JP2006162346A - Diagnostic method of amyotrophy, evaluation method of anti-amyotrophic action and screening method of anti-amyotrophic substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a diagnostic method of amyotrophy of high sensitivity, an evaluation method of anti-amyotrophic action using it and a screening method of an anti-amyotrophic substance using the evaluation method. <P>SOLUTION: The diagnosis of amyotrophy, the evaluation anti-amyotrophic action and the screening the anti-amyotrophic substance are performed using the development amount of oxidation stress inductive protein in a muscle as an index. As an example of the oxidation stress inductive protein, TNFα, pIkB, IL-8, MCP1, ICAM1 and 4-HNE modified protein are designated. The development amount of the oxidation stress inductive protein can be evaluated by measuring the amount of protein or mRNA. The same effect is also obtained using the development amount of myostatin and calcineurin in a muscle as an index. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for diagnosing muscle atrophy, a method for evaluating an antimuscular atrophy effect, and a screening method for an antimuscular atrophy substance. The present invention relates to a method for diagnosing atrophy, a method for evaluating an antimuscular atrophy using the diagnostic method, and a screening method for an antimuscular atrophy substance using the evaluation method.

  Muscle atrophy refers to a state in which muscle mass decreases due to the degradation rate of muscle protein exceeding the synthesis rate, and the muscles become thin. Muscular atrophy is roughly classified into disuse muscular atrophy caused by a state in which muscles are not used and progressive muscular atrophy caused by diseases such as amyotrophic side sclerosis (ALS). Of these, the condition that does not use muscles that cause disuse muscle atrophy can occur on a daily basis due to bed rest, cast fixation, etc., and disuse muscle atrophy is a major problem for bedridden elderly people It is. Moreover, the same symptom as muscle atrophy may occur with aging, and this is called myopathy (sarcopenia).

The mechanism by which muscle atrophy occurs is still largely unknown, but it is becoming clear that it is closely related to oxidative stress. For example, it is known that muscle atrophy occurs when vitamin E, an antioxidant, is deficient. Patent Document 1 describes that increases in thiobarbitur reaction positive substances and oxidized glutathione are observed in the atrophied muscle. On the other hand, there are reports that an increase in iron is involved in the progression of muscle atrophy. Patent Document 2 describes that muscle atrophy is inhibited by Ras activation or the action of a signal transduction molecule downstream of Ras.
JP 2001-89387 A WO 01/004354 pamphlet

  It is important to first prevent a disease such as muscular atrophy that has a major impact on daily life. For this purpose, a technique for detecting signs of muscle atrophy and the initial stage of muscle atrophy is required. As a method for detecting muscle atrophy, various enzymes leaking from the muscle, for example, creatine kinase (CK), glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), aldolase, etc. are used as marker substances. Some methods using the blood concentration as an index have been proposed, but this method is not very common. Rather, these enzymes often leak when muscles are damaged, and are unlikely to accurately reflect muscle atrophy. Furthermore, since GOT and GPT are also indicators of liver function, there is a risk of misdiagnosis as liver function disorder. Therefore, there is a need for a marker substance that can detect muscle atrophy with high sensitivity and can be useful for preventing muscle atrophy.

Also, if there is such a marker substance, by capturing the change in the amount of marker substance,
There is a possibility that the improvement state of muscle atrophy can be monitored. Furthermore, there is a possibility that the marker substance can be used as an index to evaluate the effect of the test substance on muscle atrophy or to screen for a substance effective for muscle atrophy. In particular, if there is a food material containing such a substance, it can be used to prevent muscle atrophy by eating it on a daily basis. However, no marker substance for such muscle atrophy has been found.

