseases such as Parkinson's disease.

As used herein, the term “prevention” refers to any action that can suppress or delay the onset of muscle disease by administering the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action in which symptoms are improved or benefited by administration of the pharmaceutical composition according to the present invention.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. It may additionally contain carriers or excipients necessary for the formulation. Pharmaceutically acceptable carriers, excipients and diluents that may be additionally included in the active ingredients include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, These include calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

For example, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include the extract or compound with at least one excipient, at least cotton, starch, and calcium carbonate. It is prepared by mixing sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The pharmaceutical composition of the present invention can be administered orally or parenterally (intravenously, subcutaneously, intraperitoneally, or topically) depending on the desired method, and the dosage depends on the condition and weight of the patient, the degree of the disease, and the form of the drug. It varies depending on the route and time of administration, and an appropriate form can be selected by a person skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means a reasonable amount applicable to medical treatment and an amount sufficient to treat a disease, and the criteria are the patient's disease, severity, drug activity, sensitivity to the drug, and administration time. , can be determined depending on the route of administration and excretion rate, treatment period, ingredients used together, and other matters. The pharmaceutical composition of the present invention can be administered individually or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, the dosage can be determined at a level that can minimize side effects, and this can be easily determined by a person skilled in the art. Specifically, the dosage of the pharmaceutical composition may vary depending on the patient's age, weight, severity, gender, etc., and is generally administered in an amount of 0.001 to 150 mg per 1 kg of body weight, more preferably 0.01 to 100 mg per kg of body weight every day or every other day. It can be administered 1 to 3 times a day. However, this is an example, and the dosage can be set differently as needed.

In addition, the composition of the present invention may be a food or health functional food, and in particular, the "health functional food" is manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Foods. “Functional” refers to food that is consumed for the purpose of controlling nutrients for the structure and function of the human body or obtaining useful health effects such as physiological effects.

The food or health functional food of the present invention is used in pharmaceutical dosage forms such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, tea bags, leachates, beverages, etc. for the purpose of preventing and improving muscle diseases. It can be manufactured and processed into health functional foods such as candy, jelly, and gum.

The food or health functional food composition of the present invention can be used as a food additive and can be commercialized alone or in combination with other ingredients. In addition, it is used in nutritional supplements, vitamins, electrolytes, flavoring agents, colorants and enhancers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonated beverages. Carbonating agents, etc. may be included. The above ingredients can be used alone or in combination, and can be used in combination in appropriate amounts.

The present invention relates to a composition for promoting muscle stem cell proliferation and differentiation containing farnesol. Farnesol can have the effect of promoting muscle fiber regeneration and increasing muscle mass by promoting the proliferation and differentiation of muscle stem cells.

Figure 1 schematically illustrates an experimental schedule to confirm the effect of farnesol on promoting muscle regeneration.
Figure 2 is a photograph of a cross-sectional view of muscle fibers analyzed by H&E staining 21 days (D21) after inducing muscle damage by CTX injection into the tibialis anterior (TA) of a mouse to confirm the effect of muscle fiber regeneration upon administration of farnesol. .
Figure 3 shows the change in the ratio of muscle fiber cross-sectional area to muscle fiber amount after administering farnesol to damaged mouse TA muscle (D21).
Figure 4 is a diagram comparing changes in the average cross-sectional area of muscle fibers after administering farnesol to the TA muscle (D21) of injured mice.
Figure 5 shows the results of BrdU staining to confirm the regeneration of muscle cells after administering farnesol to damaged mouse TA muscle (D3).
Figure 6 is a diagram quantitatively analyzing and comparing the BrdU staining results.
Figure 7 is a diagram confirming changes in the expression level of cell cycle regulators by performing real-time polymerase chain reaction when farnesol is administered to the damaged mouse TA muscle (D3).
Figure 8 is a diagram confirming changes in the expression level of muscle differentiation regulators (Pax7, MyoD, Myogenine, Myf5) when farnesol is administered to damaged mouse TA muscle (D3) by performing real-time polymerase chain reaction.
Figure 9 shows the amount of change in Pax7-positive satellite cells in TA muscle myofibers confirmed by Laminin/Pax7 staining after administration of farnesol to damaged mouse TA muscle (D21).
Figure 10 shows changes in Pax7-expressing cells in 26-month-old aged mice (26M) in the group administered farnesol and the group fed a normal diet, confirmed by Laminin/Pax7 staining.
Figure 11 shows the results of treating 66-year-old human-derived myoblasts with farnesol and performing BrdU staining to analyze whether farnesol promotes proliferation of aged human-derived myoblasts.
Figure 12 is a diagram quantitatively analyzing and comparing the BrdU staining results.

