JP6623162B2 - Factors involved in fatigue and their use - Google Patents

Description

  The present invention relates to a method for evaluating the degree of human fatigue and its use, a kit for evaluating the degree of fatigue, a method for measuring anti-fatigue power of an anti-fatigue substance or anti-fatigue method, and a fatigue model animal.

  Fatigue is a very familiar problem in daily life, and among modern people with much stress, many people suffer from various types of fatigue. There are two types of fatigue: “physiological fatigue”, fatigue that occurs when the body and body are subjected to physical and physical stress, and fatigue associated with some primary disease, called “pathological fatigue”. . Recently, fatigue caused by diseases of the central nervous system, such as chronic fatigue syndrome (CFS) and depression, can cause abnormal fatigue in the brain, especially as “pathological fatigue”. It is distinguished from morbid fatigue due to mechanical fatigue and diseases of peripheral organs and tissues. Pathological fatigue other than physiological fatigue and pathological fatigue is collectively referred to as “nonpathological fatigue” and occurs when fatigue load on peripheral organs and tissues is transmitted to the brain in some way, and the brain feels fatigue. On the other hand, pathological fatigue has no cause in the periphery, and feels fatigue due to some abnormality in the brain (Non-patent Document 1). That is, nonpathological fatigue can be said to be fatigue caused by peripheral organs and tissues, and pathological fatigue can be said to be fatigue caused by brain diseases. Since there is no appropriate Japanese translation for pathological fatigue and nonpathological fatigue, the names in English are used as they are in this specification. Nonpathological fatigue in the present specification represents fatigue including all physiological fatigue and pathological fatigue caused by diseases of peripheral organs and tissues. Further, unless otherwise specified, fatigue in the present specification indicates nonpathological fatigue.

  Despite fatigue being a major social problem, scientific and medical research related to fatigue was only fragmented, and was a decisive means or quantification of how to express fatigue quantitatively and objectively. No scale has been established. According to the inventors' prior patent, the fatigue measurement method (patent documents 1 and 2) using human herpesvirus in saliva can measure objective fatigue (especially nonpathological fatigue), but the measurement target is medium- to long-term. However, it was limited to measurement of fatigue and was not suitable for measurement of acute fatigue such as fatigue load test. In addition, there are problems that the mechanism of fatigue has not been elucidated and that it can be applied only to humans and cannot be applied to research and testing of fatigue using animal models. Understanding fatigue mechanisms, stress tests and animal experiments are essential factors for screening anti-fatigue substances and developing methods for preventing and recovering from fatigue, so answer these questions and measure fatigue objectively. There was a strong demand for ways to do this.

  With regard to the mechanism of fatigue, muscle fatigue has been mainly studied as a representative example of “fatigue” a while ago, and an increase in the amount of lactic acid produced in the muscle has attracted attention as an index. This is why the theory of “fatigue substance” equal lactic acid has spread. However, lactic acid is an important energy source by nature, and the theory that lactic acid inhibits muscle activity is now viewed negatively. In addition, there is a known phenomenon that pH decreases with muscle fatigue. However, these are certain phenomena that can be seen when a certain amount of stress (exercise load) is applied to the muscles, but are thought to be phenomena that remain locally as muscles. “Fatigue” is considered to be a broader and larger physiological phenomenon appearing in the living body, and it is considered that it is necessary to elucidate mechanisms other than these (Non-Patent Document 2).

  Next, chronic fatigue syndrome (CFS) was studied intensively as a mechanism of fatigue. From the CFS studies, fatigue mechanisms include oxidative stress, inflammatory cytokines, autonomic dysfunction, and immune abnormalities, which have been regarded as promising biomarkers of fatigue, particularly chronic fatigue. On the other hand, however, it has been pointed out that CFS fatigue is pathological fatigue and is not representative of fatigue in conditions not related to brain diseases. Furthermore, regarding CFS itself, theories that it is a neurological disease caused by some kind of infection and that it is appropriate to consider it as a type of depression are becoming more prominent, and the indicators obtained as biomarkers for CFS remain unchanged. Whether it can be used as an indicator of fatigue has been questioned.

  In animal experiments of fatigue, emphasis is placed on reproducing a chronic fatigue state similar to CFS, and the mechanism of fatigue in non-physiological overwork is being investigated. In the study of the fatigue mechanism, a model imitating a viral infection called an infection model may be used. However, both animal models reproduce the chronic pathological fatigue state similar to CFS. Under such circumstances, the mechanism of fatigue due to physiological conditions has hardly been elucidated (Non-Patent Documents 3 and 4).

  An important characteristic of fatigue, particularly physiological fatigue, that should be explained when studying the mechanism of fatigue is the property of being easily recovered by rest (called reversibility or easy recovery). This property is an important factor in defining the physiological phenomenon of fatigue, and is considered to be a factor necessary for elucidating the mechanism of fatigue.

Although the mechanism of fatigue is thought to be related to some kind of stress response, there are many types of stress responses and signal transmission pathways, and it is very difficult to narrow down the mechanisms and signal transmission pathways specific to fatigue. Furthermore, studies using DNA microarrays are known to change the expression of more than 1,000 genes in response to fatigue stimuli, which makes it extremely difficult to identify fatigue mechanisms and signal transduction pathways. It was expected to be.
In addition, molecular biomarker searches for fatigue using DNA microarrays are often screened based on changes in expression due to fatigue. In signal transduction, a large change does not necessarily reflect a specific change, so there was a question as to whether a fatigue-specific molecular biomarker can be searched correctly using such a search method. .
For this reason, in addition to the condition that it increases due to fatigue load, fatigue biomolecules can be induced by introduction into experimental animals, fatigue can be attenuated by inhibiting the function of the molecule, and signal transduction pathways as much as possible The condition of being upstream, and showing the relationship with the substance causing fatigue (Non-Patent Document 5) is required.

  As mentioned above, mechanisms and methods for fatigue related to muscle fatigue and CFS have been proposed, but as mentioned earlier, physiological fatigue in everyday life is something that many modern people feel. Nevertheless, there are few reports on the mechanism and objective evaluation method. Furthermore, biomarkers capable of discriminating between physiological fatigue and pathological fatigue, and pathological fatigue and nonpathological fatigue are not clear, and methods for evaluating and discriminating these have not been established. Even if the fatigue in daily life is physiological fatigue, if it is left as it is, there is a risk that it may directly lead to health problems such as mental illness and death from overwork. Although the problem of mental illness due to overwork and fatigue and the problem of death from overwork are recognized to be very important medically, economically and socially, the scientific mechanism of vital fatigue has been largely elucidated. Not. For this reason, in order to prevent these problems that have become social problems in recent years, an objective fatigue evaluation method based on the fatigue mechanism is required.

  In addition, many of the medicines such as energy drinks that flood the market or health foods are for sale with anti-fatigue effects, so the scientific support for their functionality is not limited to consumers but also to the market and society as a whole. Widely sought after. In addition, the effect of preventing or suppressing or recovering from fatigue or fatigue effects and disorders is collectively referred to as the anti-fatigue effect, but the anti-fatigue effect includes the effect of preventing or preventing fatigue (fatigue prevention effect), and It includes the effect of recovering or suppressing the generated fatigue and the effect of fatigue (fatigue recovery effect). In order to efficiently use anti-fatigue substances and anti-fatigue methods, it is necessary to evaluate the action mechanism, such as whether these have a fatigue prevention effect or a fatigue recovery effect, and use them appropriately.

  Against this background, the present inventors conducted research on the development of objective fatigue measurement methods and fatigue mechanisms (Non-Patent Documents 6 to 12). Using the unpublished know-how that only the inventor obtained by this research knows, the invention described in this patent specification was obtained.

  As described above, in order to evaluate the possibility of failure due to fatigue and take appropriate preventive measures, it is not only quantitative measurement of fatigue, but whether fatigue is physiological fatigue or pathological fatigue. It is necessary to objectively evaluate the type of fatigue, such as whether it is pathological fatigue or nonpathological fatigue, and use anti-fatigue substances and anti-fatigue methods that can appropriately deal with these types of fatigue. is there. For this reason, there is a strong demand for the development of a method that can easily and objectively evaluate the quality and amount of fatigue and its utilization method.

"Fatigue degree evaluation method and its utilization" Patent No. 4218842 (issued February 4, 2009) "Fatigue degree evaluation method and its use" Patent No. 4812708 (issued on November 9, 2011)

What is fatigue? Pathological and nonpathological fatigue. Jason LA, Evans M, Brown M, Porter N. PM & R. 2010; 2 (5): 327-31. "Mechanism of fatigue" Akira Watanabe (Ayumi of Medicine 2009; 228 (6): 598-604) "Mechanism of fatigue as revealed from research using overworked animal models" Masaaki Tanaka (Ayumi of Medicine 2009; 228 (6): 610-4) "Molecular neural mechanism of fatigue in an immunological fatigue model" Toshihiko Katanabe (Ayumi Medicine 2009; 228 (6): 615-9) "Verification of anti-fatigue effects of imidazole dipeptides from the perspective of peripheral blood gene expression profile" Seiji Nakamura, Tomohiro Sugino, Osamu Enomoto, Kenichi Matsubara, Ryo Matoba, 33rd Annual Meeting of the Molecular Biology Society of Japan (2010, Kobe), http: //www.dna-chip.co.jp/research/pdf/r4_MBSJ2010.pdf Welfare Labor Sciences Research Grants (Disability Countermeasure Research Research Project) Research Report (FY2009-23), Establishing an Objective Fatigue Diagnosis Method for Patients Complaining of Chronic Fatigue with Autonomic Nervous System Abnormalities Preparation of diagnostic guidelines for chronic fatigue, “Biological evaluation of saliva in patients with chronic fatigue”, Kazuhiro Kondo Health Labor Sciences Grant-in-Aid for Scientific Research on Disability (Comprehensive Research Project for Persons with Disabilities) Research Report (2013), Elucidation of the Pathogenesis of Chronic Fatigue Syndrome and Development of Breakthrough Diagnosis and Treatment, “HHV-6 in Saliva , Differentiation of Chronic Fatigue Syndrome from HHV-7 and Labor Fatigue ", Kazuhiro Kondo "Identification of Novel HHV-6 Latent Protein associated with Mood Disorders and Molecular Mechanism of Fatigue due to Overwork", Kazuhiro Kondo et al., International Conference on Fatigue Science 2008 Abstract S2-03 "Identification of a novel molecular mechanism and a major cause of fatigue", Kazuhiro Kondo, The 36th Congress of the International Union of Physiological Sciences 2009 Abstract RS LB-52-1 “New fatigue” Kazuhiro Kondo, Nobuyuki Kobayashi, Naomi Oka, 68th Annual Meeting of the Japanese Society of Physical Fitness (2013) Special Lecture 2 "Fatigue biomarkers: Objective fatigue measurement method", Kazuhiro Kondo, Medical Technology Journal 2013 41 (6) 583-584 “Science of“ fatigue ”at the source of psychosomatic correlation”, Kazuhiro Kondo, Psychosomatic Medicine 2014, Vol. 54, No. 9, pp. 828-833

  Although the measurement method of fatigue seems to be objective at first glance, in many cases it actually involves a recognition mechanism of fatigue in the brain, and directly measures fatigue of peripheral organs and tissues in nonpathological fatigue including physiological fatigue. It was difficult to measure.

  The cause of nonpathological fatigue is mainly in the peripheral tissues, and the cause of pathological fatigue is in the brain. For this reason, an objective fatigue measurement method should be able to easily distinguish these fatigues.

  In addition, the fatigue measurement method using human herpesvirus in saliva is one of the few methods that can objectively measure fatigue, but it was necessary to wait for accumulation of fatigue for more than one week for measurement. For efficient screening of anti-fatigue substances and anti-fatigue methods, it is required to be able to measure fatigue under a short-term fatigue load.

  Furthermore, the fatigue measurement method using human herpesvirus in saliva can be measured only in humans. For efficient screening of anti-fatigue substances and anti-fatigue methods, an objective fatigue measurement method that can be used in laboratory animals is required.

  In order to develop an anti-fatigue substance and an anti-fatigue method, an animal model of fatigue is required, and the animal is fatigued by applying various loads. However, unlike humans, laboratory animals cannot complain of their own condition, so it is impossible to determine whether a phenomenon other than fatigue has occurred due to the applied load. Therefore, there is a need for a fatigue model animal that causes only a phenomenon related to fatigue purely by introducing a substance or signal transmission related to a fatigue substance, fatigue signal transmission, or anti-fatigue effect into the animal.

  As a result of intensive studies in view of the above problems, the present inventors have found that the essential part of the phenomenon of fatigue (nonpathological fatigue or physiological fatigue) is the following phenomenon: (1) Proteins of cells constituting the organ Phosphorylation of eukaryotic initiation factor-2 alpha subunit (eIF-2α) that forms the synthesis mechanism, (2) eIF-2α in organ cells and blood cells caused by phosphorylation of eIF-2α Increase in phosphorylation signaling factor (factor that transmits eIF-2α phosphorylation signal), especially increase in activating transcription factor (ATF) 4, ATF3, C / EBP-homologous protein (CHOP), (3) ATF3 and CHOP By using an experimental system that uniquely identifies and evaluates the increase in inflammatory cytokines such as TNF-α and the recognition of fatigue in the brain caused by the increase in these inflammatory cytokines. Daily life Measuring the degree of fatigue in and completed the present invention that it is.

  Phosphorylation of eIF-2α is a phenomenon in which protein synthesis is suppressed, and ATF3 is usually famous for its function of suppressing the production of inflammatory cytokines. For this reason, it was difficult to consider the fatigue phenomenon characterized by increased production of inflammatory cytokines and the signal transduction pathways involved in eIF-2α phosphorylation and ATF3 production.

Furthermore, the present inventors, in the case of fatigue, have a factor that suppresses phosphorylation of eIF-2α and signal transduction associated therewith (eIF-2α phosphorylation signal inhibitor), particularly growth arrest and DNA damage-inducible protein 34 (GADD34), non-catalytic region of tyrosine kinase adaptor protein (Nck) 1, Nck2, constitutive repressor of eIF-2α phosphorylation (CReP), zinc finger protein 36 homolog (ZFP36), glucocorticoid-induced leucine zipper (GILZ), etc. We found that production was enhanced.
Since these eIF-2α phosphorylation signal suppressors are induced by eIF-2α phosphorylation due to fatigue, the increase in eIF-2α phosphorylation signal suppressors was slightly increased in the past. An increase has occurred. In addition, eIF-2α phosphorylation signal inhibitory factor restores or suppresses fatigue by suppressing fatigue signal transduction induced by phosphorylation of eIF-2α or eIF-2α phosphorylation, which is the true state of fatigue Has an effect. For this reason, the strong induction of the phosphorylation signal inhibitory factor of eIF-2α means that the fatigue recovery ability is strong. Therefore, the anti-fatigue power (fatigue recovery power) of the test subject organism can be evaluated using the amount of the phosphorylation signal inhibitory factor of eIF-2α as an index, and such a method is also included in the present invention. Such an anti-fatigue strength evaluation method is preferably such that if the amount of the eIF-2α phosphorylation signal inhibitory factor is significantly greater than the amount predicted from the amount of the eIF-2α phosphorylation signal transduction factor, It is characterized by evaluating that the anti-fatigue power is high. In other words, the anti-fatigue power evaluation method of the present invention provides a test subject organism when the ratio of the amount of eIF-2α phosphorylation signal inhibitor to the amount of eIF-2α phosphorylation signal transduction factor is higher than a predetermined reference value. It is characterized by evaluating that the anti-fatigue power of is high. The reference value may be a reference value set in advance for each specific combination of eIF-2α phosphorylation signal transduction factor and eIF-2α phosphorylation signal suppressor, for example, ingesting anti-fatigue substance candidates The ratio of the amount of the eIF-2α phosphorylation signal inhibitory factor to the amount of the eIF-2α phosphorylation signal transduction factor before the execution of the anti-fatigue method candidate can be used as a reference value. On the other hand, the ratio of the amount of the eIF-2α phosphorylation signal inhibitor to the amount of the eIF-2α phosphorylation signal transduction factor after ingesting the anti-fatigue substance candidate or after implementing the anti-fatigue method candidate is the anti-fatigue substance candidate It is possible to evaluate that a test subject organism showing a value higher than the value before ingesting or before anti-fatigue method candidate has increased anti-fatigue power by the anti-fatigue substance candidate or anti-fatigue method candidate.
In the present specification, “recovery of fatigue” means “the signal transmission of fatigue caused by fatigue load and the effect of reducing the effect after the fatigue load is lost”, and “the signal of fatigue caused by fatigue load”. “The function of reducing the transmission and its effect at a time when there is a fatigue load” is expressed as “inhibition of fatigue”. In addition, "to reduce the influence of fatigue by increasing the ability to prevent fatigue, recover fatigue and suppress fatigue" is called "prevention of fatigue", recovery and suppression of fatigue, It is expressed as “anti-fatigue” as a general term for prevention. In addition, the force for recovering fatigue and the force for suppressing fatigue may be combined and expressed as fatigue recovery force. The eIF-2α phosphorylation signal inhibitory factor of the present invention suppresses fatigue if acting upon fatigue loading, and recovers fatigue if acting after the end of fatigue loading. Also, increasing the action of this factor leads to fatigue prevention.

