Disuse sarcopenia animal model and use thereof

Abstract

The present invention relates to a disuse sarcopenia animal model, a method for preparing same, and a method for screening for disuse sarcopenia therapeutic agents by using the model. Unlike conventional methods (denervation, hindlimb uploading, and the like), the method for preparing a disuse sarcopenia animal model, according to the present invention, minimizes pain inflicted on animals and shortens preparation time, and thus is preferable in terms of ethics and convenience. In addition, the animal model prepared according to the method can be effectively used in the development of therapeutic agents and research on drug efficacy and the like for disuse sarcopenia.

Description

Animal model of sarcopenia and its use

[0001] The present invention relates to an animal model of sarcopenia, a method for producing the same, and a method for screening a therapeutic agent for sarcopenia using the same.

[0002] Sarcopenia is a disease characterized by a decline in muscle mass and, consequently, muscle strength. Sarcopenia can be caused by a variety of factors, including hormonal imbalances, nutritional deficiencies, inflammation, and degenerative diseases. Among these, sarcopenia and disuse muscular atrophy are known to be the most common forms of sarcopenia.

[0003] Disuse muscular atrophy can be defined as the loss of muscle tissue resulting from disuse. When social activity declines, muscle tone decreases and muscles gradually atrophy. Severe muscle wasting can occur, particularly in bedridden patients. For example, intensive care unit (ICU) patients experience rapid loss of muscle mass and respiratory muscles due to prolonged immobility, a condition known as intensive care unit-acquired weakness (ICU-AW).

[0004] Statistics show that 20 million patients worldwide receive intensive care unit (ICU) treatment each year, and nearly 30% of them exhibit ICU-acquired weakness. Despite the steady increase in ICU-acquired weakness, with an annual increase of 6 million globally and 50,000 in Korea, there is currently no safe and effective treatment available on the market. Currently, the best treatment is to restore daily functions through adequate nutrition and consistent rehabilitation exercises.

[0005] Therefore, there is an urgent need to develop effective therapeutic screening systems and animal models that can be used in these systems to develop treatments for disabling sarcopenic diseases such as ICU-acquired wasting.

[0006] In this regard, currently known animal models of disuse sarcopenia are either ethically undesirable or fail to adequately mimic disuse sarcopenia. For example, the commonly used animal model method of transection of intramuscular nerves does not demonstrate the disuse effects of long-term muscle disuse, and models based on natural aging are unlikely to demonstrate substantial differences from age-related sarcopenia models. Furthermore, hindlimb uploading, a method that suspends animals in a cage to suggest movement, places excessive stress on the animals and fails to adequately mimic disuse sarcopenia.

[0007] Accordingly, the inventors of the present invention have completed the present invention by easily producing an animal model of sarcopenia in a non-invasive manner using staples and / or sutures and confirming changes in the expression of various mRNAs or proteins in the animal model, thereby confirming its high potential for use as a model of sarcopenia in the animal model.

[0008] One object of the present invention is to provide an animal model of sarcopenia and a method for producing the same.

[0009] Another object of the present invention is to provide a method for screening an agent for treating insoluble sarcopenia.

[0010] Each description and embodiment disclosed in this invention can be applied to each other. That is, all combinations of the various elements disclosed in this invention fall within the scope of this invention. Furthermore, the scope of this invention is not limited by the specific descriptions described below.

[0011] To avoid confusion due to overlapping content, the description of redundant content will be omitted below. In other words, the content of the invention is not limited to the content described below, and the content of the invention should be interpreted based on the overall content of the invention.

[0012] In addition, the terminology used in this specification is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. It should be understood that the terms “comprise” or “have” in this specification are intended to specify the presence of a feature, number, step, operation, component, part or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts or combinations thereof.

[0013] Additionally, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries, such as those defined in such dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0014] Hereinafter, the present invention will be described in detail.

[0015] The present invention provides a method for producing an animal model of sarcopenia, comprising the step of fixing a hind limb of a rodent animal using a staple, a suture, or both, each of which integrally has a fastening pin at both ends of a bendable connecting member.

[0016] In the present invention, "disuse muscular atrophy" refers to a disease in which muscle tissue is lost due to restrictions on physical activity. Such restrictions on physical activity may include nerve damage that causes muscle weakness, weakness of a body part due to stroke, brain tumor, head trauma, or spinal disease, joint disease that limits mobility, and neuropathic pain conditions that cause avoidance of use of specific muscles. Preferably, the disuse muscular atrophy may be intensive care unit acquired weakness (ICU-AW). The above-mentioned disuse muscular atrophy includes symptoms of disuse muscular atrophy.