  The object of the present invention is to provide a new marker substance capable of detecting muscle atrophy, thereby enabling highly sensitive diagnosis of muscle atrophy, and also applicable to evaluation and screening of substances effective for muscle atrophy. The aim is to provide a technology that can

  The present inventors paid attention to oxidative stress-inducible proteins and measured the amounts of various oxidative stress-inducible proteins in muscle using an animal model of muscle atrophy. As a result, it was found that the expression levels of these oxidative stress-inducible proteins fluctuate in response to the onset, suppression, and improvement of muscle atrophy and reflect the state of muscle atrophy. Furthermore, it was found that myostatin and calcineurin also reflect the state of muscle atrophy. Furthermore, by using the animal model, it was found that the test substance can be used for evaluating the antimuscular atrophy action of a test substance and screening for a substance having an antimuscular atrophy action, thereby completing the present invention. That is, the gist of the present invention is as follows.

(1) A method for diagnosing muscle atrophy, which comprises detecting muscle atrophy using the expression level of an oxidative stress-inducing protein in muscle as an index,
(2) The method for diagnosing muscle atrophy according to (1), wherein the oxidative stress-inducible protein is a redox-sensitive protein,
(3) The method for diagnosing muscle atrophy according to (2), wherein the redox-sensitive protein is TNFα, pIκB, IL-8, MCP1 or ICAM1;
(4) The method for diagnosing muscle atrophy according to (1), wherein the oxidative stress-inducible protein is an oxidation-modified protein,
(5) The method for diagnosing muscle atrophy according to (4), wherein the oxidation-modified protein is a 4-hydroxy-2-nonenal modified protein,
(6) A method for diagnosing muscle atrophy, which comprises detecting muscle atrophy using the expression level of myostatin or calcineurin in muscle as an index,
(7) The method for diagnosing muscle atrophy according to any one of (1) to (6), wherein the expression level is a protein mass,
(8) The method for diagnosing muscle atrophy according to any one of (1) to (6), wherein the expression level is an mRNA level.
(9) A method for evaluating an antimuscular atrophy action using the method for diagnosing muscle atrophy according to any one of (1) to (8), wherein the oxidative stress is in the muscle of an animal ingested with a test substance An evaluation method of an antimuscular atrophy action characterized by evaluating an antimuscular atrophy action of a test substance using an expression level of an inducible protein, myostatin or calcineurin as an index,
(10) The method for evaluating an antimuscular atrophy action according to (9), wherein the test substance is a food material,
(11) A method for evaluating an antimuscular atrophy action of a test substance using the method for evaluating an antimuscular atrophy action described in (9) or (10), and screening a substance having a desired antimuscular atrophy action. And a screening method for antimuscular atrophy.

  According to the method for diagnosing muscle atrophy of the present invention, since the expression level of oxidative stress-inducible protein, myostatin or calcineurin is used as an index, muscle atrophy can be detected with higher sensitivity, which is useful for the prevention of muscle atrophy. Can do.

  According to the method for evaluating an antimuscular atrophy action of the present invention, the antimuscular atrophy action of a test substance such as a food material can be accurately and efficiently evaluated.

  According to the screening method for an antimuscular atrophy substance of the present invention, a substance such as a food material effective for muscle atrophy can be screened more efficiently.

  Hereinafter, the best mode for carrying out the present invention will be described in detail.

  One aspect of the method for diagnosing muscle atrophy of the present invention is to diagnose muscle atrophy using the expression level of oxidative stress-inducing protein in muscle as an index. Here, “diagnosis of muscle atrophy” not only determines whether or not muscle atrophy has developed, but also determines whether or not there is a risk of developing muscle atrophy for the purpose of preventing muscle atrophy, This includes monitoring the improvement of atrophy and recurrence.

  The “oxidative stress-inducible protein” refers to a protein induced by oxidative stress. Here, oxidative stress refers to active oxygen groups (superoxide, hydrogen peroxide, hydroxy radical, singlet oxygen, lipid radical, etc.) and active nitrogen groups (nitrogen monoxide, peroxynitrite, etc.) in cells and tissues. As a result of exceeding the antioxidant capacity, it means a state in which cells and tissues are inclined toward the oxidation side.