Among the eIF-2α phosphorylation signal suppressors, GADD34 and CReP exert anti-fatigue effects by a mechanism that releases phosphorylation of eIF-2α (promotes dephosphorylation of phosphorylated eIF-2α), Nck1 and Nck2 exert an anti-fatigue effect by a mechanism that suppresses phosphorylation of eIF-2α by PKR and the like. The anti-fatigue effect of these factors was not known until the present inventors clarified the fatigue mechanism and the fatigue signal transduction pathway in the present invention.
Among the eIF-2α phosphorylation signal inhibitors, ZFP36 and GILZ are also involved in recovery / suppression of fatigue. Among these, ZFP36, also called Tristetraprolin, is a protein that binds to ARE (AU-rich element) and promotes destabilization of ARE-RNA. ZFP36 is an intracellular protein that destabilizes TNFα mRNA and reduces its production by this function. GILZ, also called TSC22D3, is a leucine zipper-containing protein derived from glucocorticoid. GILZ is known to inhibit the DNA binding of transcription factors AP-1 and NF-kappaB, thereby blocking the signal transduction involved in these transcription factors and suppressing the anti-inflammatory signal of macrophages and dendritic cells. Yes. Both exert an anti-fatigue effect by affecting the immune phenomenon (suppressing the immune mechanism and exhibiting an anti-fatigue effect). The relationship between such fatigue and the present inventors is the fatigue signal in the present invention. It was not clear until the discovery of the transmission pathway.

  As a result of studies by the present inventors, fatigue could be generated by introducing ATF3 into animals. On the other hand, by suppressing the ATF3 production in animals using interfering RNA, fatigue could be reduced. This indicates that ATF3 is a factor responsible for fatigue. In addition, the introduction of eIF-2α phosphorylation signal transduction factor typified by ATF3 is an animal that can examine the effects of fatigue purely on biological functions, especially brain functions, without the effects of muscle movement or organ burden. This shows that the model has been established. Furthermore, it is shown that fatigue can be attenuated by suppressing eIF-2α phosphorylation signaling factor typified by ATF3. Thus, the eIF-2α phosphorylation signaling factor represented by ATF3 can act as a “factor related to the occurrence of fatigue”.

Further, the present inventors have found that an anti-fatigue effect can be obtained by inhibiting the phosphorylation signal of eIF-2α, which is a factor related to the occurrence of fatigue, with a drug.
The phosphorylation of α of eIF2 is also a factor related to stress response called integrated stress response (ISR). Signal transduction pathways and factors related to stress responses include heat shock proteins, hypothalamus-pituitary-adrenal-axis (HPA axis), catecholamines, oxidative stress, epigenetic responses, cell membrane changes, lipid changes, etc. A great many types are known. For this reason, although it is known that fatigue and stress are related, it has not been known until the present inventors which stress response has a relationship with fatigue. In addition, although there is no solid evidence, many research groups looked at oxidative stress and HPA axis as prominent stress responses related to fatigue. For these reasons, the relationship between ISR and fatigue was not noted. In addition, regarding ISR, attention has been paid to the relationship with important diseases such as immune diseases, mental illnesses, identification cards, cancer, etc., and in considering the relationship with fatigue, particularly physiological fatigue that is the object of the present invention. It was an impediment.

  In addition, the present inventors have found that salubrinal, an agent that inhibits dephosphorylation of eIF-2α, causes fatigue enhancement and fatigue recovery delay. This proves again that eIF-2α phosphorylation and eIF-2α phosphorylation signaling factor are involved in fatigue, and that eIF-2α phosphorylation signal inhibitor is involved in recovery and suppression of fatigue. It is thought that it is evidence to do. Furthermore, the fact that drugs that inhibit dephosphorylation of eIF-2α cause increased fatigue and delay fatigue recovery suggests that substances that promote dephosphorylation of eIF-2α have an anti-fatigue effect. Show. Furthermore, it is shown that the effect of anti-fatigue method and anti-fatigue substance candidate can be determined with the effect of promoting dephosphorylation of eIF-2α.

In addition, the present inventors mainly used small interfering RNA (siRNA) and microRNA (miRNA), which are factors that induce phosphorylation of eIF-2α in normal fatigue (eIF-2α phosphorylation-inducing factor), which mainly constitute interfering RNA. It was found that these RNA molecules are involved in the regulation of cell functions, and that they cause eIF-2α phosphorylation via protein kinase RNA-activated (PKR). We also found that genes related to siRNA and miRNA (interfering RNA-related factors), especially DiGeorge Syndrome Critical Region Gene 8 (Dgcr8) and Drosha, increase with fatigue.
In addition, we found that endoplasmic reticulum stress can also be an eIF-2α phosphorylation-inducing factor during fatigue as a cause of phosphorylation of eIF-2α, and factors related to endoplasmic reticulum stress, especially glucose-regulated protein 78kDa (GRP78) We also found that factors such as X-box binding protein 1 (XBP-1) increase with fatigue. In the present specification, eIF-2α phosphorylation-inducing factors related to interfering RNA and other stress-related factors such as endoplasmic reticulum stress that induces eIF-2α phosphorylation are collectively referred to as eIF-2α phosphorylation induction. Called a related factor. eIF-2α phosphorylation-related factor is related to fatigue by activating eIF-2α phosphorylation signaling factor, and interfering RNA-related factor indirectly measures eIF-2α phosphorylation-related factor It becomes an index to do. That is, eIF-2α phosphorylation induction-related factors and interfering RNA-related factors can act as “factors related to the occurrence of fatigue” in the same manner as eIF-2α phosphorylation signaling factors represented by ATF3.

Interfering RNA increase and protein kinase RNA-activated (PKR) activation observed during fatigue are caused by infection with viruses with RNA in genes such as influenza, or pseudo infection model by Poly (I: C) administration It is also common for reactions to double-stranded RNA generated in However, the main symptoms of viral infection are fever, malaise that lasts for several days, and a decrease in appetite, which is the nature of physiological fatigue. No fever, resting quickly improves fatigue, and appetite rather increases It is very different from the features such as. In addition, since the cause of CFS is thought to be due to virus infection, fatigue due to the infection model is thought to be close to fatigue of CFS. Since CFS fatigue and physiological fatigue are thought to be caused by different mechanisms, the above information could be an obstacle to link RNA present in cells with physiological fatigue.
In fact, the increase in interfering RNA causes fatigue, but this is followed by an increase in ATF3, a factor that inhibits protein synthesis and excessive cytokine production due to phosphorylation of eIF-2α, eIF- The phenomenon that the inventor has clarified the relationship with fatigue for the first time, such as an increase in 2α phosphorylation signal inhibitory factor, explains the characteristics of physiological fatigue, such as no fever and easy recovery. However, this is a phenomenon that can only be explained by the present invention of the inventor, and it is difficult to link RNA and physiological fatigue from known information.

  That is, in order to solve the above-described problem, the fatigue evaluation method according to the present invention is a signal molecule induced by phosphorylation of phosphorylated eIF-2α and eIF-2α in an organ or a blood cell. (EIF-2α phosphorylation signaling factor), a factor that suppresses phosphorylation of eIF-2α and signal transduction associated therewith (eIF-2α phosphorylation signal suppressor), a factor that induces phosphorylation of eIF-2α and the like Fatigue with the amount of at least one of the above factors selected from the group consisting of factors related to induction (factors related to induction of eIF-2α phosphorylation) and molecules involved in regulation of interfering RNA (interfering RNA-related factors) It is characterized by evaluating the degree. In addition, eIF-2α phosphorylation signal inhibitory factors can be used as biomarkers of fatigue, and at the same time, the inducibility of these factors is related to the strength of fatigue recovery. In the above method, the fatigue level and fatigue recovery ability of humans and laboratory animals can be evaluated simply and objectively, and anti-fatigue substances such as nutritional supplements such as nutritional drinks and health foods, as well as pharmaceuticals with fatigue recovery or suppression effects, It is also possible to quantitatively determine the effectiveness of the anti-fatigue method. Furthermore, it is possible to quantify the instantaneous fatigue caused by a short-time fatigue load, etc. simply, objectively and with high sensitivity.

The fatigue measurement method based on the signal transduction pathway of fatigue identified in the present invention is a nonpathological fatigue including physiological fatigue, that is, a method of measuring fatigue caused by the periphery. It is considered that the efficiency and accuracy of fatigue measurement can be improved by discriminating in advance whether the cause of this is in the periphery or in the brain or mental.
The inventors of the present invention have previously measured the fatigue measurement method using the human herpesvirus 7 (HHV-7) in saliva, which was previously invented by the inventor `` Fatigue degree evaluation method and use thereof '' (Registered No. 4812708). In humans who were determined not to be fatigued, the intensity of subjective symptoms recognized as fatigue, that is, the Visual Analog Scale (VAS) of fatigue, was found to correlate strongly with the intensity of depressive symptoms. On the other hand, there was no correlation between fatigue and depressive symptoms in humans who were determined to be tired by this test. As a result, a new diagnostic method called a simple diagnostic method for depression was found by combining VAS for fatigue and human herpesvirus 7 (HHV-7) in saliva.

  The fatigue evaluation kit according to the present invention is characterized in that it is for carrying out the above-described fatigue evaluation method in order to solve the above problems.

  In order to solve the above-described problems, the anti-fatigue substance and the method for measuring the effect of the fatigue recovery method according to the present invention can be used in animals and humans by using any of the above-described fatigue evaluation methods and fatigue evaluation kits. It is characterized by measuring anti-fatigue power of fatigue materials and fatigue recovery methods.