[0017] The rodent of the present invention may be a mouse, a rat, a hamster, a gerbil, a guinea pig, etc., and preferably a mouse.

[0018] In the present invention, the term “staple” means a locking device formed from metal or plastic wire, etc., and includes surgical fasteners made of synthetic materials and similar fasteners.

[0019] The staple of the present invention is characterized by having a fastening pin integrally provided at both ends of a bendable connecting portion. The connecting portion can be bent inwardly or outwardly depending on the direction in which force is applied. The fastening pin can be deformed inwardly or outwardly depending on the direction of the inclination. This staple has a form that is bent inwardly to secure a hind leg, and the fastening pins at both ends can be bent to restrict movement of the hind leg. It is preferable that the fastening pins at both ends of the connecting portion are bent inwardly at an arbitrary angle.

[0020] In one embodiment, the staple having a tying pin integrally formed at both ends of the bendable connecting portion may be a surgical staple. The surgical staple refers to a device capable of suturing skin at an incision site during surgical or plastic surgery, and includes staples of all sizes, including large surgical staples and small surgical staples. These staples of each size may be appropriately selected and used according to the size of the hind limb of the rodent animal model being used.

[0021] In the present invention, the term "suture" is used to comprehensively refer to medical fibers used for suturing for therapeutic or cosmetic purposes. The suture includes, but is not limited to, polymeric materials (e.g., Ethibond suture), threads, strands, fibers, wires, nylon, other windable materials, organic and inorganic materials, etc.

[0022] The method for producing an animal model of insoluble sarcopenia of the present invention includes a step of fixing a hind limb of a rodent animal using a staple, a suture, or both thereof, each of which has a tying pin integrally provided at both ends of a bendable connecting portion.

[0023] In the present invention, fixation of the hind limbs of a rodent may be performed on only one of the two hind limbs. By fixing only one of the two hind limbs in the present invention, a control experiment can be conducted within a single animal, utilizing the unfixed hind limb as a control.

[0024] The present invention reduces the number of animals used in experiments by conducting controlled experiments within a single animal, making it more ethically sound than existing methods. Furthermore, while using separate animals as a control group can lead to differences in baseline strength and muscle mass between the control and experimental groups, the present invention can conduct experiments under the assumption of a consistent baseline, eliminating these differences and offering scientific advantages.

[0025] In the present invention, the hind limb immobilization of a rodent is accomplished non-invasively, without directly damaging the muscles. Therefore, disuse sarcopenia, which can be induced by bone fractures, ligament damage, nerve damage, cachexia, etc., is blocked. Furthermore, external factors such as inflammation, cell death, or necrosis caused by muscle damage itself are blocked, allowing the creation of a disuse sarcopenia model with consistent characteristics under specific experimental conditions. This enhances reliability and accuracy in the efficacy and exploration of therapeutic candidates using this model.

[0026] In one embodiment, fixing the hind leg of the rodent is accomplished by bending the hind leg, bending a staple having a fastening pin integrally provided at both ends of a bendable connecting portion, and closing the fastening pin to fix the hind leg. The method of fixing the hind leg using the staple is different from existing hind leg fixation methods in that it is a non-invasive method of fixing the hind leg by bending and closing the fastening pin of the staple so as not to damage the skin or muscle while the hind leg is bent, rather than directly inserting the staple into the skin or muscle.

[0027] In one embodiment, fixing the hind leg of the rodent includes bending the hind leg, wrapping the hind leg with thigh skin, and fixing it by suturing. In one embodiment, fixing the hind leg of the rodent includes bending the hind leg, bending a staple having a fastening pin integrally provided at both ends of a bendable connecting portion, and closing the fastening pin to fix it, and wrapping the hind leg with thigh skin, and fixing it by suturing it with a suture.

[0028] In one embodiment, the staple used to fix the hind legs of the rodent may be a large surgical staple, characterized in that the size ranges from 5 x 2.5 mm to 10 x 6 mm. Preferably, the size ranges from 6 x 3.5 mm to 9 x 5 mm. Due to this characteristic, unlike the existing method of fixing the hind legs using staples, the hind legs of the rodent are allowed to move to some extent, thereby inducing sarcopenia more efficiently without causing skin, inflammation, rash, bedsores, etc. In other words, unlike the method of fixing strongly, some space for movement is provided to provide a similar environment related to sarcopenia, and complications or side effects caused by strong fixation are prevented.