  One of the preferred embodiments of the method for diagnosing muscle atrophy of the present invention is an embodiment in which the oxidative stress-inducible protein is a redox-sensitive protein. Redox sensitive protein is a generic term for proteins whose functions are controlled by redox reactions. For example, a redox sensitive transcription factor is activated by oxidative stress and a redox sensitive protein is induced. Preferred examples of redox sensitive proteins include TNFα (Tumor necrosis factor alpha, tumor necrosis factor α), pIκB (Phospho Inhibitor of Nuclear Factor Kappa B), IL-8 (Interleukin-1M MC, P-8). ICAM1 (Intracellular adhesion molecule 1) is mentioned.

TNFα is a kind of inflammatory cytokine and is known to have a muscle protein degradation action.
pIκB is phosphorylated IκB (Inhibitor of Nuclear Factor Kappa B). The transcription factor NF-κB (Nuclear Factor Kappa B) expressing genes such as inflammatory cytokines, chemokines, and adhesion molecules is inactive in the state of forming a complex with IκB, but oxidative stress, etc. Thus, when IκB is phosphorylated, NF-κB moves away from IκB and moves to the nucleus. This phosphorylated IκB is pIκB.
IL-8 is a chemokine that specifically attracts neutrophils. In animals such as rats, CINC1 (cytokine-induced neutrophic chemotractant-1) corresponds to IL-8. In addition, the protein corresponding to IL-8 in mice is called KC, and the name of the protein corresponding to IL-8 varies depending on the animal species. In the present specification, IL-8 includes proteins corresponding to IL-8 of other animal species such as CINC1 and KC in addition to human IL-8.
MCP1 is a chemokine that specifically attracts monocytes (macrophages).
ICAM1 is a kind of inflammatory mediator and is expressed in muscle tissue and vascular endothelium. ICAM1 is also a kind of adhesion molecule, and adheres to circulating phagocytes and has an action of promoting tissue infiltration.

Other examples of redox sensitive proteins include IL-6 (Interleukin-6), IL-1β (Interleukin-1β), and IFN-γ (Interferon-γ). These redox sensitive proteins are also transcriptionally controlled by the transcription factor NF-κB.
As described above, redox-sensitive proteins include proteins exhibiting various activities such as cytokines, chemokines, and adhesion molecules.

  In another preferred embodiment of the method for diagnosing muscle atrophy of the present invention, the oxidative stress protein is an oxidatively modified protein. Here, the “oxidation-modified protein” refers to a protein in which some amino acid residues are modified by reacting with active oxygen or the like. Examples of amino acid residue modifications include 4-hydroxy-2-nonenal (4-HNE), acreolein (ACR), hexanoyllysine (Hexanoyl-lysine). ), Nitrotyrosine (Nitro-tyrosine) and the like.

  In still another preferred embodiment of the method for diagnosing muscle atrophy of the present invention, the oxidation-modified protein is a 4-HNE-modified protein.

  4-HNE is a kind of aldehyde that is a degradation product of lipid peroxide and is known to be generated by an increase in oxidative stress. As a result, it is known that 4-HNE is added and modified protein (4-HNE modified protein) is generated.

  In another aspect of the method for diagnosing muscle atrophy of the present invention, muscle atrophy is diagnosed using the expression level of myostatin or calcineurin in muscle as an index. Both myostatin and calcineurin are said to be associated with muscle hypertrophy. That is, myostatin is known to be a growth factor that suppresses muscle growth, and is a member of the TGF-β superfamily that is also called GDF8 (Growth and Differentiation Factor 8). Calcineurin is also called protein phosphatase 2B, and is known to have an action of promoting muscle hypertrophy and differentiation.

  The above-described redox-sensitive protein, 4-HNE-modified protein, myostatin and calcineurin are all known substances, but the present inventors have revealed for the first time that their expression level in muscle reflects the state of muscle atrophy. I made it.

  The specimen used for the method for diagnosing muscle atrophy of the present invention is preferably muscle, but may be any other tissue or blood as long as it reflects the expression level of oxidative stress-inducing protein in the muscle. . As the test material, an extract can be used when the sample is muscle or tissue, and plasma or serum can be used when the sample is blood.