Specifically, in order to solve the above problems, the following inventions are provided more specifically.
[1] A method for evaluating the degree of fatigue, comprising evaluating the degree of fatigue of a test organism using the amount of a eukaryotic translation initiation factor 2α (eIF-2α) phosphorylation-related factor in a cell as an index.
[2] The method for evaluating fatigue level according to [1], wherein if the amount of the eIF-2α phosphorylation-related factor is large, the fatigue level is evaluated to be high.
[3] The fatigue evaluation method according to [2], wherein the amount of the eIF-2α phosphorylation-related factor is measured as the mRNA expression level or protein production amount of the eIF-2α phosphorylation-related factor.
[4] The fatigue according to any one of [1] to [3], characterized in that if the amount of the eIF-2α phosphorylation-related factor is large, it is evaluated that nonpathological fatigue has occurred in the test subject organism. Degree evaluation method.
[5] The eIF-2α phosphorylation-related factor is selected from the group consisting of an eIF-2α phosphorylation signal transduction factor, an eIF-2α phosphorylation signal inhibitor, an eIF-2α phosphorylation induction-related factor, and an interfering RNA-related factor. The fatigue evaluation method according to any one of [1] to [4].
[6] The eIF-2α phosphorylation signaling factor is at least one selected from the group consisting of phosphorylated eIF-2α, activating transcription factor (ATF) 3, ATF4, and C / EBP-homologous protein (CHOP) The fatigue evaluation method according to [5], characterized in that:
[7] The above eIF-2α phosphorylation signal inhibitory factor is growth arrest and DNA damage-inducible protein 34 (GADD34), non-catalytic region of tyrosine kinase adaptor protein (Nck) 1, Nck2, constitutive repressor of eIF-2α phosphorylation (CReP), zinc finger protein 36 homolog (ZFP36), and glucocorticoid-induced leucine zipper (GILZ).
[8] The eIF-2α phosphorylation induction-related factor is an RNA molecule involved in cell function regulation, including small interfering RNA (siRNA) and microRNA (miRNA) constituting interfering RNA, protein kinase RNA-activated (PKR), and It is at least one selected from the group consisting of factors related to endoplasmic reticulum stress that induces phosphorylation of eIF-2α including glucose-regulated protein 78kDa (GRP78) and X-box binding protein 1 (XBP-1), The interfering RNA-related factor is at least one selected from the group consisting of DiGeorge Syndrome Critical Region Gene 8 (Dgcr8) and Drosha, [5].
[9] The fatigue evaluation according to any one of [1] to [8], wherein the cell is at least one selected from blood, liver, heart, muscle, brain, skin and pancreas. Method.
[10] A fatigue evaluation kit including means for carrying out the fatigue evaluation method according to any one of [1] to [9].
[11] The kit according to [10], wherein the kit can be used to evaluate the degree of fatigue of the test organism using the amount of eIF-2α phosphorylation-related factor in the collected cells as an index.
[12] Assessing the quality of fatigue characterized by evaluating whether the test subject's fatigue is nonpathological fatigue or pathological fatigue using the amount of eIF-2α phosphorylation-related factor in the cell as an index Method.
[13] The method for evaluating the quality of fatigue according to [12], wherein the eIF-2α phosphorylation-related factor is an eIF-2α phosphorylation signal transduction factor or an eIF-2α phosphorylation signal inhibitor.
[14] (i) The amount of eIF-2α phosphorylation-related factor in cells collected from the cells of the subject organism before the subject organism ingests the anti-fatigue substance candidate or performs the anti-fatigue method candidate. Measuring a pre-intake eIF-2α phosphorylation-related factor amount measuring step,
(Ii) Post-ingestion eIF that measures the amount of eIF-2α phosphorylation-related factor in cells collected from the subject organism after the subject organism has ingested the anti-fatigue substance candidate or implemented the anti-fatigue method candidate -2α phosphorylation-related factor amount measurement step,
(Iii) Ingestion of the anti-fatigue substance candidate obtained by the step of measuring the amount of eIF-2α phosphorylation-related factor before ingestion in (i) and the step of measuring the amount of eIF-2α phosphorylation-related factor after ingestion of (ii) Based on the measurement results of changes in the amount of eIF-2α phosphorylation-related factor before and after the implementation of the anti-fatigue method candidate, ingestion of the anti-fatigue substance candidate or eIF-2α phosphorylation-related in cells before and after the implementation of the anti-fatigue method candidate EIF-2α phosphorylation-related factor amount change calculating step for calculating a change in factor amount;
(Iv) The amount of eIF-2α phosphorylation-related factor in the cell before and after the intake of the anti-fatigue substance candidate or the implementation of the anti-fatigue method candidate obtained by the step of calculating the change in the amount of eIF-2α phosphorylation-related factor in (iii) An anti-fatigue substance candidate or anti-fatigue method candidate anti-fatigue effect evaluation method, comprising: an anti-fatigue power measurement step of measuring an anti-fatigue power in a living body of the anti-fatigue substance candidate or anti-fatigue method candidate based on the change of
[15] (i) Collected from test subject organisms that have ingested anti-fatigue substance candidates or anti-fatigue method candidates and from test subject organisms that have not ingested anti-fatigue substance candidates or anti-fatigue method candidates EIF-2α phosphorylation-related factor amount measuring step for measuring the amount of eIF-2α phosphorylation-related factor in each cell;
(Ii) The change in the amount of eIF-2α phosphorylation-related factor caused by the intake of the anti-fatigue substance candidate or the implementation of the anti-fatigue method candidate obtained by the step of measuring the amount of eIF-2α phosphorylation-related factor in (i) A step of calculating a change in the amount of eIF-2α phosphorylation-related factor in the cell based on the measurement result, calculating a change in the amount of eIF-2α phosphorylation-related factor in the cell depending on whether the anti-fatigue substance candidate is ingested or whether the anti-fatigue method candidate is implemented. When,
(Iii) eIF-2α phosphorylation-related factor in cells depending on whether the anti-fatigue substance candidate is ingested or the anti-fatigue method candidate is obtained by the step of calculating the change in the amount of eIF-2α phosphorylation-related factor in (ii) An anti-fatigue substance candidate or an anti-fatigue method candidate anti-fatigue effect evaluation method, comprising: an anti-fatigue force measurement step of measuring an anti-fatigue force in a living body of the anti-fatigue substance candidate or anti-fatigue method candidate based on a change in amount.
[16] Anti-fatigue substance candidates using the fatigue evaluation method according to any one of [1] to [9] and the fatigue evaluation kit according to any one of [10] or [11] Alternatively, the anti-fatigue effect evaluation method according to any one of [14] and [15], wherein the anti-fatigue effect of an anti-fatigue method candidate is measured.
[17] The anti-fatigue effect evaluation method according to any one of [14] to [16], for screening an anti-fatigue substance or an anti-fatigue method.
[18] A method for evaluating anti-fatigue power, comprising evaluating the anti-fatigue power of a test organism using the amount of the eIF-2α phosphorylation signal inhibitor described in [7] in a cell as an index.
[19] If the amount of the eIF-2α phosphorylation signal inhibitory factor is greater than the amount predicted from the amount of the eIF-2α phosphorylation signal transduction factor described in [6], the anti-fatigue power is evaluated to be high. The anti-fatigue power evaluation method according to [18], wherein
[20] The amount of the eIF-2α phosphorylation signal suppressor and the amount of the eIF-2α phosphorylation signal transduction factor are expressed as the mRNA expression level of the eIF-2α phosphorylation signal suppressor and the eIF-2α phosphorylation signal transduction factor, or The anti-fatigue power evaluation method according to [19], which is measured as protein production.
[21] (i) The amount of eIF-2α phosphorylation signal inhibitory factor and eIF in cells collected from the subject organism before the subject organism ingests the anti-fatigue substance candidate or performs the anti-fatigue method candidate A pre-intake measurement step to measure the amount of -2α phosphorylation signaling factor;
(Ii) The amount of eIF-2α phosphorylation signal inhibitory factor and eIF-2α phosphorylation in cells collected from the test organism after the test organism has ingested the anti-fatigue substance candidate or implemented the anti-fatigue method candidate A post-ingestion measuring step to measure the amount of signaling factor; and
(Iii) Inhibition of eIF-2α phosphorylation signal before and after the intake of the anti-fatigue substance candidate or the implementation of the anti-fatigue method candidate obtained by the pre-intake measurement step of (i) and the post-intake measurement step of (ii) Based on the measurement results of the amount of the factor and the change in the amount of the eIF-2α phosphorylation signaling factor, the eIF-2α phosphorylation signal inhibitor in the cell before and after the intake of the anti-fatigue substance candidate or the implementation of the anti-fatigue method candidate Calculating the amount and change in eIF-2α phosphorylation signaling factor; and
(Iv) The amount of eIF-2α phosphorylation signal inhibitory factor and eIF-2α phosphorylation signal in the cell before and after the intake of the anti-fatigue substance candidate or the implementation of the anti-fatigue method candidate obtained by the calculation step of (iii) An anti-fatigue substance candidate or anti-fatigue method candidate anti-fatigue, including an anti-fatigue force measurement step that measures the anti-fatigue power in the living body of the anti-fatigue substance candidate or anti-fatigue method candidate based on a change in the amount of the transfer factor Effectiveness evaluation method.
[22] (i) eIF-2α in each cell collected from cells of the test subject organism in which the test subject organism has taken the anti-fatigue substance candidate or implemented the anti-fatigue method candidate and not A measurement step of measuring the amount of phosphorylation signal inhibitor and the amount of eIF-2α phosphorylation signaling factor;
(Ii) The amount of eIF-2α phosphorylation signal inhibitory factor and eIF-2α phosphorylation signal transduction by ingestion of the anti-fatigue substance candidate or implementation of the anti-fatigue method candidate obtained by the measurement step of (i) Based on the measurement result of the change in the amount of the factor, the amount of the eIF-2α phosphorylation signal inhibitory factor in the cell and the eIF-2α phosphorylation signal transduction factor depending on the ingestion of the anti-fatigue substance candidate or the presence or absence of the anti-fatigue method candidate. A calculation step for calculating a change in quantity;
(Iii) The amount of eIF-2α phosphorylation signal inhibitory factor and eIF-2α phosphorylation in cells depending on the intake of the anti-fatigue substance candidate obtained by the calculation step of (ii) or the implementation of the anti-fatigue method candidate An anti-fatigue substance candidate or an anti-fatigue method including an anti-fatigue power measurement step of measuring intake of the anti-fatigue substance candidate or an anti-fatigue power in the living body of the anti-fatigue method candidate based on a change in the amount of a signal transduction factor Candidate anti-fatigue effect evaluation method.
[23] A fatigue model animal, wherein fatigue is caused by introducing at least one of the genes according to [6] or [8] or a gene product thereof.
[24] A fatigue model animal characterized by causing fatigue by eIF-2α phosphorylation.
[25] A fatigue model animal, characterized by introducing an ATF3 gene or a gene product thereof to cause fatigue.
[26] An anti-fatigue model animal characterized by imparting anti-fatigue ability by introducing at least one of the genes according to [7] or a gene product thereof.
[27] An anti-fatigue model animal characterized by imparting anti-fatigue ability by suppressing eIF-2α phosphorylation and signal transmission associated therewith.
[28] A fatigue model animal, wherein the anti-fatigue effect is suppressed by inhibiting the function of at least one gene product of the genes according to [7].
[29] A method for evaluating the quality of fatigue, comprising determining whether a subject's fatigue is nonpathological fatigue or pathological fatigue using the amount of eIF-2α phosphorylation-related factor in cells as an index.
[30] The amount of the eIF-2α phosphorylation-related factor according to any one of [6] to [8] in the cells collected from the subject, although the subject's subjective fatigue is strong [29] The method for evaluating the quality of fatigue according to [29], wherein if it is not so much, it is determined that the subject's fatigue is pathological fatigue.
[31] Cause of subject's mental state and / or subjective fatigue, characterized by evaluating subject's mental state and / or cause of subjective fatigue, using amount of human herpesvirus in saliva as an index How to evaluate.
[32] If the amount of human herpesvirus in the saliva of the subject is small compared to the subject's subjective fatigue, the subject's subjective fatigue is evaluated as being due to depressive symptoms, 31].
[33] If it is determined that the subject is a healthy subject and the subject is not fatigued by fatigue measurement based on the amount of human herpesvirus in the saliva of the subject, the subject's fatigue is due to depressive symptoms The method according to [31] to [32], wherein the method is determined.
[34] The subject is a healthy subject and the subject has a strong feeling of subjective fatigue even though it is determined that the subject is not fatigued by fatigue measurement based on the amount of human herpesvirus in the saliva of the subject The method according to any one of [31] to [33], wherein the subject is determined to be in a depressed state.
[35] The method according to any one of [31] to [34], wherein the amount of human herpesvirus is measured as the amount of human herpesvirus DNA or the amount of human herpesvirus protein.
[36] The method according to any one of [31] to [35], wherein the human herpesvirus is at least one selected from human herpesvirus 6 and human herpesvirus 7.
[37] An anti-fatigue substance or an anti-fatigue method that exerts an anti-fatigue effect by suppressing the effect of a signal transduction pathway related to eIF-2α phosphorylation.
[38] An anti-fatigue substance candidate or an anti-fatigue method characterized by evaluating the strength of an anti-fatigue effect using as an index the strength of an effect of suppressing the effect of a signal transduction pathway related to phosphorylation of eIF-2α Candidate anti-fatigue effect evaluation method.
[39] An anti-fatigue substance or an anti-fatigue method that exhibits an anti-fatigue effect by promoting dephosphorylation of eIF-2α.
[40] Method for evaluating anti-fatigue effect of anti-fatigue substance candidate or anti-fatigue method candidate, comprising evaluating strength of anti-fatigue effect using strength of effect of promoting dephosphorylation of eIF-2α as an index .
[41] An anti-fatigue agent (fatigue recovery agent, fatigue inhibitor, or fatigue preventive agent) containing a substance selected from the group consisting of the following as an active ingredient:
(I) a substance that inhibits the expression or function of at least one of the factors described in [6] or [8];
(Ii) a substance that enhances expression or function of at least one of the factors according to [7]; and (iii) at least one of the factors according to [7].
[42] An anti-fatigue agent (fatigue recovery agent, fatigue inhibitor, or fatigue preventive agent) containing a substance selected from the group consisting of the following as an active ingredient:
(A) a substance that inhibits phosphorylation of eIF-2α (eIF-2α phosphorylation inhibitor);
(B) a substance that promotes dephosphorylation of eIF-2α (eIF-2α dephosphorylation promoter); and (c) a substance that suppresses the activity of a signal transduction pathway induced by phosphorylation of eIF-2α (eIF -2α phosphorylation signal inhibitor).
[43] The anti-fatigue agent according to [41] or [42], wherein the fatigue is nonpathological fatigue including physiological fatigue.
[44] The anti-fatigue agent according to [41] or [42], wherein the fatigue is caused by peripheral organs or tissues.
[45] The anti-fatigue agent according to [41] or [42], wherein the fatigue is mental fatigue or physical fatigue due to lack of sleep.
[46] A method of reducing fatigue (recovering or suppressing fatigue), comprising a step selected from the group consisting of:
(I) Ingesting an anti-fatigue substance that exhibits an effect of inhibiting the expression or function of at least one of the factors described in [6] or [8], or a step of performing an anti-fatigue method that exhibits the effect And (ii) ingesting an anti-fatigue substance exhibiting an effect of enhancing the expression or function of at least one of the factors described in [7], or performing an anti-fatigue method exhibiting the effect.
[47] Reducing fatigue (recovering or suppressing fatigue), including a step of ingesting an anti-fatigue substance that exhibits an effect selected from the group consisting of: how to:
(A) inhibits phosphorylation of eIF-2α;
(B) promotes the dephosphorylation of eIF-2α; and (c) suppresses the activity of signal transduction pathways induced by the phosphorylation of eIF-2α.
[48] The method according to [46] or [47], wherein the fatigue is nonpathological fatigue including physiological fatigue.
[49] The method according to [46] or [47], wherein the fatigue is caused by peripheral organs or peripheral tissues.
[50] The method according to [46] or [47], wherein the fatigue is mental fatigue or physical fatigue due to lack of sleep.
[51] Use of an anti-fatigue substance selected from the group consisting of the following in the manufacture of an anti-fatigue agent (fatigue recovery agent or fatigue inhibitor):
(I) a substance that inhibits the expression or function of at least one of the factors described in [6] or [8];
(Ii) a substance that enhances expression or function of at least one of the factors according to [7]; and (iii) at least one of the factors according to [7].
[52] Use of an anti-fatigue substance selected from the group consisting of the following in the manufacture of an anti-fatigue agent (fatigue recovery agent or fatigue inhibitor):
(A) a substance that inhibits phosphorylation of eIF-2α (eIF-2α phosphorylation inhibitor);
(B) a substance that promotes dephosphorylation of eIF-2α (eIF-2α dephosphorylation promoter); and (c) a substance that suppresses the activity of a signal transduction pathway induced by phosphorylation of eIF-2α (eIF -2α phosphorylation signal inhibitor).
[53] An anti-fatigue substance selected from the group consisting of the following for use in reducing fatigue (recovering or suppressing fatigue):
(I) a substance that inhibits the expression or function of at least one of the factors described in [6] or [8];
(Ii) a substance that enhances expression or function of at least one of the factors according to [7]; and (iii) at least one of the factors according to [7].
[54] An anti-fatigue substance selected from the group consisting of the following for use in reducing fatigue (recovering or suppressing fatigue):
(A) a substance that inhibits phosphorylation of eIF-2α (eIF-2α phosphorylation inhibitor);
(B) a substance that promotes dephosphorylation of eIF-2α (eIF-2α dephosphorylation promoter); and (c) a substance that suppresses the activity of a signal transduction pathway induced by phosphorylation of eIF-2α (eIF -2α phosphorylation signal inhibitor).
[55] A method of manufacturing an anti-fatigue device for performing an anti-fatigue substance (for example, an anti-fatigue agent) or an anti-fatigue method, including any of the following steps:
(1) a step of evaluating the strength of the effect of inhibiting phosphorylation of eIF-2α by the anti-fatigue substance or the anti-fatigue method;
(2) a step of evaluating the strength of the effect of promoting dephosphorylation of eIF-2α by the anti-fatigue substance or the anti-fatigue method;
(3) a step of evaluating the strength of the anti-fatigue substance or the anti-fatigue method for suppressing the effect of a signal transduction pathway related to phosphorylation of eIF-2α; or (4) [14] to [16 ] The process of evaluating the anti-fatigue effect of the said anti-fatigue substance or the said anti-fatigue method by the method in any one of [21] or [22].

  The method for assessing fatigue level according to the present invention, a kit for assessing fatigue level, a method for using the same, and a method for measuring the anti-fatigue substance (anti-fatigue effect) of anti-fatigue substances and anti-fatigue methods are to collect a subject's organ, blood or saliva. As a result, the nonpathological fatigue of the subject can be quantitatively evaluated. It is also possible to distinguish between nonpathological fatigue and pathological fatigue. Furthermore, the method and kit according to the present invention are not only simple, but also do not require long-term restraint, so that the subject does not feel pain or annoyance. In addition, the method and the like are simple for the practitioner and are very easy to handle for both the subject and the practitioner. Therefore, it can be used for screening methods of anti-fatigue substances and anti-fatigue methods, and in vivo evaluation and in vitro evaluation of foods with anti-fatigue ability, and is a very useful technique.

  In the present invention, a method using a molecular biomarker related to eIF-2α phosphorylation can be used for animals and cultured cells as well as humans. The present invention also provides an animal model capable of professionally analyzing physiological fatigue. As a result, screening of anti-fatigue substances and anti-fatigue methods can be performed much more efficiently than in human tests, and it is possible to contribute to the development of better products.