[0029] In the present invention, the step of immobilizing the hind limbs of the rodent animal may be maintained for at least 3.5 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 21 days, 28 days, 35 days or more. The number of days of immobilizing the hind limbs may be applied differently depending on the severity of the desired disuse sarcopenia.

[0030] The method for producing an animal model of sarcopenia of the present invention may further include a step of determining that the animal model of sarcopenia of the present invention has been produced when the muscle weight of the fixed hind limb is reduced by 5% or more in weight compared to the muscle weight of the non-fixed hind limb.

[0031] If necessary, the production of an animal model can be determined by identifying a characteristic of a decrease in the expression level of any one or more selected from the group consisting of MyoD, HGF, IGF, and MGF; or an increase in the expression level of any one or more selected from the group consisting of TNF-α, IL-1b, IL-6, Caspase-3, MuRF-1, and Atrogin-1, compared to the expression level of a marker in a non-fixed hindlimb.

[0032] The present invention provides an animal model of insoluble sarcopenia produced by the above production method.

[0033] The animal model of sarcopenia of the present invention is characterized by a decrease in the expression level of at least one selected from the group consisting of MyoD, HGF, IGF, and MGF in the immobilized hind limb compared to a normal animal; and / or an increase in the expression level of at least one selected from the group consisting of TNF-α, IL-1b, IL-6, Caspase-3, MuRF-1, and Atrogin-1.

[0034] In the present invention, the term “MyoD” is classified as a primary myogenic regulatory factor among muscle transcription factors, and plays an essential role in muscle formation because it induces proliferated somatic cells into myoblasts.

[0035] In the present invention, the terms “HGF, IGF and MGF” are muscle growth factors that stimulate mTOR (mammalian Target of Rapamycin), which stimulates muscle protein synthesis, thereby promoting the Akt / Pkb signaling system and thereby increasing muscle growth rate.

[0036] That is, the above MyoD, HGF, IGF and MGF are factors that contribute to muscle formation and growth, and their expression levels are reduced in an animal model of sarcopenia compared to normal animals.

[0037] The terms "TNF-α, IL-1b, and IL-6" used herein refer to proinflammatory cytokines that directly induce muscle wasting by promoting the degradation of myofibrillar proteins and reducing protein synthesis. Specifically, they activate various intracellular factors, inducing ubiquitin-dependent protein degradation and apoptosis. Chronic low-grade inflammation caused by an increase in these proinflammatory cytokines is known to cause sarcopenia.

[0038] In the present invention, the term “Caspase-3” is encoded by the CASP3 gene and is a factor that plays an important role in the execution stage of cell death by promoting the degradation of cellular proteins.

[0039] In the present invention, “MuRF-1” refers to a signaling substance that decomposes muscle proteins, and it is generally known that muscle atrophy progresses when MuRF-1 is activated.

[0040] In the present invention, “Atrogin-1” is a muscle-specific F-box protein that is involved in the process of decomposing muscle proteins, and is induced in the early stage of muscle atrophy, so it increases prior to the decrease in muscle mass.

[0041] That is, at least one selected from the group consisting of TNF-α, IL-1b, IL-6, Caspase-3, MuRF-1, and Atrogin-1 is a sarcopenia marker, and its expression level increases in an animal model of disuse sarcopenia compared to a normal animal.

[0042] The present invention comprises the steps of: (a) fixing a hind leg of a rodent using a staple, a suture, or both, each of which integrally has a fastening pin at both ends of a bendable connecting member;

[0043] (b) a step of raising a rodent while maintaining the above hind leg fixation for at least 5 days;

[0044] (c) a step of treating the candidate substance to the animal of step (b); and

[0045] (d) a step of measuring the expression level of mRNA or protein indicators related to sarcopenia or measuring the hind limb weight for an animal treated with the candidate substance of step (c); a method for screening a treatment agent for insoluble sarcopenia is provided.

[0046] The mRNA or protein indicator associated with sarcopenia in the above step (d) may be at least one selected from the group consisting of MyoD, HGF, IGF, MGF, TNF-α, IL-1b, IL-6, Caspase-3, MuRF-1, and Atrogin-1.