  In the method for diagnosing muscle atrophy of the present invention, there are at least two embodiments depending on the measurement target when measuring the expression level of the oxidative stress-inducible protein. One embodiment is an embodiment in which the measurement target is the oxidative stress-inducible protein itself, and the amount of the oxidative stress-inducible protein is used as the expression level. In the present embodiment, since the amount of oxidative stress-inducing protein is directly measured, muscle atrophy can be diagnosed more accurately. In addition, when the oxidative stress-inducible protein is a protein that has been modified after translation, such as a 4-HNE-modified protein, it is particularly preferable to use this embodiment. The method for measuring the protein mass is not particularly limited as long as it can specifically measure the target oxidative stress-inducing protein. For example, various immunoassays, Western blotting, mass spectrometry, chromatography, etc. can be used. it can. In particular, when an antibody against the target oxidative stress-inducing protein has been obtained, immunoassay or Western blotting is preferably used.

  According to the immunoassay, the amount of oxidative stress-inducing protein can be accurately measured even in a sample containing a lot of contaminants. Examples of immunoassays include classical methods such as precipitation, agglutination, and hemolysis, which directly or indirectly measure antigen-antibody conjugates, and enzyme immunoassays (EIA) with increased detection sensitivity in combination with labeling methods. , Radioimmunoassay (RIA), fluorescent immunoassay (FIA) and the like. The antibody specific for the oxidative stress inducible protein may be monoclonal or polyclonal.

  When measuring the amount of oxidative stress-inducing protein by Western blotting, for example, the test material is subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the target oxidative stress-inducing protein. Then, the whole protein is transferred to the membrane, and an antibody against the oxidative stress inducing protein is used to detect the oxidative stress inducing protein on the membrane. When separation of oxidative stress-inducing proteins is insufficient with SDS-PAGE alone, two-dimensional electrophoresis combined with isoelectric focusing (IEF) can also be used. The antibodies specific for these oxidative stress inducible proteins may be monoclonal or polyclonal.

  When the amount of oxidative stress-inducible protein is measured by mass spectrometry, any of matrix-assisted laser desorption / ionization (MALDI) and electrospray ionization (ESI) can be used as the ionization method. Although applicable, MALDI is preferred because it produces less multivalent ions. In particular, according to MALDI-TOF-MS combined with a time-of-flight mass spectrometer (TOF), the amount of the marker substance can be measured more accurately. Furthermore, an oxidative stress-inducing protein can be immobilized on a carrier and subjected to mass spectrometry. For example, using a protein chip that uses a substrate as a carrier, surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is performed. In addition, the amount of oxidative stress-inducible protein can be measured more accurately.

  In the case of measuring the amount of oxidative stress-inducing protein by chromatography, for example, a method by liquid high performance chromatography (HPLC) can be used. That is, the amount of the oxidative stress-inducing protein in the sample can be measured by subjecting the sample to HPLC to separate the target oxidative stress-inducing protein and measuring the peak area of the chromatogram.

  Another embodiment of the method for diagnosing muscle atrophy of the present invention is an embodiment in which the measurement target is mRNA of an oxidative stress-inducible protein, and the transcription level of the gene is measured. In this embodiment, the transcription stage of the oxidative stress-inducible protein gene can be captured, and highly sensitive diagnosis is possible. The method for measuring the amount of mRNA is not particularly limited as long as it can specifically measure the amount of mRNA of the target oxidative stress-inducing protein, and real-time PCR (RT-PCR), quantitative PCR, Northern blotting, etc. are used. be able to. In particular, RT-PCR is preferable because the measurement time is short and even a very small amount of mRNA can be measured. In addition, any probe or primer may be used for these methods as long as it can specifically hybridize to a gene transcription product of each oxidative stress-inducing protein or amplify the gene transcription product. By using a probe or primer labeled by an appropriate method, the amount of mRNA can be measured. In the method for diagnosing muscle atrophy of the present invention, either the protein mass or the mRNA amount may be measured, but both may be measured when more accuracy is required. Further, the number of oxidative stress-inducing proteins whose expression level is measured may be one or plural.