  According to the kit for assessing fatigue level according to the present invention, for example, by measuring the amount of eIF-2α phosphorylation-related factor in blood collected from a subject and calculating the amount, there is a fatigue suppression or recovery effect. Evaluate the effectiveness of medicines, foods and anti-fatigue methods. In addition, by comprehensively measuring the types and amounts of eIF-2α phosphorylation-related factors that have changed, it is possible to determine at which stage the substance or method acts, whether it has fatigue suppression effect, fatigue recovery effect Detailed examination such as whether it has is possible. That is, it is possible to easily and quantitatively determine the efficacy and efficacy in the living body of a pharmaceutical or food that has a fatigue suppression or recovery effect.

  According to the method of the present invention, the anti-fatigue substance or anti-fatigue method acts on human fatigue symptoms at which stage of the fatigue generation mechanism and how much improvement effect is obtained, that is, anti-fatigue substance or The anti-fatigue power possessed by the anti-fatigue method can be measured simply, reliably and quantitatively. This makes it possible to scientifically obtain a stronger and more reliable anti-fatigue effect by using a combination of anti-fatigue substances and anti-fatigue methods that exhibit anti-fatigue effects with different mechanisms.

  The present invention provides a method, a kit, and a method for using the method for simply and quantitatively measuring and evaluating the degree of fatigue in daily life. For this reason, according to the present invention, it is possible to objectively know the degree of fatigue in daily life, and it is possible to avoid the occurrence of various diseases that are accumulated and caused without knowing fatigue. Furthermore, it is possible to easily determine the depressive symptoms that trigger suicide, which is one of the biggest causes of death due to the accumulation of fatigue, so overwork death and suicide caused by continuing to work without being aware of fatigue. It is also possible to reduce the occurrence rate.

  Furthermore, according to the present invention, the amount of anti-fatigue power in the living body, such as pharmaceuticals and foods that provide fatigue recovery, nourishing tonics and nutritional supplements, and health appliances and electrical appliances that are effective for fatigue recovery, which are supplied to the market Can provide information to consumers and society. This information can be used as a guideline for consumers to select anti-fatigue foods, medicines, and health equipment that are effective in preventing fatigue and nourishing. The present invention is very useful and has a strong social impact.

2 is a photograph of Western blotting showing phosphorylation of eIF-2α and PKR and enhancement of ATF4 protein production due to fatigue in Example 1, and a graph quantifying each band. It is a figure which shows the increase in ATF3 in various organs by the mental fatigue in Example 2. FIG. It is a figure which shows the increase in ATF3 in various organs by the physical fatigue in Example 3. FIG. It is a figure which shows the measurement of the physical fatigue by ATF3 using the human peripheral blood in Example 4. FIG. It is a figure which shows the increase in CHOP in the liver by the physical fatigue and mental fatigue in Example 5. It is a figure which shows the measurement of the physical fatigue by CHOP using the human peripheral blood in Example 6. FIG. It is a figure which shows the induction | guidance | derivation of the fatigue by ATF3 introduction | transduction in Example 7. FIG. It is a figure which shows the reduction effect of the fatigue | exhaustion by the expression suppression of ATF3 in Example 8. It is a figure which shows the gene expression of ATF3 and inflammatory cytokine by ATF3 introduction | transduction in Example 9. It is a figure which shows the continuation of FIGS. FIG. 10 shows interference RNA induction by fatigue in Example 10. It is a figure which shows the continuation of FIG. FIG. 10 shows the induction of interfering RNA-related molecules by fatigue in Example 11. It is a figure which shows the measurement of the fatigue by the interference RNA related molecule | numerator in the human blood in Example 12. FIG. It is a figure which shows the measurement of the fatigue by the endoplasmic reticulum stress related molecule | numerator in the human blood in Example 13. FIG. It is a figure which shows that there is almost no gene expression of ATF3 by intraperitoneal administration of Poly (I: C) in Example 14. It is a figure which shows the increase in the eIF-2 (alpha) phosphorylation inhibitory factor in various organs by the mental fatigue and physical fatigue in Example 15. It is a figure which shows the continuation of FIG. It is a figure which shows the measurement of the fatigue by the eIF-2 (alpha) phosphorylation inhibitory factor in the human blood in Example 16. It is a figure which shows the fatigue recovery method using the eIF-2 (alpha) phosphorylation signal transduction factor and the eIF-2 (alpha) phosphorylation signal inhibitory factor in Example 17, and the effect determination of an anti-fatigue component. It is a figure which shows the fatigue measurement by the fatigue related factor of the chronic fatigue syndrome patient in Example 18. FIG. It is a figure which shows the fatigue measurement result of the depression disorder patient in Example 19. FIG. It is a figure which shows the simple diagnosis method of the depression state by the combination of fatigue VAS and the human herpesvirus 7 (HHV-7) in saliva in Example 20. It is a figure which shows the increase in ZFP36 in each organ by mental fatigue in Example 21. FIG. It is a figure which shows the increase in TSC22D3 in each organ by mental fatigue in Example 22. FIG. It is a figure which shows the increase in ZFP36 in each organ by the physical fatigue in Example 23. It is a figure which shows the increase in TSC22D3 in each organ by the physical fatigue in Example 24. It is a figure which shows the increase in ZFP36 in the human peripheral blood by mental fatigue in Example 25. It is a figure which shows the increase in the forced swimming movement distance of the mouse | mouth by the eIF-2 (alpha) phosphorylation signal inhibitor ISRIB in Example 26. It is a figure which shows the reduction | decrease of the forced swimming movement distance of the mouse | mouth by eIF-2 (alpha) dephosphorylation inhibitor salubrinal in Example 27.

  Hereinafter, although the fatigue evaluation method, kit, and utilization method concerning this invention are demonstrated, this invention is not limited to this.

  In this specification, “fatigue” is a temporary physical / mental function and physical qualitative or quantitative decrease in physical strength that occurs when a physical or mental load is continuously applied. It is classified into physical fatigue and morbid fatigue. As used herein, “physiological fatigue” refers to the qualitative or quantitative nature of temporary physical and mental work abilities seen when a healthy person is given physical or mental loads continuously. It means a serious decline phenomenon. “Pathologic fatigue” means fatigue associated with diseases such as chronic fatigue syndrome, mental illness, central nervous system disease, heart disease, hepatitis, anemia, various infectious diseases, and malignant tumors.

  Among `` pathological fatigue '', fatigue due to diseases of the central nervous system such as chronic fatigue syndrome (CFS) and depression is something that the brain feels abnormally tired even though there is no fatigue load, “Pathological fatigue” is distinguished from physiological fatigue and other pathological fatigue. Pathological fatigue other than physiological fatigue and pathological fatigue is called "nonpathological fatigue" and occurs when fatigue in peripheral organs and tissues is transmitted to the brain in some way and the brain feels fatigue. Pathological fatigue, on the other hand, has no cause in the periphery, and feels fatigue due to abnormalities in the brain. That is, nonpathological fatigue can be said to be fatigue caused by peripheral organs and tissues, and pathological fatigue can be said to be fatigue caused by brain diseases. Nonpathological fatigue in this specification represents fatigue including all physiological fatigue and pathological fatigue in which peripheral organs and tissues cause fatigue.

  “Physiological fatigue” is further classified into “acute fatigue” and “continuous fatigue”. As used herein, “acute fatigue” means transient fatigue that is recovered by appropriate rest. “Sustained fatigue” means fatigue over a long period in which fatigue has accumulated due to incomplete daily recovery. The term “medium- to long-term fatigue” in this specification means acute fatigue before reaching the above-mentioned persistent fatigue.

  “Acute fatigue” and “persistent fatigue” are classified into “mental fatigue” and “physical fatigue”, respectively. “Mental fatigue” in this specification means not only complicated calculations, memories, or psychological activities such as thinking, but also emotional and intentional feelings such as the feeling of working with patience, tension, or time. It means fatigue, including lack of sleep, eye strain, and mental stress, caused by excessive demand for activity. In addition, “physical fatigue” in this specification means fatigue caused by performing physical work or exercise. Furthermore, “fatigue load” in this specification means giving the fatigue.

  In this specification, “fatigue level” refers to a body / body with excessive physical and mental activities or “fatigue” caused by the effects of the disease resulting from the various “fatigue” listed above. Degree of mental function and physical strength.

  In this specification, “fatigue” is a feeling that the brain feels fatigue resulting from the various types of “fatigue” mentioned above, and has a unique discomfort and rest such as “fatigue” and “fatigue”. It is a sense accompanied by the desire to seek.

  The present invention is intended for all of the various types of “fatigue” mentioned above, and among them, nonpathological fatigue is preferable. Among nonpathological fatigue, acute fatigue or medium to long-term fatigue is preferable. Note that nonpathological fatigue includes fatigue due to illness, such as fatigue of cancer patients, and measurement of such fatigue is one of the important uses of the present invention. In addition, the subject of the present invention is preferably pathological fatigue of pathological fatigue associated with depressive symptoms and mental illness.

  Conditions for optimal fatigue-related factors (fatigue-related factors) for carrying out the present invention include factors related to human and animal nonpathological fatigue, and ease of measurement. .

  Fatigue-related factors that satisfy the above conditions include eIF-2α phosphorylation signaling factor (phosphorylated eIF-2α, ATF3, ATF4, CHOP), eIF-2α phosphorylation signal inhibitory factor (GADD34, Nck1, Nck2, CReP , ZFP36, GILZ), eIF-2α phosphorylation-related factors (interfering RNAs such as siRNA and miRNA, PKR, GRP78, XBP-1), and interfering RNA-related factors (Dgcr8, Drosha) Factors and human herpesviruses (HHV-6, HHV-7) are considered suitable. In this specification, the term “gene” used for these factors means a concept including both the protein or interfering RNA molecule that is the factor or a polynucleotide encoding them, and the protein or interfering RNA. When referring to both a molecule and the polynucleotide, the factor may be expressed as a “gene or gene product”.

  In addition, these factors increase the amount of mRNA or protein in cells due to fatigue load (if the factor is an interfering RNA molecule such as siRNA or miRNA), the amount of RNA in humans increases in blood cells. By measuring the amount of mRNA or protein (or the amount of interfering RNA), and in animal models, the amount of mRNA or protein (or the amount of interfering RNA) in blood or various organs, changes associated with fatigue can be measured easily. be able to. Regarding human herpesviruses, it is known that viral DNA is released into saliva. Therefore, it can be easily measured by measuring viral DNA in saliva.

  Furthermore, eIF2α and some eIF-2α phosphorylation-related factors include factors that induce the occurrence of fatigue by phosphorylation and are related to the anti-fatigue effect by dephosphorylation. By measuring the degree of phosphorylation in various organs in blood, biopsy, pathological specimens, and forensic specimens in humans, and measuring the degree of phosphorylation in blood and various organs in animal models, changes due to fatigue can be easily measured. can do.

  The outline of the present invention will be briefly described below. In the outline of the method described here, there are many parts common to the kit and the usage method described later.

(1) Fatigue degree evaluation method The present inventors measured the amount of the fatigue-related factor present in cells of blood collected from a subject in humans, blood collected in animals or various organs and tissues in animals. By doing so, it was found that the degree of fatigue of humans and animals can be measured easily and quantitatively. Regarding herpes virus, only humans are indicated, and saliva is used for measurement. This method not only requires a large-scale apparatus, but also has a short time for blood collection in humans, so there are few time constraints for the subject, and it is a very simple method for the person who performs the method.

  The fatigue-related factors that are the subject of the present invention include eIF-2α phosphorylation signaling factor (phosphorylated eIF-2α, ATF3, ATF4, CHOP), eIF-2α phosphorylation signal inhibitor (GADD34, Nck1, Nck2 , CReP, ZFP36, GILZ), eIF-2α phosphorylation-related factors (siRNA, miRNA and other interfering RNAs, PKR, GRP78, XBP-1), interfering RNA-related factors (Dgcr8, Drosha), human herpesvirus (HHV- 6, HHV-7) is preferred.

  The cells to be the subject of the present invention may be at least one selected from blood and various organs and tissues, but blood is suitable for humans and various organs for laboratory animals.

  The blood collection method is preferably blood collection using a Paxgene RNA blood collection tube in consideration of the convenience of measurement using mRNA. In animal models, a method using an RNA protecting agent is desirable.

  The measurement of the amount of the fatigue-related factor in the cells may be a conventionally known method, and specific methods and conditions can be appropriately set by those skilled in the art. There are a method for measuring the amount of RNA molecules such as mRNA, siRNA, miRNA, and a method for measuring the amount of protein.

  As a method for measuring the amount of mRNA of the fatigue-related factor, for example, RNA is purified from blood cells, and a primer and a probe for real-time PCR specific to each fatigue-related factor are used. And a method for measuring the amount of mRNA by Real-time PCR. Similarly, the amount of RNA molecules such as siRNA and miRNA can be measured using primers and probes for real-time PCR specific to the RNA molecule. Examples of viral DNA include a method in which DNA in saliva is purified and measured by a virus-specific Real-time PCR method (reference materials: Patent No. 4218842, Patent No. 4812708). In the present invention, the real-time PCR method is preferred.

  Examples of a method for measuring the amount of the fatigue-related factor protein include an immunoassay method (immunoassay method) using an antibody against a viral protein. As a typical immunoassay method, for example, a sandwich ELISA method is cited (reference: J. Clin. Microbiol. 1983 May; 17 (5): 942-4. “Typing of herpes simplex virus isolates by enzyme-linked immunosorbent”. assay: comparison between indirect and double-antibody sandwich techniques. "Gerna G, Battaglia M, Revello MG, Gerna MT.).

Further, in the fatigue level measurement method according to the present invention, if the amount of fatigue-related factors in the cells collected from the test target organism is larger or smaller, it can be evaluated that the level of fatigue of the organism is higher. It is preferable to evaluate that the fatigue level of the subject and the test animal is higher when the amount of the fatigue-related factor in the cell is larger. This is derived from the fact that the expression level of the fatigue-related factor in the cells increases in accordance with the increase in the fatigue level of the subject and the test animal, as shown in the examples described later. The amount of the fatigue-related factor in the cells is preferably evaluated by comparison with a predetermined reference value (control value). That is, preferably, in the method for measuring fatigue level according to the present invention, if the amount of the fatigue-related factor in the cells collected from the test target organism is significantly greater than a predetermined reference value, the fatigue level of the test target organism is determined. It is characterized by being evaluated as high. In one embodiment of the present invention, the reference value may be determined after receiving a fatigue load, after taking sufficient rest (preferably immediately after rest), after taking an anti-fatigue substance, or after performing an anti-fatigue method. It is the amount of the fatigue-related factor in cells collected from the test subject organism. Alternatively, the reference value is collected from an organism that is the same species as the subject organism and is not fatigued, rested, ingested an anti-fatigue substance, or has undergone an anti-fatigue method. The amount of the fatigue-related factor in the cells thus obtained may be used, and the reference value may be a value measured in advance.
In the case of human herpesvirus, as shown in the examples described later, it is important to compare with subjective fatigue.

  Furthermore, it will be apparent to those skilled in the art that part or all of the fatigue evaluation method according to the present invention can be performed using a conventionally known arithmetic device (information processing device) such as a computer. For example, the fatigue level evaluation method according to the present invention includes a measurement step for measuring the amount of the fatigue-related factor in blood cells, and evaluates the fatigue level of the subject according to the measurement result of the amount of the fatigue-related factor in blood cells. In other words, an arithmetic unit can be used particularly for the evaluation process.