[0047] The screening method for the insoluble sarcopenia treatment agent of the present invention may further include a step of selecting a candidate substance that increases or decreases the expression level of an mRNA or protein indicator related to sarcopenia in an animal treated with the candidate substance in step (d) compared to a control group that is not treated with the candidate substance.

[0048] For an animal treated with the above candidate substance, if the expression level of any one or more selected from the group consisting of MyoD, HGF, IGF, and MGF increases compared to a control group not treated with the candidate substance, the candidate substance may be selected as a therapeutic agent candidate.

[0049] For an animal treated with the above candidate substance, if the expression level of any one or more selected from the group consisting of TNF-α, IL-1b, IL-6, Caspase-3, MuRF-1, and Atrogin-1 is reduced compared to a control group not treated with the candidate substance, the animal may be selected as a therapeutic agent candidate.

[0050] For animals treated with the above candidate substance, if the decrease in hind limb weight is recovered, the candidate substance may be selected as a therapeutic agent candidate.

[0051] In the present invention, the step of measuring the expression level of the mRNA or protein indicator related to sarcopenia may be performed by measuring using any one selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), western blot, flow cytometry (FACS), and whole cell patch clamp assay, but is not limited thereto. The above measurement methods are known and thus are not described herein.

[0052] The method for producing an animal model of sarcopenia according to the present invention, unlike conventional methods (denervation, hindlimb uploading, etc.), uses a non-invasive method and performs controlled experiments within a single animal, thereby minimizing animal suffering and shortening production time, and reducing the number of animals used in experiments, making it desirable from the perspectives of ethics and convenience. Furthermore, since it is easy to maintain consistent experimental conditions, a model with consistent characteristics can be produced, thereby providing highly accurate and reliable data for the development of treatments for sarcopenia and research on drug efficacy.

[0053] Figure 1 is a diagram showing the production of an ICU-AW animal model according to methods 1 to 5 using staples and / or sutures, and the change in calf muscle weight of the animal model.

[0054] Figure 2 is a diagram confirming that the size of the calf muscles is reduced by fixing the leg with staples or sutures according to methods 1, 3, and 5.

[0055] Figure 3 is a diagram confirming that the weight and size of the calf muscles are reduced by fixing the mouse's legs with staples according to method 1.

[0056] Figure 4 is a diagram showing the change in the expression level of a muscle transcription factor (MyoD) measured by qPCR after 2 and 4 weeks of fixing the leg with staples according to Method 1.

[0057] Figure 5 is a diagram showing the change in the expression level of muscle growth factors (HGF, IGF, MGF) measured by qPCR after 2 and 4 weeks of fixing the leg with staples according to Method 1.

[0058] Figure 6 is a graph showing the change in the expression level of inflammatory cytokines (TNF-α, IL-1b, IL-6) measured after 2 and 4 weeks of fixing the leg with staples according to Method 1.

[0059] Figure 7 is a diagram showing the change in the expression level of a cell death factor (Caspase-3) measured by qPCR after 2 and 4 weeks of fixing the leg with staples according to Method 1.

[0060] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0061] Example 1. Preparation of an ICU-AW (Intensive Care Unit acquired weakness) animal model and confirmation of changes in calf muscle weight in the animal model.

[0062] ICU-AW animal models were created using the following five methods using surgical staples or sutures (left side of Fig. 1).

[0063] Method 1: Bend the hind legs of a 3-5 month old normal mouse and secure the entire leg with a large surgical staple (I in Fig. 1).

[0064] Method 2: After bending the hind legs of a 3-5 month old normal mouse, wrap and secure the legs using two small surgical staples (Ⅱ in Fig. 1).

[0065] Method 3: After bending the hind legs of a normal mouse aged 3 to 5 months, modify the small surgical staples to widen the gap and then wrap and secure the entire leg (Ⅲ in Fig. 1).

[0066] Method 4: After bending the hind leg of a normal mouse aged 3 to 5 months, pull the thigh skin and fix it by suturing it three times using surgical suture (Ⅳ in Fig. 1).

[0067] Method 5: After bending the hind leg of a 3-5 month old normal mouse, the thigh skin was pulled and sutured three times using surgical sutures, and the excess skin was pulled and fixed twice with small surgical staples (V in Fig. 1).