  Although the present invention relates to a method for diagnosing muscle atrophy, it can capture the state of muscle hypertrophy using the same means, that is, the expression level of oxidative stress-inducing protein, myostatin or calcineurin in muscle as an index. There is a possibility.

  The method for diagnosing muscle atrophy of the present invention can also be used for the development and quality control of medical devices. For example, when developing a medical device for suppressing muscle atrophy or promoting muscle hypertrophy by applying a stimulus such as heat, cooling, electricity, or physical stimulus, the muscle atrophy diagnosis method of the present invention The effect of suppressing muscle atrophy and promoting muscle hypertrophy by using the medical device can be evaluated.

  The method for evaluating the antimuscular atrophy action of the present invention uses an animal to ingest a test substance and uses the expression level of an oxidative stress-inducing protein, myostatin or calcineurin in the muscle of the animal as an index. Here, the “antimuscular atrophy action” refers to an action for treating, improving, suppressing and / or preventing muscle atrophy. There is no restriction | limiting in particular as an animal used with the evaluation method of the antimuscular atrophy action of this invention, For example, a mouse | mouth, a rat, a rabbit etc. can be used. Examples of the test substance in the method for evaluating an antimuscular atrophy action of the present invention include a drug substance and a food material, but a food material is preferable. That is, by evaluating food materials by the method for evaluating antimuscular atrophy of the present invention, it can be used for the development of functional foods. In addition, there is a possibility that the effect of promoting muscle hypertrophy as well as the antimuscular atrophy action can be evaluated by the method for evaluating the antimuscular atrophy action of the present invention.

  The method for evaluating the antimuscular atrophy action of the present invention can be carried out, for example, as follows. First, rats are divided into two groups, and one group is fed with a normal diet and the other group is fed with a feed containing a test substance. After rearing for a while, the left limb sciatic nerve is cut in each group of rats to inactivate the left lower limb muscle, causing disuse muscle atrophy in the left foot. Next, the soleus muscle of the left leg of each group is extracted, and the protein amount of the oxidative stress-inducing protein in the soleus muscle is measured by immunoassay or the like. When the group containing the test substance-containing sample has a smaller amount of oxidative stress-inducing protein, it is evaluated that the test substance is more effective in suppressing muscle atrophy. According to this method, muscle atrophy cannot be detected even if muscle weight or muscle protein is used as an index. Can be evaluated.

  The present invention relates to a method for screening an antimuscular atrophy substance, which allows an animal to ingest a test substance, and uses the expression level of an oxidative stress-inducing protein, myostatin or calcineurin in the muscle of the animal as an index. The action is evaluated. Here, the “antimuscular atrophy substance” refers to a substance having an effect of treating, improving, suppressing and / or preventing muscle atrophy, that is, a substance having an antimuscular atrophy action. According to the screening method for an antimuscular atrophy substance of the present invention, an antimuscular atrophy substance can be screened more efficiently and accurately. In particular, when the test substance is a food material, a functional food having an antimuscular atrophy action can be produced using the screened food material as a main raw material. And by eating such a functional food on a daily basis, it is possible to suppress the onset of muscle atrophy and prevent muscle atrophy. In addition, the screening method for an antimuscular atrophy substance of the present invention may be able to screen not only an antimuscular atrophy substance but also a substance that promotes muscle hypertrophy.

  The screening method for an antimuscular atrophy substance of the present invention can be performed, for example, as follows. First, rats are divided into groups corresponding to the number of test substances, and each group of rats is fed with a feed containing each test substance and reared. After rearing for a while, the left limb sciatic nerve is cut in each group of rats to inactivate the left lower limb muscle, causing disuse muscle atrophy in the left foot. Next, the soleus muscle of the left leg of each group is extracted, and the protein amount of the oxidative stress-inducing protein in the soleus muscle is measured by immunoassay or the like. And the test substance given to the group which showed a smaller value is selected as a substance effective in suppression of muscle atrophy. According to this method, muscle atrophy cannot be detected even if muscle weight or muscle protein is used as an index. Even if the soleus muscle is removed at an early stage after nerve cutting, muscle atrophy can be detected. Substances can be screened.