(2) Fatigue degree evaluation kit Next, the fatigue degree evaluation kit according to the present invention will be described. The fatigue evaluation kit according to the present invention is a kit for evaluating fatigue in humans or animals. That is, the kit may be any kit for carrying out the fatigue evaluation method according to the present invention described in the above section (1). Specifically, it may be a kit including means for measuring the amount of the fatigue-related factor in the cells of the test organism. The means for measuring the amount of the fatigue-related factor in the cells in the present invention may be any means necessary for carrying out a conventionally known measurement method. The means necessary for carrying out the conventionally known measurement method is specifically, for example, necessary for carrying out the method for measuring the amount of the fatigue-related factor in the cells described in the above section (1). Such as a probe, a primer, or an antibody for specifically measuring the amount of a fatigue-related factor that is a subject of the present invention. The fatigue evaluation kit according to the present invention may include means for collecting cells of a test organism. In addition, even if the label attached to the packaging material or the attached document indicates that it can be used to evaluate the fatigue level of the subject using at least one of the fatigue-related factors in the cells as an indicator. Good.

  Further, a conventionally known arithmetic device such as a computer may be used for the fatigue evaluation using the fatigue evaluation kit according to the present invention.

(3) Fatigue level evaluation method and usage method of fatigue level evaluation kit As described above, before and after ingesting anti-fatigue substances by test organisms using the fatigue level evaluation method and fatigue level evaluation kit according to the present invention or anti-fatigue Measure the amount of the above fatigue-related factors in the cells of the test organism before and after performing the method to quantify the anti-fatigue power (anti-fatigue effect) of the anti-fatigue substance or anti-fatigue method candidate in the test organism Can be measured and evaluated automatically. Furthermore, both of these methods and kits are not only simple, but also have the advantage that they are very easy to handle for both the subject and the practitioner because they do not require a large-scale device or long-term restraint.

Therefore, a method for measuring the anti-fatigue substance (anti-fatigue effect) of an anti-fatigue substance candidate or an anti-fatigue method candidate using either the fatigue evaluation method or the fatigue evaluation kit according to the present invention is also included in the present invention. It is. The anti-fatigue substance candidate or anti-fatigue method candidate anti-fatigue power measurement method is, for example,
(I) The fatigue-related factor before ingestion that measures the amount of the fatigue-related factor in cells collected from the subject organism before the test subject ingests the anti-fatigue substance candidate or conducts the anti-fatigue method candidate. A quantity measuring step;
(Ii) The amount of the fatigue-related factor after ingestion that measures the amount of the fatigue-related factor in cells collected from the test subject after the test organism has ingested the anti-fatigue substance candidate or implemented the anti-fatigue method candidate Measuring process;
(Iii) Ingestion of the anti-fatigue substance candidate or anti-fatigue method candidate obtained by the step of measuring the amount of fatigue-related factors before ingestion of (i) and the step of measuring the amount of fatigue-related factors after ingestion of (ii) Based on the measurement result of the change in the amount of the fatigue-related factor before and after, the change in the amount of the fatigue-related factor that calculates the change in the amount of the fatigue-related factor in the cell before and after the intake of the anti-fatigue substance candidate or the implementation of the anti-fatigue method candidate A calculation process;
(Iv) Based on the change in the fatigue-related factor amount in the cell before and after the intake of the anti-fatigue substance candidate or the implementation of the anti-fatigue method candidate obtained by the calculation step of the fatigue-related factor amount change in (iii) In other words, the method includes an anti-fatigue strength measurement step of measuring an anti-fatigue strength in a living body of a substance candidate or an anti-fatigue method candidate.

  In the method for measuring anti-fatigue power of an anti-fatigue substance candidate or anti-fatigue method candidate of the present invention, a test organism that has administered such an anti-fatigue substance candidate or implemented an anti-fatigue method candidate, and the anti-fatigue substance candidate It is also possible to adopt a method for carrying out the above anti-fatigue power measurement method in a test organism that has not been administered or that has not been subjected to anti-fatigue method candidates.

  A person skilled in the art can appropriately set the method of ingesting the anti-fatigue substance candidate in the test organism or the implementation method of the anti-fatigue method candidate. “Ingestion” in the present specification means not only intake of the substance as a food or drink in a test organism, but also oral administration and parenteral administration of the substance.

  “Anti-fatigue” in the present specification means an effect of recovery, suppression or prevention of fatigue. The anti-fatigue mechanism may be any mechanism that reduces fatigue as a result, may be one that prevents fatigue, or may promote recovery or suppression of fatigue. The anti-fatigue effect is brought about not only by the intake of anti-fatigue substances but also by the implementation of an anti-fatigue method. “Anti-fatigue substance” is a molecule that can be taken into the body by any method that has the effect of recovering, suppressing, or preventing fatigue, and is a drug, nutritional component, scent component, gas molecule, liquid molecule, and biological function complementing substance. including. “Biological function-complementing substance” means a substance that adjusts the balance of the living body, and includes substances having a function of enhancing immune function. "Anti-fatigue method" means exercise methods such as gymnastics, massage, treatment including acupuncture, folk remedies including aromatherapy and hypnosis, furniture including bedding, etc., which have the effect of restoring, suppressing or preventing fatigue, Includes the use of anti-fatigue devices including appliances such as health appliances, lighting, air conditioners and personal computers.

  The fatigue evaluation method and the fatigue evaluation kit according to the present invention can be used, for example, as a screening method for anti-fatigue substances and anti-fatigue methods. That is, the screening method for anti-fatigue substances and anti-fatigue methods according to the present invention is a method for screening anti-fatigue substances and anti-fatigue methods using any one of the above fatigue evaluation methods and fatigue evaluation kits. Well, the specific method, conditions, etc. are not limited.

  According to the above screening method, for example, a test organism can be orally ingested with a food that seems to be usable as an anti-fatigue food, and a food that actually shows excellent anti-fatigue ability in vivo can be selected simply and objectively. Can do. Therefore, the anti-fatigue substances and anti-fatigue foods obtained by the screening method have been proven to be effective in the living body and can be highly evaluated in the market. The anti-fatigue method can be selected in vivo as well, and can be highly evaluated in the market.

  The new anti-fatigue substance and the new anti-fatigue method according to the present invention may be those obtained by the above screening method, and the anti-fatigue substance and the anti-fatigue method obtained by the above screening method are also included in the present invention. Such anti-fatigue substance or anti-fatigue method is preferably (i) an eIF-2α phosphorylation signaling factor, an eIF-2α phosphorylation induction related factor or an interfering RNA related factor (ie, factors related to the occurrence of fatigue) And / or (ii) the expression or function of at least one of eIF-2α phosphorylation signal inhibitory factor (ie, a factor related to recovery or suppression of fatigue). The anti-fatigue effect can be exhibited by strengthening.

  The anti-fatigue effect is preferably exerted by suppressing the effects of signal transduction pathways involving eIF-2α phosphorylation, for example, promoting eIF-2α dephosphorylation, inhibiting eIF-2α phosphorylation Or exerted by inhibiting the activity of signal transduction pathways induced by eIF-2α phosphorylation. Accordingly, the strength of the anti-fatigue effect of the anti-fatigue substance or anti-fatigue method according to the present invention is the effect of suppressing the effect of the signal transduction pathway related to phosphorylation of eIF-2α by the anti-fatigue substance or anti-fatigue method. Can be evaluated as an index. Anti-fatigue substances that exhibit such effects can be useful as anti-fatigue agents, for example, substances that suppress the activity of signal transduction pathways induced by phosphorylation of eIF-2α (eIF-2α phosphorylation signal inhibitors ) ISRIB, a substance that inhibits phosphorylation of eIF-2α (eIF-2α phosphorylation inhibitor) GSK2606414, etc., but the effects of signal transduction pathways related to phosphorylation of eIF-2α There is no particular limitation as long as the substance to be suppressed is contained as an active ingredient.

  The present invention also suppresses the effects of signal transduction pathways related to phosphorylation of eIF-2α (eg, inhibits phosphorylation of eIF-2α, promotes dephosphorylation of eIF-2α, or eIF- 2) Reducing fatigue, including ingesting anti-fatigue substances that exhibit an effect that suppresses the activity of signal transduction pathways induced by 2α phosphorylation, or implementing an anti-fatigue method that exhibits that effect. It also relates to methods of recovery or suppression). The fatigue targeted by the anti-fatigue substance and anti-fatigue method according to the present invention and the method of reducing fatigue (recovering or suppressing fatigue) according to the present invention is preferably nonpathological fatigue including physiological fatigue, That is, fatigue caused by peripheral organs and tissues, such as mental fatigue or physical fatigue due to lack of sleep.

  In addition, as fatigue becomes a social problem, anti-fatigue substances, anti-fatigue foods, and anti-fatigue methods with anti-fatigue functions have been increasing with types and quantities, and the anti-fatigue power of these foods and methods has been increased appropriately. Development of a method for evaluation is also strongly demanded. However, according to the fatigue evaluation method, fatigue evaluation kit, and usage thereof according to the present invention, this requirement can be met.

  Further, the fatigue evaluation method shown in the present invention is a method of discriminating between nonpathological fatigue and pathological fatigue (method of evaluating the quality of fatigue), that is, the cause of the fatigue phenomenon in the periphery, brain or mental It can be used as a method for discriminating whether or not there is. In addition, by combining this method with the measurement of subjective fatigue such as VAS, it is possible to detect depression in healthy individuals, and a simple and objective evaluation method for mental health and mental care of workers. It becomes.

  Embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible. Embodiments obtained by appropriately combining the disclosed technical means are also included in the technical scope of the present invention. .

  All prior art documents cited herein are hereby incorporated by reference.

  We studied the phosphorylation of eIF-2α in liver cells and the related factors by applying fatigue load to mice.

(1) Method A mouse (C57BL / 6NCrSlc) was placed in a cage with 1 cm of water for 24 hours. Livers were extracted from mice immediately after loading and mice not subjected to loading as a control group and frozen. Ultrasonic crush the liver in Cell Lysate, centrifuge the lysate with Ultra-15 centrifugal filter unit Ultracel-100 (MILLIPORE), flow through with Ultra-15 centrifugal filter unit Ultracel-30 (MILLIPORE) Centrifuge and concentrate the lysate. Lysates were separated by SDS-PAGE, and PKR (phospho T451) antibody (ab4818) (abcam) (dilution ratio: 1/1000), Anti-PKR antibody [YE350] (ab32052) (abcam) (dilution ratio: 1/1000) ), Phospho-eIF-2α (Ser51) (119A11) antibody (Cell Signaling) (dilution ratio: 1/1000), Anti-EIF2S1 (Bio Matrix Research Inc) (dilution ratio: 1/100), Anti-ATF4 antibody ( ProteinTech Group, Inc) (dilution ratio: 1/100), Anti-ACTB (Bio Matrix Research Inc) (dilution ratio: 1/200) as the primary antibody and X-ray using the VECTASTAIN ABC kit (VECTOR) Detected with film. The density of each band in the X-ray film image was quantified with ImageJ 1.38 (National Institutes of Health).

(2) Results
It was found that the 24-hour fatigue load increased the production of ATF4, a signal factor induced by phosphorylation of eIF-2α in the liver cells (P-eIF2 in Fig. 1) and eIF-2α phosphorylation. . Causes of enhanced phosphorylation of eIF-2α include factors such as endoplasmic reticulum stress, ultraviolet light stimulation, and oxidative stress. Protein kinase RNA-activated (PKR) phosphorylation (P-PKR in Fig. 1) Was observed at the same time, indicating that some RNA is involved in phosphorylation of eIF-2α (FIG. 1).
Fatigue is thought to be an alarm that informs the danger of a living body, but its content is a signal that informs the living body of too much work or lack of rest and commands the living body to "rest". Phosphorylation of eIF-2α is a “rest” signal that forcibly stops cellular protein synthesis, and this finding that fatigue signals are also exerted at the cellular level is in good agreement with the concept of fatigue, It is thought to form the basis of fatigue.

  Using the mouse insomnia model, the increase in ATF3 in the organ due to mental fatigue due to insomnia was measured.

(1) Method Place the mouse (C57BL / 6NCrSlc) in a cage with 1 cm of water, and immediately after loading (4h, 6h, 8h, 12h, 24h, 36h loading) and as a control group no mouse (0h) ), The organs were removed over time (n = 2-14) and frozen. Frozen organs were homogenized, RNA was purified using BioRobot EZ1 workstation and EZ1 RNA Universal Tissue Kit (QIAGEN, Inc.), and cDNA was synthesized using PrimeScript RT reagent Kit (Takara Bio, Inc.). ATF3 and GAPDH mRNA were measured using Applied Biosystems 7300 real-time PCR System (Applied Biosystems). The measurement was performed in duplicate under the following conditions.
Real-time PCR conditions: FastStart TaqMan Probe Master (ROX) (Roche Diagnostics) 12.5 μl, PCR forward primer (100 μM) 0.225 μl, PCR reverse primer (100 μM) 0.225 μl, TaqMan probe (10 μM) 0.625 μl, cDNA 2 μl PCR-grade water 9.425 μl, total 25 μl. Initial step 95 ° C 10 minutes, then 95 ° C 10 seconds, 60 ° C 31 seconds 45 cycles.
The probe and primer sequences are as follows.
ATF3 probe, 5'-FAM- CGCCTCAGACTTGGTGACTGACATCTCCA-TAMRA-3 '(SEQ ID NO: 1)
ATF3 forward primer, 5'- AGTCAGTTACCGTCAACAACAGA-3 '(SEQ ID NO: 2)
ATF3 reverse primer, 5'- CCGCCTCCTTTTCCTCTCATC-3 '(SEQ ID NO: 3)
GAPDH probe, 5'-FAM- TGGTCACCAGGGCTGCCATTTGCA-TAMRA-3 '(SEQ ID NO: 4)
GAPDH forward primer, 5'- TGAAGGTCGGTGTGAACGG-3 '(SEQ ID NO: 5)
GAPDH reverse primer, 5'- CCATGTAGTTGAGGTCAATGAAGG-3 '(SEQ ID NO: 6)
Data analysis was performed using Sequence Detection Software version 1.4 (Applied Biosystems). The values in the figure are shown as mean ± standard error.

(2) Results
ATF3 increased in insomnia of about 8 hours, and was found to be useful for measuring the mental labor fatigue experienced daily. In addition, the increase in ATF3 was diverse in the brain, muscle, heart, and liver, and it was found that the degree of fatigue in these organs could be individually evaluated (Fig. 2).

  Using a forced swimming model of mice, the increase in ATF3 was measured in various organs immediately after physical fatigue and after rest.

(1) Method Forcibly swim a mouse (C57BL / 6NCrSlc) in a pool (22cm in diameter) with water deep enough to prevent your feet from reaching, and immediately after loading (30 minutes, 1 hour, 2 hours) Load) and as a control group, organs were removed over time (n = 2-12) from mice not subjected to load (0 hour) and frozen. Furthermore, after performing forced swimming (FS) for 2 hours, after giving a rest for 2 hours in a normal cage, the organ was removed and frozen (in FIG. 3, FS 2h + rest 2h). ATF3 and GAPDH mRNA were quantified by the TaqMan PCR method in the same manner as the experiment shown in Example 2. The values in the figure are shown as mean ± standard error.

(2) Results In 1 hour and 2 hours of swimming, an increase in ATF3 was observed in various organs such as brain, muscle, heart and liver. In addition, with a rest of about 2 hours, ATF3 decreased rapidly. This indicates that physical fatigue and recovery in various organs can be quantitatively measured by measuring ATF3. In addition, since swimming in this fatigue model is performed without applying a load such as a weight, it is considered that the fatigue load does not deviate from the level of daily exercise load (FIG. 3).

  Using human peripheral blood specimens, fatigue measurements were performed using ATF3.