Hereinafter, examples will be given in detail to explain the present specification in detail. However, the embodiments according to the present specification may be modified into various other forms, and the scope of the present specification is not to be construed as being limited to the embodiments described in detail below. The embodiments of this specification are provided to more completely explain the present specification to those with average knowledge in the art.

Example 1. Animal experiment

Three-month-old female C57BL/6J mice were obtained from the Korea Basic Science Research Institute. Mice were provided with food and water ad libitum. Water and food intake was measured once a week for each cage. Rearing facilities were maintained at 23°C with a 12-hour light/dark cycle.

To analyze the regenerative ability of farnesol in muscle cells, 10 μM Cardiotoxin (CTX; Sigma-Aldrich) was injected into the TA muscle of 3-month-old young mice. Farnesol (350 mg/kg) was administered orally for 31 or 13 days (10 days + 21 days pretreatment or 3 days after CTX injury), and the vehicle was 5% DMSO diluted in normal saline containing 0.85% NaCl. used. To confirm the regenerative ability of muscle cells in aged mice, mice injected with CTX were fed food containing farnesol or mock instead of oral administration. (Figure 1)

Example 2. qRT-PCR

For verification of RNA-seq, cDNA was subjected to qRT-PCR using Thermal Cycler Dice Real Time System (TaKaRa) and SYBR premix EX Taq (TaKaRa) according to the manufacturer's recommended protocol. Relative expression of mRNA for the genes investigated was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression.

Example 3. Cryosections, histology and immunostaining analysis

For immunostaining, muscle sections were fixed, permeabilized, and processed for incubation with primary antibodies against Pax7 (DSHB), BrdU (abcam), and Laminin (Abcam), followed by incubation with fluorescently conjugated secondary antibodies. Images were captured on a confocal laser scanning microscope LSM710 or Nikon ECLIPS TE-2000U using NIS-Elements F software (Nikon).

For hematoxylin and eosin (H&E) staining, frozen sections were processed for staining with Mayer's hematoxylin and eosin (BBC Biomedical).

Experimental Example 1. Strengthening effect of muscle fiber regeneration

The muscle regenerative ability of mice was confirmed by the method of Example 1. Mice were pretreated with Vehicle or farnesol (350 mg/kg) for 10 days, and the TA muscle was injected with CTX along with continuous farnesol administration. The TA muscle was confirmed at the time points indicated in Figure 1, 3 days (CTX D3) and 21 days (CTX D21) after causing damage by CTX. To label proliferating cells in regenerating muscles, BrdU (100 μg/g) was injected intraperitoneally 2 days after injury, and muscles were harvested the following day. After differentiation, the cells were confirmed under a microscope, the cells were pulverized, and Western blot analysis was performed using differentiation marker Myogenin and MHC (myosin heavy chain) antibodies.

Figure 2 shows the results of H&E staining of TA muscle tissue 21 days after administration of Vehicle and farnesol, respectively, and injection of CTX. It can be seen that the muscle fibers of damaged muscles in the farnesol-administered group recovered compared to the control group, and the cross-sectional area increased further.

In addition, changes in the cross-sectional area of muscle fibers and the average cross-sectional area of TA muscle were quantitatively confirmed as shown in Figures 3 and 4. Although there were differences depending on the administered concentration, it was confirmed that the muscle fiber area in the TA muscle increased on average when farnesol was administered.