  The screening method for an antimuscular atrophy substance of the present invention is also useful in the field of livestock, for example, by feeding a livestock containing a screened antimuscular atrophy substance to livestock to suppress muscle atrophy or promote muscle hypertrophy. The meat yield per head can be increased.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

1. Construction of animal model of disuse muscle atrophy 9 weeks old male male Wistar rats were divided into 3 groups of 6 rats, each of which was allowed to freely eat normal diet, vitamin E (VE) supplemented diet, and coenzyme Q (CoQ) supplemented diet And raised. As the VE-added food, food containing 25 times the amount of α-tocopherol (500 IU / kg) as compared with the normal food was used. In addition, as the CoQ-added food, 100 mg of CoQ added per 1 kg of the normal food was used. One week after breeding, the left limb sciatic nerve was cut for each group of rats to make the left lower limb muscle inactive. Thereafter, after further breeding for 2 weeks, each group of rats was sacrificed, and the soleus, one of the lower limb muscles, was removed from the left foot (denervated limb) and the right foot (control limb).

  First, the muscle weight of the extracted soleus was measured with a microbalance. Furthermore, the amount of muscle protein was measured by the following procedure. First, the extracted soleus muscle was homogenized, and 50 mg was suspended in lysis buffer (Sigma). After stirring for 1 hour, centrifugation (14000 rpm, 10 minutes, 4 ° C.) was performed, and the supernatant was recovered to obtain a protein extract. The protein concentration in the obtained supernatant was measured by the Bradford method and used as the amount of muscle protein. FIG. 1 (a) shows the measurement result of muscle weight, and FIG. 1 (b) shows the measurement result of muscle protein amount. That is, the left foot denervated in any group was significantly lower in both muscle weight and muscle protein than the normal right foot (p <0.05). This confirmed that disuse muscular atrophy developed by denervation. In addition, the group fed the VE-added diet or CoQ-added diet had significantly higher muscle weight and muscle protein in the left foot than the group fed the normal diet (p <0.05). Thereby, it was confirmed that disuse muscle atrophy can be suppressed or improved by giving a VE-added meal or a CoQ-added meal. Based on the above, an animal model for the onset, suppression, and improvement of disuse muscle atrophy was constructed.

2. Measurement of each oxidative stress-inducible protein (1)
Using the protein extract prepared in 1 above as a sample, the concentrations of 4-HNE modified protein, ICAM1, TNFα, and pIκB were measured by Western blotting according to the following procedure. First, 20 μg of the protein extract was subjected to SDS-PAGE, and the entire protein was transferred to the membrane. The membrane was immersed in PBST containing 5% skim milk (PBS + 0.05% TWEEN20) for 1 hour to block. Next, the membrane was immersed overnight in a primary antibody solution (primary antibody dissolved in PBST containing 5% skim milk). Next, the membrane was immersed for 60 minutes in a secondary antibody solution (secondary antibody dissolved in PBST containing 5% skim milk). Next, the membrane was chemiluminescentd and developed into a film, and the target band was detected. The emission intensity of the band was measured by NIH image and used as the concentration of each band. The primary antibodies were manufactured by ALEXIS Biochemical (Part No. 210-767-R100) for 4-HNE-modified protein, SantaCluz (Part No. sc-8439) for ICAM1, and SantaCluz for TNFα. A product manufactured by SantaCluz (product number sc-8404) was used for the product (product number sc-1348) and pIκB. In addition, as the secondary antibody, a peroxidase-labeled anti-rabbit IgG antibody (Anti-rabbit IgG horseradish peroxidase) was used. The product number NA934V manufactured by Amersham Bioscience was used.