[0068] Mice manufactured according to each method were observed in the same environment for 7 days, and changes in calf muscle weight were observed. As a result, the greatest decrease in muscle weight was observed when the hind limbs of the mice were immobilized according to Method 1 (right side of Figure 1).

[0069] Example 2. Confirmation of changes in muscle size in the ICU-AW animal model

[0070] After fixing the hind legs of the mice according to methods 1, 3, and 5, the staples and sutures were removed after 7 days, and the mice were sacrificed, and the gastrocnemius muscles of both calves were removed. When the size of the contralateral muscle of the non-fixed leg was compared with the ipsilateral muscle of the fixed leg, it was confirmed that the size of the ipsilateral muscle was significantly reduced compared to the contralateral muscle (Fig. 2). In particular, it was confirmed that the muscle weight was reduced the most when the hind legs of the mice were fixed according to method 1.

[0071] Example 3. Confirmation of changes in muscle weight and size in the ICU-AW animal model manufactured according to Method 1.

[0072] After 28 days of fixing the hind legs of the mouse according to Method 1, the staples were removed, the mouse model was sacrificed, and the gastrocnemius muscles of both calves were excised. The contralateral muscles of the non-fixed leg and the ipsilateral muscles of the fixed leg of the mouse model were photographed and measured in size, and it was confirmed that the size of the ipsilateral muscles was significantly reduced compared to the contralateral muscles (Fig. 3).

[0073] That is, it was confirmed that sarcopenia was effectively induced by Method 1 by observing a decrease in muscle weight and size in the mouse model manufactured according to Method 1.

[0074] Example 4. Confirmation of expression levels of mRNA or proteins related to sarcopenia according to immobilization time.

[0075] After fixing the hind limbs of mice for 4 weeks according to Method 1, the expression levels of muscle transcription factors, muscle growth factors, cytokines, apoptosis factors, and sarcopenia markers were measured using the qPCR method, and the expression levels at 2 and 4 weeks were compared.

[0076] Specifically, RNA was extracted from fixed hindlimb muscle cells using the AccuPrep® Universal RNA Extraction Kit (Bioneer) to perform qPCR. SuperScript was purchased from Invitrogen. TM cDNA was synthesized using IV Reverse Transcriptase, and the protein expression level was confirmed by quantitative real-time polymerase chain reaction (qPCR) using PowerSYBR green PCR Master Mix from Applied biosystems.

[0077] (1) Muscle transcription factor (MyoD) and muscle growth factors (HGF, IGF, MGF)

[0078] The verification results for the above factors are shown in Figures 4 and 5.

[0079] Specifically, MyoD, HGF, IGF, and MGF showed a gradual decrease in expression levels over time. This suggests that the muscle regeneration capacity of the immobilized leg is gradually decreasing and muscle atrophy is progressing.

[0080] (2) Inflammatory cytokines (TNF-α, IL-1b, IL-6)

[0081] The verification results for the above factors are shown in Figure 6.

[0082] Specifically, TNF-α, IL-1b, and IL-6 showed high expression levels overall, and the expression levels increased rapidly in the 4th week compared to the 2nd week. In other words, it was confirmed that the longer the fixation time, the higher the level of pro-inflammatory cytokines.

[0083] (3) Cell death factor (Caspase-3)

[0084] The verification results for the above factors are shown in Figure 7.

[0085] Specifically, Caspase-3 expression was more than three times higher than that of the control group in both weeks 2 and 4. This indicates that apoptosis is actively occurring in the fixed leg muscles.

[0086] (4) Sarcopenia markers (MuRF-1, Atrogin-1)

[0087] MuRF-1 and Atrogin-1 showed a rapid increase on the third day after fixation. This confirmed that fixation according to Method 1 resulted in muscle loss.

[0088] In summary, the expression levels of muscle transcription factors (MyoD) and muscle growth factors (HGF, IGF, MGF) decreased by fixation in Method 1, and the expression levels of inflammatory cytokines (TNF-α, IL-1b, IL-6), apoptotic factors (Caspase-3), and sarcopenia markers (MuRF-1, Atrogin-1) increased.

[0089] That is, by confirming that the expression of various factors related to sarcopenia significantly changed in the mouse model manufactured according to Method 1, it was confirmed that fixing the legs with staples is one of the methods that can successfully induce sarcopenia.