(1) Method Using a cycle ergometer (Aerobike 75XL2 ME; Combi Wellness Co.) for a healthy person (n = 2), cycling anaerobic for 4 hours at anaerobic threshold (AT) of 80% went. Before and after loading, blood was collected in a PAXgene Blood RNA blood collection tube (Japan BD). Blood RNA was purified using PAXgene Blood RNA Kit (QIAGEN, Inc.), and cDNA was synthesized using PrimeScript RT reagent Kit (Takara Bio, Inc.). ATF3 and β-actin mRNA in blood was quantified using a Applied Biosystems QuantStudio real-time PCR System (Applied Biosystems) with a TaqMan Array under the following conditions. Probe and primer set: ATF3 (Hs00231069_m1), ACTB (Hs01060665_g1); per port, TaqMan Gene Expression Master Mix (Applied Biosystems) 50 μl, cDNA 10 μl, PCR-grade water 40 μl. PCR conditions: Initial step: 50 ° C 2 minutes, then 95 ° C 10 minutes, 95 ° C 15 seconds, 60 ° C 1 minute 40 cycles. Data analysis was performed using ExpressionSuite Software version v1.0.3 (Applied Biosystems). The values in the figure are shown as mean ± standard error.

(2) Results It was confirmed that ATF3 in human peripheral blood samples increased due to exercise fatigue load. As a result, it was shown that the fatigue measurement by ATF3 is possible in the test using human peripheral blood (FIG. 4).

  Mental fatigue due to insomnia using the mouse insomnia model and physical fatigue using the forced swimming model were measured by increasing the expression of CHOP in the liver.

(1) Method CHOP and GAPDH mRNA in the mouse liver (n = 2 to 4) subjected to fatigue load was quantified by the same method as the experiments shown in Examples 2 and 3. The probe and primer sequences are as follows.
CHOP probe, 5'-FAM-TCCTCTGTCAGCCAAGCTAGGGACGC-TAMRA-3 '(SEQ ID NO: 7)
CHOP forward primer, 5'-CCTATATCTCATCCCCAGGAAACG-3 '(SEQ ID NO: 8)
CHOP reverse primer, 5'-TGTGCGTGTGACCTCTGTTG-3 '(SEQ ID NO: 9)
The values in the figure are shown as mean ± standard error.

(2) Results An increase in the expression level of CHOP in the liver was observed at 2 hours of swimming (FS 2h in the left graph of FIG. 5) and 8 hours and 24 hours of insomnia (8h and 24h in the right graph of FIG. 5). This indicates that physical fatigue and mental fatigue in organs can be quantitatively measured by measuring the amount of CHOP expression (FIG. 5).

  Using human peripheral blood specimens, fatigue measurement by CHOP was performed.

(1) Method CHOP (probe, primer set: Hs00358796_g1) and ACTB mRNA in blood before and after bicycle rowing (n = 2) and ACTB were measured by the same method as the experiment shown in Example 4. The values in the figure are shown as mean ± standard error.

(2) Results It was confirmed that the amount of CHOP expression in human peripheral blood specimens increased due to exercise fatigue load. Thereby, it was shown that fatigue measurement by CHOP is possible in a test using human peripheral blood (FIG. 6).

  It was confirmed that fatigue was induced by ATF3.

(1) Method
i) Construction of ATF3 expression plasmid vector
A synthetic gene (Mm ATF3-opt (SEQ ID NO: 64)) of mouse ATF3 (Mm ATF3) optimized for Codon Usage for mice was prepared, and the pEGFP-N1 expression vector (pEGFP (-)-) with the GFP coding region removed. The resulting plasmid was called Mm ATF3-opt / pEGFP (-)-N1.

ii) Transient expression of ATF3 in vivo by Hydrodynamic delivery system TransIT (r) Hydrodynamic Delivery System (TaKaRa) was used to transiently express ATF3 in vivo in mice. The method followed a standard protocol. MTF ATF3-opt / pEGFP (−)-N1 was used as an ATF3 expression plasmid.

iii) Behavioral testing of ATF3 in vivo transiently expressed mice
An open field test for 10 minutes was performed 0, 4, 8, and 16 hours after the in vivo administration of the ATF3 expression plasmid Mm ATF3-opt / pEGFP (−)-N1 to the mouse, and the moving distance was measured by TopScan (CleverSys). Statistical significance (* p <0.05, ** p <0.01, *** p <0.001) was calculated using Welch's t test.

(2) Results
In ATF3-expressing mice, spontaneous movement decreased significantly 4 to 8 hours after plasmid introduction, and recovery of locomotor activity was observed 16 hours after introduction. Therefore, it was shown that the expression of ATF3 caused fatigue in the mice, and the brain felt fatigue, which decreased the locomotor activity. Moreover, it was shown that the fatigue induced by ATF3 recovered in a short time and had the characteristics of physiological fatigue (FIG. 7).

  We investigated whether suppression of ATF3 expression by ATF3-specific interfering RNA can reduce fatigue.

(1) Method Mice (C57BL / 6NCrSlc) were bred in a cage with a rotating pulley (Clea Japan). The number of rotations of the pulley was recorded by a computer system (CLEA Japan) with 3 rotations per rotation. In the morning, Stealth RNAi siRNA against ATF3 (MSS273298) (Life Technologies) 40 μg and Luciferase expression vector (pLuc-TK) 1 μg expressed with TK promoter using TransIT-QR Hydrodynamic Delivery Solution (Mirus Bio LCC.) Introduced by hydrodynamic method (n = 6). As controls, Stealth RNAi siRNA Negative Control Hi GC (12935-400) 40 μg (Life Technologies) and pLuc-TK 1 μg were introduced (n = 2). After the introduction, the mice were placed in a cage with 1 cm of water for 8 hours and then returned to the rotating cage to measure the spontaneous momentum. The amount of night exercise on the day of loading was calculated as a percentage of the previous day. After measuring locomotor activity, the liver was removed from the mouse, sonicated, and Luciferase activity was measured by the Luciferase Reporter Assay System (Promega). Mice that did not show Luciferase activity were excluded from the analysis because they failed to introduce the gene by hydrodynamic method. The values in the figure are shown as mean ± standard error (FIG. 8).

(2) Results As a result of measuring the spontaneous momentum of the mouse, it was found that the amount of spontaneous momentum was large in those into which interfering RNA (siRNA) specific for ATF3 was introduced. This result indicates that inhibition of ATF3 production suppresses fatigue, indicating that ATF3 plays an important role in the true state of fatigue (FIG. 8).

  We observed changes in fatigue-related factors with the introduction of ATF3.

(1) Method The livers of 4 mice each of 0, 4, 8, and 16 hours after in vivo transformation by the hydrodynamic delivery system of Mm ATF3-opt / pEGFP (-)-N1 as in Example 7 were collected, The gene expression levels of ATF3 and inflammation-related factors were compared. FIG. 9 shows A) gene expression of endogenous ATF3, B) gene expression of inflammatory cytokine IL-1β, C) gene expression of inflammatory cytokine IL-6, and D) gene expression of inflammatory cytokine TNF-α. Show. The bar graph shows the average value, and the error bar shows the standard error. Statistical significance (* p <0.05) was calculated using Welch's t test.
Primer and probe sequences are as follows.
ATF3:
Probe: 5'-TCCTCAATCTGGGCCTTCAGCTCAGCAT-3 '(SEQ ID NO: 10)
Primer-F: 5'-TGTCGAAACAAGAAAAAGGAGAAGA-3 '(SEQ ID NO: 11)
Primer-R: 5'-GCAGGTTGAGCATGTATATCAAATG-3 '(SEQ ID NO: 12)
IL-1β:
Probe; 5'-TGCTCTCATCAGGACAGCCCAGGTCA-3 '(SEQ ID NO: 13)
Primer-F: 5'-CCTGAACTCAACTGTGAAATGCC-3 '(SEQ ID NO: 14)
Primer-R: 5'-CTGCTGCGAGATTTGAAGCTG-3 '(SEQ ID NO: 15)
IL-6:
Probe: 5'-ACCACTCCCAACAGACCTGTCTATACCACT-3 '(SEQ ID NO: 16)
Primer-F: 5'-CCGGAGAGGAGACTTCACAGA-3 '(SEQ ID NO: 17)
Primer-R: 5'-GTTGTTCATACAATCAGAATTGCCATT-3 '(SEQ ID NO: 18)
TNF-α:
Probe: 5'-ATTCGAGTGACAAGCCTGTAGCCCACG-3 '(SEQ ID NO: 19)
Primer-F: 5'-CCAGACCCTCACACTCAGATCAT-3 '(SEQ ID NO: 20)
Primer-R: 5'-CCTCCACTTGGTGGTTTGCTAC-3 '(SEQ ID NO: 21)

(2) Results
Since ATF3-opt and mouse endogenous ATF3 have the same protein amino acid sequence, but have different DNA base sequences, we investigate whether ATF3 has the ability to induce ATF3 by Real-time PCR. It is possible. As a result of this examination, as shown in FIG. 8A, it was found that ATF3 induces the expression of ATF3 and that the effect is relatively short. This indicates that although ATF3 temporarily exerts a large ATF3 expression-inducing effect by positive feedback, the effect is quickly eliminated and the fatigue recovers in a short time. It is shown that.

  In addition, regarding inflammatory cytokines that are thought to transmit fatigue to the brain and generate a feeling of fatigue, it was found that TNF-α mainly works in physiological fatigue involving ATF3. It was also found that IL-1β and IL-6, which are thought to play a major role in CFS fatigue, are rather lowered. ATF3 is known to act in a suppressive manner with respect to IL-1β and IL-6, and by suppressing IL-1β and IL-6, which cause chronic persistent fatigue in CFS, It is thought that it provides easy recovery and reversibility, which are characteristics of fatigue (FIG. 9).

  Fatigue substances that cause generalized physiological fatigue have not been identified so far. Since the fatigue substance is an alarm that informs the living body that the living body is overworked or lacked in rest, the trigger for the fatigue alarm is considered to be a waste product or a metabolite accumulated in the living body due to life activity. In addition, since there was a possibility that RNA was related to this phenomenon from Example 1, interfering RNA having an important function in biological regulation was examined as a fatigue substance candidate. Interfering RNA includes siRNA and miRNA, but there are many molecular species, which usually exert their effects by binding specifically to mRNA. The inventor has focused on the total amount of interfering RNA in the cell that is unrelated to the function of the original interfering RNA. We investigated the possibility that fatigue increases the total amount of interfering RNA and causes phosphorylation of eIF-2α via PKR.

(1) Method From a mouse heart and liver (n = 1) subjected to fatigue loading in the same manner as in the experiments shown in Examples 2 and 3, RNA containing micro RNA (miRNA) was miRNAeasy isolation Kit (Qiagen) And purified. Using RT2 miRNA First Strand Kit (SABioscoences), reverse transcription was performed from miRNA contained in 100 ng of each organ RNA, and a total of 10 μl of cDNA was synthesized according to the standard protocol. A total of 10 ml of a reaction solution was prepared consisting of 100 μl of diluted cDNA obtained by adding 90 μl of RNase-free water to the cDNA, 5.0 ml of 2X RT2 SYBR Green PCR Master Mix, and 4.9 ml of water. 25 μl of the reaction solution was dispensed into RT2 miRNA PCR Array (SABioscoences), and miRNA was quantified comprehensively under the following conditions.
PCR conditions: Initial step: 95 ° C 10 minutes, then 95 ° C 15 seconds, 60 ° C 31 seconds, 72 ° C 30 seconds 40 cycles.
Data was analyzed on the PCR Array Data Analysis Web Portal (http://www.sabiosciences.com/pcrarraydataanalysis.php).

(2) Results FIG. 10 shows a representative example of the analysis results of various interfering RNAs. In both the liver (FIG. 10-1) and the heart (FIG. 10-2), various interfering RNAs under fatigue load are shown. An increase was shown. The total amount of interfering RNA calculated simultaneously increased as well. In addition, an increase in interfering RNA was observed both in exercise fatigue (FS 2h in FIG. 10) and mental fatigue (8h and 24h in FIG. 10). This indicates that the fatigue-causing substance that is the first trigger of physiological fatigue is interfering RNA (FIG. 10).

  Because interfering RNA is laborious and expensive to measure, we searched for molecular biomarkers that accurately reflect the increase in interfering RNA and can be measured easily. Candidates are Dgcr8 and Drosha, enzymes involved in the production of interfering RNA.

(1) Method Dgcr8, Drosha and GAPDH mRNA in mouse liver (n = 2) subjected to fatigue loading was quantified in the same manner as the experiments shown in Examples 2 and 3. The probe and primer sequences are as follows.
Dgcr8 probe, 5'-FAM- TGCGAAGAATAAAGCTGCCCGAGCCA-TAMRA-3 '(SEQ ID NO: 22)
Dgcr8 forward primer, 5'-CGGATCTGGAACTGCAAGCA-3 '(SEQ ID NO: 23)
Dgcr8 reverse primer, 5'-GTTCTTCACTGTCTTTAGGCTTCTC-3 '(SEQ ID NO: 24)
Drosha probe, 5'-FAM- AGTTCCTCTGCTACCTTGGCTTGCGT-TAMRA-3 '(SEQ ID NO: 25)
Drosha forward primer, 5'- ACAGAGTACTTGTTCATTCATTTCCC-3 '(SEQ ID NO: 26)
Drosha reverse primer, 5'- TGTCGTTGGTGATGGCATACTC-3 '(SEQ ID NO: 27)
The values in the figure are shown as mean ± standard error.

(2) Results The expression levels of DGCR8 and Drosha, enzymes involved in the production of interfering RNA, increase due to exercise fatigue (FS 2h in FIG. 11) and mental fatigue (8h, 12h, 24h in FIG. 11). I understood. As a result, it was shown that these were also biomarkers that could be used for the measurement of physiological fatigue (FIG. 11).

  Fatigue measurements were performed with Dgcr8 and Drosha using human peripheral blood samples.

(1) Method Dgcr8 (probe, primer set: Hs00256062_m1), Drosha (probe, primer set: Hs00203008_m1) and ACTB before and after bicycle rowing (n = 2) in the same manner as the experiment shown in Example 4 MRNA was quantified. The values in the figure are shown as mean ± standard error.

(2) Results It was confirmed that Dgcr8 and Drosha in human peripheral blood samples increased due to exercise fatigue load. Thereby, it was shown that the fatigue measurement by Dgcr8 or Drosha is possible in the test using human peripheral blood (FIG. 12).

  Using human peripheral blood specimens, fatigue measurements with GRP78 and XBP-1 were performed.

(1) Method GRP78 (probe, primer set: Hs00607129_gH), XBP1 (probe, primer set: Hs00231936_m1) and ACTB before and after bicycle rowing (n = 2) in the same manner as the experiment shown in Example 4 mRNA was quantified. The values in the figure are shown as mean ± standard error.

(2) Results It was confirmed that the expression levels of GRP78 and XBP-1 in human peripheral blood samples increased due to exercise fatigue load. As a result, it was shown that fatigue tests using GRP78 and XBP-1 were possible in tests using human peripheral blood (FIG. 13).

  We examined whether ATF3 expression can distinguish physiological fatigue from Poly (I: C) peritoneal administration model, which is a model of fatigue due to infection and CFS.

(1) Method
The livers of 3 mice each 6 hours after the intraperitoneal administration of Poly (I: C) were collected, and the gene expression levels of ATF3 were compared. In addition, ATF3 gene expression of 4 mice subjected to 2 hours of forced swimming is shown as FS 2hr.