Experimental Example 2. Effect of strengthening muscle cell proliferation ability

2-1) Changes in muscle cell proliferation

In addition, to confirm the effect of farnesol on muscle cell proliferation and regeneration, TA muscles treated with Vehicle or farnesol were immunostained with BrdU (green) on the 3rd day (D3) after inducing damage by CTX. The results were confirmed. As a result, as shown in Figure 5, it was confirmed that the amount of differentiated muscle cells increased when farnesol was administered. Figure 6 shows quantitatively the percentage of BrdU positive cells in the muscle cell field. (n =4)

2-2) Confirmation of expression levels of factors related to stem cell proliferation and differentiation

In order to confirm changes in the expression levels of cell proliferation-related factors in muscle cells upon administration of farnesol, the expression levels of related factors were confirmed through qRT-PCR. As cell cycle regulators, the expression levels of Ccnb1, Ccnb2, Ccnd1, Ccne1, Ccnf, Ccna2, Mcm3, Mcm6, Mcm7, and AurkB were confirmed. In the same manner as the above experimental example, CTX was injected into the TA muscle of mice to induce muscle damage, and then treated with Vehicle or farnesol on the 3rd day (D3), and changes in the expression levels of each cell cycle regulator were confirmed. . As a result, as shown in Figure 7, it was confirmed that the expression of factors that regulate the cell cycle mostly increased.

In addition, as a result of confirming the expression of muscle differentiation regulatory factors (MRFs) using the above method, the expression rate of muscle differentiation regulatory factors such as Pax7, MyoD, Myog, and Myf5 was confirmed to be significantly higher in the farnesol-administered group. From the above results, it can be seen that muscle differentiation can be promoted when farnesol is administered.

Experimental Example 3. Effect of promoting proliferation of mouse muscle satellite cells (in-vivo)

Vehicle and farnesol were injected into the TA muscle of mice 21 days after CTX was injected to induce muscle damage, and the expression of Pax7, an activator of muscle satellite cells, was checked. Figure 9 shows the results of immunostaining confirming this, and 21 days (D21) after CTX injury, in the TA muscles of mice treated with Vehicle or farnesol, there were more Pax7+ cells in the muscle fiber plasma membrane indicated by laminin (red). A lot has been confirmed. In other words, since more muscle satellite cells expressing Pax7 were observed in the group administered farnesol, this result shows that farnesol increased the number of muscle satellite cells.

In addition, the experiment was conducted in the same manner in the case of old mice. In 26-month-old mice, the expression of Pax7, a muscle satellite cell activator, was confirmed in the TA muscles of mice fed a normal diet and mice administered farnesol by performing Laminin and Pax7 fluorescence staining. As a result, as shown in Figure 10, it was found that administration of farnesol increased the expression of Pax7. Additionally, in order to analyze the effect of farnesol on the proliferation of aging stem cells, 66-year-old human-derived myogenic cells were treated with DMSO or farnesol and then BrdU staining was performed to analyze differences in cell proliferation. As shown in Figures 11 and 12, it was confirmed that the proliferation of senescent myogenic cells was increased by farnesol compared to the control group (DMSO). From the above results, it can be seen that farnesol affects not only the regeneration caused by muscle damage, but also the increase in muscle satellite cells in aged muscles.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims, not the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (9)

A composition for promoting muscle stem cell proliferation and differentiation containing farnesol. According to paragraph 1,
A composition wherein the muscle stem cells are normal muscle stem cells, aged muscle stem cells, or muscle stem cells damaged by disease or stimulation. According to paragraph 1,
The composition is characterized in that it promotes the activity and expression of muscle stem cell proliferation and differentiation factors. According to paragraph 3,
A composition wherein the muscle stem cell differentiation factor is at least one selected from the group consisting of Pax7, MyoD, Myog, and Myf5. According to paragraph 1,
The composition is characterized in that it promotes the activity and expression of cell cycle regulatory factors. According to clause 5,
A composition characterized in that the cell cycle regulator is at least one selected from the group consisting of Ccnb1, Ccnb2, Ccnd1, Ccne1, Ccnf, Ccna2, Mcm3, Mcm6, Mcm7 and AurkB. A composition for muscle fiber regeneration containing farnesol. In clause 7,
The composition is characterized in that it regenerates damaged or aged muscles by increasing the cross-sectional area of muscle fibers. According to any one of claims 1 to 8,
The composition is characterized in that it has a preventive or therapeutic effect on muscular dystrophy, Lou Gehrig's or Parkinson's disease through proliferation and differentiation of muscle stem cells or muscle fiber regeneration.