Fig. 2 (a) shows the measurement result of 4-HNE modified protein, Fig. 2 (b) shows the measurement result of ICAM1, Fig. 2 (c) shows the measurement result of TNFα, and Fig. 2 (d) shows the measurement result of pIκB. Indicates. In each graph, the vertical axis represents a value obtained by quantifying the detected band with the background value, and the relative value to the control. In other words, when a normal diet, a VE-added diet, or a CoQ-added diet is consumed, when the muscle (left foot) causing disuse muscle atrophy is compared with the normal muscle (right foot), disuse muscle atrophy The concentration of any oxidative stress-inducing protein of 4-HNE-modified protein, ICAM1, TNFα, and pIκB was significantly higher in the muscles (left foot) that had developed (p <0.05). pIκB was not detected in normal muscle (right foot). In addition, when comparing muscles (left foot) that caused disuse muscle atrophy when taking a normal diet, a VE-added diet, or a CoQ-added diet, the concentrations of 4-HNE-modified protein, TNFα, and pIκB are: It was significantly lower when both the VE-added diet and the CoQ-added diet were consumed than when the normal diet was consumed (p <0.05). Moreover, the concentration of ICAM1 was significantly lower when the CoQ-added diet was ingested than when the normal diet was ingested (p <0.05). In particular, the concentration of pIκB was significantly different, reflecting the degree of disuse muscle atrophy with high sensitivity.
As described above, by using the expression levels of 4-HNE-modified protein, ICAM1, TNFα, and pIκB in the muscle as an index, it is possible to detect disuse muscle atrophy, and to suppress or improve disuse muscle atrophy. The possibility of monitoring the condition was suggested.

3. Quantification of each oxidative stress-inducible protein (2)
Using the protein extract prepared in 1 above as a sample, the concentrations of CINC1 and MCP1 were measured by ELISA. A CIBL kit was used for CINC1 measurement, and a BIOSOURCE kit was used for MCP1 measurement. FIG. 3A shows the CINC1 measurement result, and FIG. 3B shows the MCP1 measurement result. In each graph, the vertical axis represents the value measured in units of pg / mg protein × 10 ⁻² relative to the control. In other words, when a normal diet, a VE-added diet, or a CoQ-added diet is consumed, when the muscle (left foot) causing disuse muscle atrophy is compared with the normal muscle (right foot), disuse muscle atrophy The concentration of the oxidative stress-inducing protein of CINC1 and MCP1 was significantly higher in the muscles (left foot) that had developed (p <0.05). In addition, when comparing muscles (left foot) that caused disuse muscle atrophy when a normal diet, a VE-added diet, or a CoQ-added diet were consumed, the concentrations of CINC1 and MCP1 were VE-added and CoQ-added diets. When either of these was ingested, it was significantly lower (p <0.05) than when ingested the normal diet.
From the above, by using the expression level of CINC1 (IL-8) or MCP1 in muscle as an index, it is possible to detect disuse muscle atrophy and to monitor the suppression or improvement of disuse muscle atrophy. The possibility was suggested.