Intraperitoneal administration of Poly (I: C):
Poly (I: C) sodium salt (Sigma-Aldrich) was diluted with endotoxin-free physiological saline (Otsuka Pharmaceutical Co., Ltd.) for intraperitoneal administration of Poly (I: C) to mice, 0, 1, 2 mg / ml An aqueous solution of Poly (I: C) was prepared. Poly (I: C) aqueous solution was administered to the abdominal cavity of 6-week-old C57BL / 6NCrSlc mice at 0, 5, 10 mg / kg (about 100 μl). The mice after intraperitoneal administration were returned to the breeding cage.

Gene expression analysis of Poly (I: C) peritoneally administered mice:
As a result of confirming the ATF3 gene expression level over time after Poly (I: C) intraperitoneal administration, it was found that the expression level reached the maximum after 6 hours (data not shown). Therefore, the mouse liver was collected 6 hours after the intraperitoneal administration of Poly (I: C) to the mouse. Liver RNA was purified, and the amount of ATF3 mRNA was quantified by real-time RT-PCR. The β-actin gene was used as a reference gene. For comparison, the ATF3 expression level of mice subjected to 2-hour forced swimming (FS) was also measured. Primer probe sequences used in real-time RT-PCR are as follows.
ATF3:
Probe: 5'-TCCTCAATCTGGGCCTTCAGCTCAGCAT-3 '(SEQ ID NO: 10)
Primer-F: 5'-TGTCGAAACAAGAAAAAGGAGAAGA-3 '(SEQ ID NO: 11)
Primer-R: 5'-GCAGGTTGAGCATGTATATCAAATG-3 '(SEQ ID NO: 12)
The bar graph shows the average value, and the error bar shows the standard error. Statistical significance (** p <0.01) was calculated using Welch's t test. Statistical significance (* p <0.05, ** p <0.01, *** p <0.001) was calculated using Welch's t test.

(2) Results Although ATF3 was strongly induced by 2 hours of FS, the expression level of ATF3 6 hours after Poly (I: C) intraperitoneal administration was remarkably low. Another study confirms that ATF3 induced after Poly (I: C) intraperitoneal administration is highest after 6 hours. In addition, according to another embodiment, it has been found that ATF3 increases as much as FS of 2 hours in mental fatigue. For this reason, it was shown that the fatigue observed in the Poly (I: C) peritoneal administration model considered to be a model of CFS and virus infection is different from physiological fatigue (FIG. 14).

  We examined whether mental fatigue and physical fatigue can be evaluated by increasing eIF-2α phosphorylation inhibitory factor in various organs.

(1) Method GADD34, Nck1, Nck2, CReP, and GAPDH mRNA in mouse heart and liver (n = 2 to 4) subjected to fatigue load were quantified in the same manner as the experiments shown in Examples 2 and 3. . The probe and primer sequences are as follows.
ADD34 probe, 5'-FAM- TCTCAGCGAAGTGTACCTTCCGAGCT-TAMRA-3 '(SEQ ID NO: 28)
GADD34 forward primer, 5'-GGCGGCTCAGATTGTTCAAAG-3 '(SEQ ID NO: 29)
GADD34 reverse primer, 5'-CAGCAAGGAAATGGACTGTGAC-3 '(SEQ ID NO: 30)
Nck1 probe, 5'-FAM- CACCAGGCACCAGGCAGAAATGGCAT-TAMRA-3 '(SEQ ID NO: 31)
Nck1 forward primer, 5'-GCTGGCAATCCTTGGTATTATGG-3 '(SEQ ID NO: 32)
Nck1 reverse primer, 5'-CCCTTGTGCTTTTAGTGATACTGAG-3 '(SEQ ID NO: 33)
Nck2 probe, 5'-FAM- TCCTCGCCCAGTGACTTCTCCGTGT-TAMRA-3 '(SEQ ID NO: 34)
Nck2 forward primer, 5'-CGACTTCCTCATTAGGGACAGC-3 '(SEQ ID NO: 35)
Nck2 reverse primer, 5'-CACCTTGAAGTGCTTGTTTCTCC-3 '(SEQ ID NO: 36)
CReP probe, 5'-FAM- AGGCTCTTGCTGGAGAAAGATACACCCA-TAMRA-3 '(SEQ ID NO: 37)
CReP forward primer, 5'- AAGAAGATAATTGTCCAGGCTGTG-3 '(SEQ ID NO: 38)
CReP reverse primer, 5'- CTCATCACCACTTATATAATACTCAGTAAC-3 '(SEQ ID NO: 39)
The values in the figure are shown as mean ± standard error.

(2) Results eIF-2α phosphorylation inhibitory factor (GADD34, Nck1 in the heart and liver of mice) due to the stress of mental fatigue (FS 2h in FIG. 15) and exercise fatigue (8h, 12h, 24h in FIG. 15). , Nck2, and CReP) were found to increase. From this, it was shown that mental fatigue and physical fatigue can be evaluated by the eIF-2α phosphorylation inhibitory factor (FIG. 15).

  We examined whether the fatigue of subjects can be evaluated by the increase of eIF-2α phosphorylation inhibitor in blood cells.

(1) Method In the same manner as the experiment shown in Example 4, blood GADD34 (probe, primer set: Hs00169585_m1), Nck1 (probe, primer set: Hs01592377_m1), Nck2 before and after bicycle rowing (n = 2) (Probe, primer set: Hs02561903_s1), CReP (probe, primer set: Hs00262481_m1), and ACTB mRNA were quantified. The values in the figure are shown as mean ± standard error.

(2) Results It was found that eIF-2α phosphorylation inhibitory factors (GADD34, CReP, Nck1) in human blood cells increase due to exercise fatigue. This showed that eIF-2α phosphorylation inhibitory factor can evaluate mental fatigue and physical fatigue (FIG. 16).

eIF-2α phosphorylation signaling factor or eIF-2α phosphorylation signal suppressor, or both using ATF3 as a representative of eIF-2α phosphorylation signaling factor and GADD34 as a representative of eIF-2α phosphorylation signal inhibitor We examined whether it was possible to determine the effects of anti-fatigue substances and anti-fatigue methods by combining the above.
Hiking is an anti-fatigue method that is well known as a method of recovering from fatigue, while fatigue occurs during early mountain climbing. Imidapeptide is a preparation containing a high concentration of imidazole dipeptide extracted from chicken breast meat, and is an anti-fatigue substance that has been confirmed to have an anti-fatigue effect from various studies. We examined how the effects of such anti-fatigue substances and anti-fatigue methods were judged by the eIF-2α phosphorylation signaling factor ATF3 and the eIF-2α phosphorylation signal inhibitor GADD34.

(1) Method Blood was collected before and after mountain climbing in the group that climbed the mountain in an overpace (fast climbing group) (n = 5) and the group that climbed slowly with a break (hiking group) (n = 8). In the same manner as in the experiment shown in Example 4, RNA in blood before and after climbing was purified to synthesize cDNA.
In addition, in a group in which healthy subjects (n = 6) were imidated with Imida Peptide 240 (Japanese preventive medicine) 1 day / day for 1 week, blood was collected 2 weeks before and after ingestion, RNA in blood was purified, and cDNA was obtained. Synthesized.
The amount of mRNA of ATF3, GADD34 and GAPDH was measured using Applied Biosystems 7300 real-time PCR System (Applied Biosystems). The measurement was performed in duplicate under the following conditions.
Real-time PCR conditions: FastStart TaqMan Probe Master (ROX) (Roche Diagnostics) 12.5 μl, PCR forward primer (100 μM) 0.225 μl, PCR reverse primer (100 μM) 0.225 μl, TaqMan probe (10 μM) 0.625 μl, cDNA 2 μl PCR-grade water 9.425 μl, total 25 μl. Initial step 95 ° C 10 minutes, then 95 ° C 10 seconds, 60 ° C 31 seconds 45 cycles.
The probe and primer sequences are as follows.
ATF3 probe, 5'-FAM-CCCTCGGGGTGTCCATCACAAAAGCC-TAMRA-3 '(SEQ ID NO: 40)
ATF3 forward primer, 5'-CGCTGGAATCAGTCACTGTCAG-3 '(SEQ ID NO: 41)
ATF3 reverse primer, 5'-CCTTCTTCTTGTTTCGGCACTTTG-3 '(SEQ ID NO: 42)
GADD34 probe, 5'-FAM- GCCTTTAGGGGAGTCTCAGGGTCCG-TAMRA-3 '(SEQ ID NO: 43)
GADD34 forward primer, 5'-GCCCAGAAACCCCTACTCATG-3 '(SEQ ID NO: 44)
GADD34 reverse primer, 5'-AGGAAATGGACAGTGACCTTCTC-3 '(SEQ ID NO: 45)
GAPDH probe, 5'-FAM-CGCCCAATACGACCAAATCCGTTGACTCC-TAMRA-3 '(SEQ ID NO: 46)
GAPDH forward primer, 5'- CACCATGGGGAAGGTGAAGG-3 '(SEQ ID NO: 47)
GAPDH reverse primer, 5'-CAATATCCACTTTACCAGAGTTAAAAGC-3 '(SEQ ID NO: 48)
Data analysis was performed using Sequence Detection Software version 1.4 (Applied Biosystems). The values in the figure are shown as mean ± standard error.

(2) Results
In the determination with the eIF-2α phosphorylation signaling factor ATF3 alone, it was found that the ratio of ATF3 was decreased from the beginning due to hiking and imidapepide. From this, it was found that if ATF3 decreases before and after use, it can be determined that the corresponding anti-fatigue substance candidate or anti-fatigue method candidate has an anti-fatigue effect. Even with the eIF-2α phosphorylation signal inhibitor GADD34 alone, strong induction of GADD34 was observed corresponding to the anti-fatigue effect of imidapeptide.
According to the comprehensive judgment of eIF-2α phosphorylation signaling factor ATF3 and eIF-2α phosphorylation signal inhibitor GADD34, early hill climbing shows strong induction of GADD34, but the effect of ATF3 has not been counteracted. It has been shown that the effect of GADD34 exceeds that of ATF3, thereby reducing ATF3 and exerting anti-fatigue effects. Moreover, in the imidapeptide, it was shown that the imidapeptide exerts strong anti-fatigue effect by strongly inducing GADD34 and strongly suppressing ATF3 (FIG. 17).

  The evaluation of pathological fatigue by fatigue-related factors is shown by taking CFS patients as an example.

(1) Method
Blood was collected from CFS patients (n = 79) and healthy subjects (n = 69), and ATF3, CHOP, GADD34, Nck1, Nck2, CReP, and GAPDH mRNA were quantified in the same manner as the experiment shown in Example 17. . CHOP, Nck1, Nck2, and CReP probes and primer sequences are as follows.
CHOP probe, 5'-FAM-TCCTCCTCAGTCAGCCAAGCCAGAGA-TAMRA-3 '(SEQ ID NO: 49)
CHOP forward primer, 5'-CCTATGTTTCACCTCCTGGAAATG-3 '(SEQ ID NO: 50)
CHOP reverse primer, 5'-GTGACCTCTGCTGGTTCTGG-3 '(SEQ ID NO: 51)
Nck1 probe, 5'-FAM- CATCAGCAGGAGATGCAGAATCTGGCACAC-TAMRA-3 '(SEQ ID NO: 52)
Nck1 forward primer, 5'-GCTCGGAAAGCATCTATTGTGAAA-3 '(SEQ ID NO: 53)
Nck1 reverse primer, 5'- ATGTTGAGGTCATAGAGACGTTCC-3 '(SEQ ID NO: 54)
Nck2 probe, 5'-FAM- TCTCCCCGTGCTCGCTGGTGAAGAT-TAMRA-3 '(SEQ ID NO: 55)
Nck2 forward primer, 5'-GAGCGGAAGAACAGCCTGAAG-3 '(SEQ ID NO: 56)
Nck2 reverse primer, 5'- GGCCCTGACGAGGTAGAGC-3 '(SEQ ID NO: 57)
CReP probe, 5'-FAM-TGTGTCTTCCTCCAGAAAGAACGTCACGCT-TAMRA-3 '(SEQ ID NO: 58)
CReP forward primer, 5'-GAAAGTGAATGTCCAGACTCGGTA-3 '(SEQ ID NO: 59)
CReP reverse primer, 5'- ACTCAGTAACTTCTTCAAGGAAGGT-3 '(SEQ ID NO: 60)
The values in the figure are shown as median values. Comparison between the two groups was performed by Mann-Whitney U test.

(2) Results
Although all patients with CFS complained of strong subjective fatigue, no increase in fatigue-related factors in blood cells was observed, unlike physiological fatigue. Contrary to physiological fatigue, a significant decrease was observed for GADD34 and Nck1, which are eIF-2α phosphorylation inhibitors. This indicates that measurement of fatigue-related factors is useful for distinguishing between physiological fatigue and pathological fatigue (FIG. 18).

  The evaluation of pathological fatigue by fatigue-related factors is shown by taking depression patients as an example.

(1) Method Blood was collected from patients with depressive disorder (Dep) (n = 19) and healthy subjects (n = 18), and ATF3, GADD34 and GAPDH (BET) in blood were collected in the same manner as the experiment shown in Example 4. Probe and primer set: Hs99999905_m1) mRNA was quantified. The values in the figure are shown as median values. Comparison between the two groups was performed by Mann-Whitney U test.

(2) Results Despite the fact that depression patients often complain of subjective fatigue, no increase in fatigue-related factors in blood cells was observed unlike physiological fatigue. A significant decrease was observed for GADD34, an eIF-2α phosphorylation inhibitor, as opposed to physiological fatigue. This indicates that measurement of fatigue-related factors is also useful for distinguishing between physiological fatigue and pathological fatigue even in depression (FIG. 19).

  In fatigue associated with life, particularly labor, there are problems of the relationship between fatigue and depression, and a decrease in work efficiency due to depressive symptoms due to fatigue. The present inventors have already invented an objective fatigue evaluation method using human herpesvirus in saliva as an objective fatigue evaluation method. We examined whether it was possible to obtain information about the mental state of healthy workers by this method.

(1) Method Saliva was collected from healthy workers (n = 41) into a saliva collection tube Salivette (Sarstedt AG & Co.). After collection, it was centrifuged at 1,700 g for 5 to 15 minutes at 4 ° C., and the flow-through was stored at −80 ° C. until analysis. The virus DNA was purified from 400 μl of saliva using BioRobot EZ1 workstation and EZ1 virus mini kit v2.0 (QIAGEN, Inc.). DNA was eluted in 90 μl of elution buffer. HHV-7 DNA in saliva was quantified by duplicate using TaqMan PCR method using Applied Biosystems 7300 real-time PCR System (Applied Biosystems). PCR conditions are as follows: Premix Ex Taq (Perfect Real Time) (Takara Bio Inc.) 25 μl, PCR forward primer (100 μM) 0.45 μl, PCR reverse primer (100 μM) 0.45 μl, TaqMan probe (10 μM) 50 μl total of 1.25 μl, Rox reference dye 1 μl, viral DNA 5 μl, PCR-grade water 16.85 μl. Initial step 95 ° C 30 seconds, then 95 ° C 5 seconds, 60 ° C 31 seconds 50 cycles.
The probe and primer sequences for quantifying HHV-7 are as follows.
HHV-7 probe, 5′-FAM-CCTCGCAGATTGCTTGTTGGCCATG-TAMRA-3 ′ (SEQ ID NO: 61)
HHV-7 forward primer, 5′-CGGAAGTCACTGGAGTAATGAC-3 ′ (SEQ ID NO: 62)
HHV-7 reverse primer, 5′-CCAATCCTTCCGAAACCGAT-3 ′ (SEQ ID NO: 63)
Data analysis was performed using Sequence Detection Software version 1.4 (Applied Biosystems).
The low fatigue group and the high fatigue group were separated by drawing a boundary line of the amount of HHV-7 DNA in saliva at 100,000 copies / ml or 40,000 copies / ml. Depressive symptoms were evaluated by Beck Depression Inventory (BDI), and subjective fatigue was evaluated by visual analogue scale (VAS).