Claims

1. A method for producing an animal model of sarcopenia, comprising the step of fixing a hind limb of a rodent animal using a staple, a suture, or both, each of which has a fastening pin integrally provided at both ends of a bendable connecting member.

2. A method for producing an animal model of sarcopenia with disuse, wherein fixing the hind legs of the rodent animal in paragraph 1 is performed on only one of the two hind legs.

3. A method for producing an animal model of sarcopenia with insoluble fibers, wherein in paragraph 1, fixing the hind legs of the rodent animal is performed by bending the hind legs, and then closing and fixing a staple having fastening pins integrally provided at both ends of a bendable connecting portion by bending the staples.

4. A method for producing an animal model of sarcopenia with insoluble fibers, wherein fixing the hind legs of the rodent animal in the first paragraph is performed by bending the hind legs, wrapping the hind legs with thigh skin, and suturing them with a suture to fix them.

5. A method for producing an animal model of insoluble sarcopenia, characterized in that the staple having a fastening pin integrally provided at both ends of the bendable connecting portion in the first paragraph is a surgical staple.

6. A method for producing an animal model of sarcopenia with insoluble fibers, wherein fixing the hind legs of the rodent in the first paragraph includes bending the hind legs and then closing and fixing the hind legs by bending staples having fixing pins integrally formed at both ends of a bendable connecting portion, and wrapping the hind legs with thigh skin and suturing them with a suture to fix them.

7. A method for producing an animal model of sarcopenia according to claim 1, wherein the rodent animal is any one selected from the group consisting of a mouse, a rat, a hamster, a gerbil, and a guinea pig.

8. A method for producing an animal model of sarcopenia with dissolution, wherein the step of fixing the hind limb of the rodent animal in paragraph 1 is maintained for at least 3.5 days.

9. A method for producing an animal model of dissoluble sarcopenia, further comprising a step of determining that the animal model of dissoluble sarcopenia has been produced when the muscle weight of the fixed hind limb is reduced by 5 wt% or more compared to the muscle weight of the non-fixed hind limb in the second paragraph.

10. An animal model of insoluble sarcopenia manufactured by a manufacturing method according to any one of claims 1 to 9.

11. In claim 10, the animal model is characterized by a decrease in the expression level of any one or more selected from the group consisting of MyoD, HGF, IGF and MGF in the fixed hind limb compared to a normal animal; or an increase in the expression level of any one or more selected from the group consisting of TNF-α, IL-1b, IL-6, Caspase-3, MuRF-1 or Atrogin-1, an animal model of sarcopenia with disuse. 12.(a) A step of fixing a hind leg of a rodent using a staple, a suture, or both, each having a fastening pin integrally provided at both ends of a bendable connecting member; (b) a step of raising a rodent while maintaining the hind limb fixation for at least 5 days; (c) a step of treating the animal of step (b) with a candidate substance; and (d) a step of measuring the expression level of mRNA or protein indicators related to sarcopenia or measuring the weight of hind limbs in an animal treated with the candidate substance of step (c); a screening method for a therapeutic agent for insoluble sarcopenia, comprising:

13. A method for screening a therapeutic agent for insoluble sarcopenia in claim 12, wherein the mRNA or protein indicator related to sarcopenia in step (d) includes at least one selected from the group consisting of MyoD, HGF, IGF, MGF, TNF-α, IL-1b, IL-6, Caspase-3, MuRF-1, and Atrogin-1.

14. A method for screening for an insoluble sarcopenia treatment agent, further comprising: a step of selecting a candidate substance that increases MyoD, HGF, IGF, and MGF by comparing the expression level of mRNA or protein indicators related to sarcopenia in an animal treated with the candidate substance in step (d) with that of a control group that was not treated with the candidate substance; 15. A method for screening for a treatment agent for insoluble sarcopenia, further comprising: a step of selecting a candidate substance that reduces TNF-α, IL-1b, IL-6, Caspase-3, MuRF-1, and Atrogin-1 by comparing the expression levels of mRNA or protein indicators related to sarcopenia in an animal treated with the candidate substance in step (d) with that of a control group that is not treated with the candidate substance.

16. A method for screening for a treatment agent for a disease related to muscle weakness, wherein in the 12th paragraph, the expression level of the mRNA or protein indicator related to sarcopenia in the step (d) is measured by any one selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), western blot, flow cytometry (FACS), and whole cell patch clamp assay.

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