4). Quantification of myostatin and calcineurin The amount of myostatin and calcineurin mRNA in muscle was measured by the following procedure. First, RNA was extracted from the muscle homogenized in the above 1 using an RNeasy kit (Qiagen). Next, cDNA was synthesized from the extracted RNA by reverse transcription using an RT-PCR kit (Takara Bio Inc.). Next, PCR was performed using the obtained cDNA as a template to obtain an amplified DNA fragment. As primers, oligonucleotides represented by SEQ ID NO: 1 (sense) and SEQ ID NO: 2 (antisense) are used for myostatin, and SEQ ID NO: 3 (sense) and SEQ ID NO: 4 (antisense) are used for calcineurin. Oligonucleotides were used. The amount of the obtained amplified DNA fragment was quantified with GeneAmp5700 (Applied Biosystem) and converted to the amount of mRNA. FIG. 4 (a) shows the measurement results of myostatin mRNA, and FIG. 4 (b) shows the measurement results of calcineurin mRNA. In each graph, the vertical axis is a relative value with respect to the control. In other words, the amount of myostatin mRNA compared to the muscles with disuse muscle atrophy (left foot) and normal muscles (right foot), regardless of whether the normal diet, VE supplemented diet, or CoQ supplemented diet was consumed. The muscle (left foot) that caused disuse muscular atrophy was significantly higher (p <0.05). On the other hand, the calcineurin mRNA level when comparing the normal diet, the VE-added diet, and the CoQ-added diet was compared with the muscle (left foot) that caused disuse muscle atrophy and the normal muscle (right foot). The muscle (left foot) that caused disuse muscular atrophy was significantly lower (p <0.05). In addition, when comparing muscles (left foot) that caused disuse muscle atrophy when taking a normal diet, a VE-added diet, or a CoQ-added diet, the amount of myostatin mRNA is It was significantly lower (p <0.05) than when the normal diet was ingested. On the other hand, when comparing muscles (left foot) that caused disuse muscle atrophy when a normal diet, a VE-added diet, or a CoQ-added diet was consumed, the amount of calcineurin mRNA was not significantly different.
Based on the above, disuse muscle atrophy can be detected by using the expression level of myostatin or calcineurin in the muscle as an index, and the suppression or improvement of disuse muscle atrophy can be monitored for myostatin Was suggested.

FIG. 1A is a graph showing the measurement result of muscle weight in each group, and FIG. 1B is a graph showing the measurement result of muscle protein amount in each group. FIG. 2 (a) is a graph showing the measurement result of 4-HNE modified protein in each group, FIG. 2 (b) is a graph showing the measurement result of ICAM1 in each group, and FIG. 2 (c) is a graph showing the measurement result of each group. Fig. 2 (d) is a graph showing the measurement results of pIκB in each group. FIG. 3A is a graph showing the CINC1 measurement results in each group, and FIG. 3B is a graph showing the MCP1 measurement results in each group. FIG. 4A is a graph showing the measurement results of myostatin mRNA in each group, and FIG. 4B is a graph showing the measurement results of calcineurin mRNA in each group.

Claims (11)

  A method for diagnosing muscle atrophy, comprising detecting muscle atrophy using the expression level of an oxidative stress-inducing protein in muscle as an index.   The method for diagnosing muscle atrophy according to claim 1, wherein the oxidative stress-inducing protein is a redox-sensitive protein.   The method for diagnosing muscle atrophy according to claim 2, wherein the redox-sensitive protein is TNFα, pIκB, IL-8, MCP1 or ICAM1.   The method for diagnosing muscle atrophy according to claim 1, wherein the oxidative stress-inducible protein is an oxidatively modified protein.   The method for diagnosing muscle atrophy according to claim 4, wherein the oxidation-modified protein is a 4-hydroxy-2-nonenal modified protein.   A method for diagnosing muscle atrophy, comprising detecting muscle atrophy using the expression level of myostatin or calcineurin in muscle as an index.   The method for diagnosing muscle atrophy according to any one of claims 1 to 6, wherein the expression level is a protein mass.   The method for diagnosing muscle atrophy according to any one of claims 1 to 6, wherein the expression level is an mRNA level.   A method for evaluating an antimuscular atrophy action using the method for diagnosing muscle atrophy according to any one of claims 1 to 8, comprising an oxidative stress-inducing protein in the muscle of an animal fed with a test substance, myostatin Alternatively, a method for evaluating an antimuscular atrophy effect, comprising evaluating an antimuscular atrophy effect of a test substance using an expression level of calcineurin as an index.   The method for evaluating an antimuscular atrophy action according to claim 9, wherein the test substance is a food material.   An antimuscular atrophy characterized by evaluating an antimuscular atrophy of a test substance using the method for evaluating an antimuscular atrophy according to claim 9 or 10, and screening a substance having a desired antimuscular atrophy. Substance screening method.

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