(2) Results Correlation between subjective fatigue level (VAS) and score (BDI) indicating depression in the low fatigue level group (FIG. 20 left) and high group (FIG. 20 right) was examined (FIG. 20). Shows an example with 100,000 copies / ml as the boundary).
As a result, surprisingly, in the group where the degree of fatigue was determined to be low in the method for measuring fatigue with HHV-7 in saliva, the subject's awareness even when the subject's depressive symptoms did not lead to a morbid state. It was found that there was a fairly strong correlation between mental fatigue and depressive symptoms. The correlation coefficients were 0.73 and p <0.01 when the boundary line was 40,000 copies / ml, and 0.69 and p <0.001 when the boundary line was 100,000 copies / ml. When fatigue was high, there was no correlation between subjective fatigue level and depressive symptoms (FIG. 20).
The objective observation of the mental state of healthy workers was an outcome that could not have been predicted from previous studies, including studies on fatigue assessment methods. Therefore, it was considered to be a screening method for depression and depression which is very simple and does not require specialist diagnosis (FIG. 20).

  Using a mouse insomnia model, the increase in ZFP36 in organs due to mental fatigue due to insomnia was measured.

(1) Method In the same apparatus as the experimental apparatus shown in Example 2, ZFP36 of the liver, heart, muscle (n = 4) and brain (n = 6) of mice (C57BL / 6NCrSlc) subjected to fatigue load and β-actin (ACTB) mRNA was quantified using the TaqMan PCR method. The fatigue loading time was 2 or 24 hours for the liver, 4 or 24 hours for the heart and muscle, and 4 or 8 hours for the brain. The mRNA expression of each gene was evaluated by the ratio to the amount of β-actin mRNA. The probe and primer sequences are as follows.
ZFP36 probe, 5'-FAM- TGAGCCATGACCTGTCATCCGACCACG -TAMRA-3 '(SEQ ID NO: 65)
ZFP36 forward primer, 5'- TCTGCCATCTACGAGAGCCTC -3 '(SEQ ID NO: 66)
ZFP36 reverse primer, 5'-GAGTCCGAGTTTATGTTCCAAAGTC -3 '(SEQ ID NO: 67)
ACTB probe, 5'-FAM- CACACCCGCCACCAGTTCGCCATG -TAMRA-3 '(SEQ ID NO: 68)
ACTB forward primer, 5'- CGCGAGCACAGCTTCTTTG -3 '(SEQ ID NO: 69)
ACTB reverse primer, 5'-CGACCAGCGCAGCGATATC -3 '(SEQ ID NO: 70)

(2) Results
ZFP36 increased significantly even in insomnia of 2 to 24 hours, and it was found that ZFP36 increased during the mental labor fatigue experienced on a daily basis. From this, it was found that ZFP36 exerts an anti-fatigue effect by suppressing signal transduction associated with phosphorylation of eIF-2α due to fatigue in mental fatigue. It was also found that ZFP36 can be used to measure fatigue as a biomarker of mental fatigue. In addition, it was found that the increase in ZFP36 was diverse in the liver, heart, muscle, and brain, and the fatigue-suppressing effect and the degree of fatigue in these organs could be individually evaluated. Statistical significance was determined by t-test, and in any organ, p <0.05 between the control group (sleep) and mental fatigue load group (insomnia) (FIG. 21A: liver, heart, muscle, FIG. 21B: brain).

  Using a mouse insomnia model, the increase in TSC22D3 (GILZ) in the organ due to mental fatigue due to insomnia was measured.

(1) Method In the same manner as in the experiment shown in Example 21, the TSC22D3 and β of the liver, heart, and muscle (n = 4) of the mouse (C57BL / 6NCrSlc) subjected to fatigue load were subjected to the fatigue load. -Actin (ACTB) mRNA was quantified using TaqMan PCR. TSC22D3 probe and primer sequences are as follows.
TSC22D3 probe, 5'-FAM-TTCTTCACGAGGTCCATGGCCTGCTCA-TAMRA-3 '(SEQ ID NO: 71)
TSC22D3 forward primer, 5'- GTGGTGGCCCTAGACAACAAG -3 '(SEQ ID NO: 72)
TSC22D3 reverse primer, 5'- CCTCTCTCACAGCGTACATCAG -3 '(SEQ ID NO: 73)

(2) Results
TSC22D3 was also significantly increased at 4 and 24 hours of insomnia, and it was found that TSC22D3 was increased during the mental labor fatigue experienced daily. From this, it was found that TSC22D3 exerts an anti-fatigue effect by suppressing signal transduction associated with phosphorylation of eIF-2α due to fatigue in mental fatigue. We also found that TSC22D3 can be used to measure fatigue as a biomarker of mental fatigue. In addition, the increase in TSC22D3 was diverse in the liver, heart, and muscle, and it was found that the fatigue-suppressing effect and the degree of fatigue in these organs can be individually evaluated. Statistical significance was determined by t-test, and p <0.05 between the control group (sleep) and the mental fatigue load group (insomnia) in any organ (FIG. 22).

  The increase in ZFP36 immediately after physical fatigue was measured using a forced swimming model of mice.

(1) Method In the same manner as the experiment shown in Example 3, ZFP36 and β-actin (ACTB) of liver, heart, brain, peripheral blood (n = 6) of mice subjected to fatigue load (C57BL / 6NCrSlc) ) MRNA was quantified using the TaqMan PCR method. The fatigue loading time was 1 hour for the liver, heart, and brain, and 30 minutes for peripheral blood. The mRNA expression of each gene was evaluated by the ratio to the amount of β-actin mRNA. Probe and primer sequences are as in Example 21.

(2) Results
It was found that ZFP36 increased significantly even during exercise load of about 30 minutes to 1 hour, and that ZFP36 increased during the physical labor fatigue experienced on a daily basis. From this, it was found that, in physical fatigue, ZFP36 exerts an anti-fatigue effect by suppressing signal transduction associated with phosphorylation of eIF-2α due to fatigue. It was also found that ZFP36 can be used to measure fatigue as a biomarker of physical fatigue. In addition, the increase in ZFP36 was diverse in the liver, heart, brain, and peripheral blood, and it was found that the fatigue suppression effect and the degree of fatigue in these organs can be individually evaluated. Statistical significance was determined by t-test, and p <0.05 between the pre-exercise group and the post-exercise group in any organ (FIG. 23A: liver, heart, FIG. 23B: peripheral blood, brain).

  Using a forced swimming model of mice, the increase in TSC22D3 immediately after physical fatigue was measured.

(1) Method In the same manner as in the experiment shown in Example 23, TSC22D3 and β-actin (ACTB) of heart, brain and peripheral blood (n = 6) of mice subjected to fatigue load (C57BL / 6NCrSlc) mRNA was quantified using the TaqMan PCR method. The fatigue loading time was 1 hour for the heart and brain, and 30 minutes for peripheral blood. The mRNA expression of each gene was evaluated by the ratio to the amount of β-actin mRNA. Probe and primer sequences are as in Example 22.

(2) Results
It was found that TSC22D3 increased significantly even during exercise loads of about 30 minutes to 1 hour, and that TSC22D3 increased during physical labor fatigue experienced on a daily basis. From this, it was found that TSC22D3 exerts an anti-fatigue effect by suppressing signal transduction accompanying phosphorylation of eIF-2α due to fatigue in physical fatigue. It was also found that TSC22D3 can be used to measure fatigue as a biomarker of physical fatigue. In addition, the increase in TSC22D3 was diverse in the heart, brain, and peripheral blood, and it was found that the fatigue-suppressing effect and the degree of fatigue in these organs can be individually evaluated. Statistical significance was determined by t-test, and p <0.05 between the pre-exercise group and the post-exercise group in any organ (FIG. 24).

  ZFP36 was measured using a human peripheral blood sample.

(1) MethodPatients diagnosed with sleep apnea syndrome (SAS), which is considered to have significant mental fatigue due to lack of sleep (Figure 25: A, B, n = 56; C, n = 13) and Healthy subjects (FIG. 25: A, B, n = 21; C, n = 18) were used as subjects. Blood was collected in a PAXgene Blood RNA blood collection tube (Japan BD). Blood RNA was purified using PAXgene Blood RNA Kit (QIAGEN, Inc.), and cDNA was synthesized using PrimeScript RT reagent Kit (Takara Bio, Inc.). ATF3, Nck1, ZFP36 and β-actin (ACTB) mRNA in blood were quantified with a TaqMan Array using Applied Biosystems QuantStudio real-time PCR System (Applied Biosystems) under the following conditions. Probe and primer set: ATF3 (Hs00231069_m1), Nck1 (Hs01592377_m1), ZFP36 (Hs00185658_m1), ACTB (Hs01060665_g1); TaqMan Gene Expression Master Mix (Applied Biosystems) 50 μl per port, cDNA 10 μl, PCR-grade water 40 μl. PCR conditions: Initial step: 50 ° C 2 minutes, then 95 ° C 10 minutes, 95 ° C 15 seconds, 60 ° C 1 minute 40 cycles. Data analysis was performed using ExpressionSuite Software version v1.0.3 (Applied Biosystems). The values in FIG. 25 are shown as mean ± standard error. * P <0.05, *** P <0.001, **** P <0.0001, Welch's t test.

(2) Results
SAS confirmed that ATF3, Nck1, and ZFP36 in human peripheral blood samples were increased compared to healthy subjects (controls). As a result, it was shown that it is possible to measure fatigue with ATF3, Nck1, and ZFP36 in a test using peripheral blood of a human who suffers from sleep deprivation or mental fatigue (FIG. 25).

Measurement of anti-fatigue effect by inhibition of signal transduction pathway related to phosphorylation of eIF-2α by ISRIB Integrated stress response inhibitor (ISRIB) exerts effects such as memory enhancement by inhibiting ISR pathway It was known to do. The present inventors examined whether a substance having an ISR inhibitory function such as ISRIB has an anti-fatigue effect for the purpose of supporting the discovery by the present inventors and developing an anti-fatigue drug.

(1) Method
In the mouse (n = 4) intraperitoneally injected with ISRIB 2.5 mg / kg and the control mouse (n = 4) injected with the solvent alone, using the same mouse forced swimming experimental apparatus as in Example 3, forced swimming 50 minutes The distance of movement of the mouse for 10 minutes later (movement distance of 50 to 60 minutes after the start of forced swimming) was measured with TopScan (CleverSys).

(2) Results
The movement distance of the mice injected intraperitoneally with ISRIB and the control mice injected with solvent alone for 10 minutes after 50 minutes of forced swimming was significantly longer in the ISRIB group. From this result, it can be seen that the fatigue of forced swimming was reduced by ISRIB, and that swimming at a longer distance became possible even after 50 minutes. Statistical significance was determined by the Mann-Whitney U test with p <0.05 between the control and ISRIB groups (FIG. 26).

  This indicates that phosphorylation of eIF-2α leads to increased fatigue, and substances that suppress signal transduction pathways related to phosphorylation of eIF-2α are useful as anti-fatigue substances such as anti-fatigue drugs. And anti-fatigue effects of anti-fatigue substances such as anti-fatigue drugs and anti-fatigue devices can be determined with the effect of suppressing signal transduction pathways related to phosphorylation of eIF-2α. It is thought to show that there is.

Suppression of fatigue recovery mechanism by inhibition of eIF-2α dephosphorylation by Salubrinal Inhibits dephosphorylation of eIF-2α, a fatigue recovery mechanism involving GADD34 and CreP, which are the aforementioned eIF-2α phosphorylation signal suppressors We found the fatigue enhancement effect of the drug salubrinal.

(1) Method
10 minutes after the start of forced swimming in mice (salubrinal) injected with 100 μl of 37.5 μl of 20 mg / ml salubrinal stock solution in 3 ml of DMSO and mice injected with 100 μl of DMSO as a solvent (control) alone (control) The 10-minute movement distance (movement distance from 10 minutes to 20 minutes after the start of forced swimming) was measured using the same experimental system as in Example 26.

(2) Results
The distance traveled for 10 minutes after 10 minutes of forced swimming between the mice injected intraperitoneally with Salubrinal and the control mice injected with solvent alone was significantly shorter in the salubrinal group. From this result, it is considered that the fatigue suppression and recovery of forced swimming was suppressed by salubrinal, and the travel distance in forced swimming decreased. Statistical significance was determined by t-test, and p <0.05 between the control group and salubrinal group (FIG. 27).
This result shows that dephosphorylation of eIF-2α has anti-fatigue effects, especially fatigue suppression / recovery effects, and substances that promote dephosphorylation of eIF-2α are anti-fatigue substances such as anti-fatigue agents. The usefulness and eIF-2α dephosphorylation effects indicate that anti-fatigue substances such as anti-fatigue drugs and anti-fatigue methods such as anti-fatigue devices can be evaluated for anti-fatigue effects. It is considered a thing.

  The fatigue evaluation method according to the present invention can be used for elucidation of stress and fatigue mechanism, and can develop a stress relieving method, evaluate the degree of fatigue, and evaluate the mental surface associated with fatigue. In addition, by using the present invention, quantification (evaluation) of the effects of health foods, anti-fatigue health foods, foods for specified health use, nutrition drinks and biological function supplements, electrical appliances, furniture, folk remedies, etc. Is possible. Therefore, the present invention can be used in a wide range of fields such as the medical industry, the pharmaceutical industry, the health food industry, and the health equipment industry.

Claims (3)

An anti-fatigue power evaluation method characterized by evaluating the anti-fatigue power of a test target organism using the amount of a eukaryotic translation initiation factor 2α (eIF-2α) phosphorylation signal inhibitor in a cell as an index, The anti-fatigue power is characterized by evaluating that the anti-fatigue power is high if the amount of the eIF-2α phosphorylation signal inhibitor is larger than the amount predicted from the amount of the eIF-2α phosphorylation signal transduction factor. Evaluation method:
Here, the eIF-2α phosphorylation signal inhibitor is growth arrest and DNA damage-inducible protein 34 (GADD34), non-catalytic region of tyrosine kinase adaptor protein (Nck) 1, Nck2, constitutive repressor of eIF-2α phosphorylation (CReP), zinc finger protein 36 homolog (ZFP36), and glucocorticoid-induced leucine zipper (GILZ), and the eIF-2α phosphorylation signaling factor is phosphorylated eIF- It is at least one selected from the group consisting of 2α, activating transcription factor (ATF) 3, ATF 4, and C / EBP-homologous protein (CHOP).   The amount of eIF-2α phosphorylation signal suppressor and the amount of eIF-2α phosphorylation signal transduction factor are expressed as the amount of mRNA expression or protein production of eIF-2α phosphorylation signal suppressor and eIF-2α phosphorylation signal transduction factor. The anti-fatigue strength evaluation method according to claim 1, measured as follows. About a subject having subjective fatigue, using the amount of human herpesvirus in saliva as an index , a method for evaluating the cause of the subject 's subjective fatigue,
(1) a step of measuring the DNA level of human herpesvirus 7 in the saliva of the subject;
(2) When the amount of DNA is equal to or lower than a boundary line for determining that the subject's fatigue level is low, the subject's subjective fatigue is evaluated as a depressive symptom , wherein the boundary line is The amount of DNA of human herpesvirus 7 in saliva is 100,000 copies / ml or less;
Including methods.

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