Pharmaceutical composition for preventing or treating diseases associated with phospholipid hydroperoxide-dependent cell death

Abstract

To provide techniques for preventing or treating diseases associated with phospholipid-hydroperoxide-dependent cell death.SOLUTION: Disclosed is a pharmaceutical composition for preventing or treating diseases associated with phospholipid-hydroperoxide-dependent cell death, the composition comprising cells or culture of a species of the genus Enterococcus and vitamin E or derivative thereof as effective ingredients. Also disclosed is a food composition for preventing or treating diseases associated with phospholipid-hydroperoxide-dependent cell death containing cells or culture of a species of the genus Enterococcus and vitamin E or derivative thereof as effective ingredients.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, a food composition for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, and a method for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death. [Background technology]

[0002] Sudden death is defined as death that occurs within 24 hours of onset. Most sudden cardiac deaths are sudden cardiac deaths. Most sudden cardiac deaths are caused by myocardial infarction due to arteriosclerosis, but there are also cases of sudden cardiac death that are not caused by abnormalities in the vascular system (sudden death due to idiopathic cardiomyopathy). The mechanism of sudden death due to idiopathic cardiomyopathy has not been elucidated, and currently there are no methods of prevention or treatment.

[0003] Phospholipid-hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a type of antioxidant enzyme. The inventors previously demonstrated that mice with cardiac-specific GPx4 deficiency were embryonic lethal, but that the mice could survive if their mothers were fed a diet high in vitamin E, and that the mice could survive even after weaning if they were fed a diet high in vitamin E. Furthermore, they suffered sudden death due to heart failure when the diet high in vitamin E was replaced with a normal diet (see, for example, Patent Documents 1 and 2).

[0004] Furthermore, the inventors previously established an experimental system in which mice lacking GPx4 specifically in the heart were subjected to an exercise load and their mortality rate or exercise capacity was measured (see Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6652761 [Patent Document 2] Patent No. 6253013 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a technique for preventing or treating diseases associated with phospholipid hydroperoxide-dependent cell death. [Means for solving the problem]

[0007] The present invention includes the following aspects. [1] A pharmaceutical composition for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, comprising a bacterium or culture of the genus Enterococcus, and vitamin E or a derivative thereof as active ingredients. [2] The pharmaceutical composition according to [1], wherein the disease associated with phospholipid hydroperoxide-dependent cell death is heart failure or cardiac fatigue. [3] The pharmaceutical composition according to [1] or [2], wherein the Enterococcus bacterium is Enterococcus faecium, Enterococcus hirae, Enterococcus gallinarum, Enterococcus durans, Enterococcus raffinosus, Enterococcus psedoavium, Enterococcus hermanniensis, Enterococcus rivorum, Enterococcus thailandicus, Enterococcus asini, Enterococcus avium, Enterococcus villorum, Enterococcus lactis, Enterococcus ratti, or Enterococcus feacalis. [4] A food composition for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, containing as an active ingredient the cells or culture of an Enterococcus bacterium and vitamin E or a derivative thereof. [5] A method for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death (excluding medical acts on humans), comprising the step of administering to a subject the cells or culture of an Enterococcus bacterium and vitamin E or a derivative thereof.

Effects of the Invention

[0008] The present invention can provide a technique for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death.

Brief Description of the Drawings

[0009] [Figure 1] 1(a) is a diagram illustrating the experimental schedule in Experimental Example 1. FIG. 1(b) is a graph showing the change in survival rate of mice in each group in Experimental Example 1. [Diagram 2] 1(a) is a diagram illustrating the experimental schedule in Experimental Example 2. FIG. 1(b) is a graph showing the change in survival rate of mice in each group in Experimental Example 2. [Diagram 3] 1(a) is a diagram illustrating the experimental schedule in Experimental Example 3. FIG. 1(b) is a graph showing the types and proportions of bacteria detected in Experimental Example 3. [Figure 4] 1(a) and (b) are diagrams illustrating the experimental schedule in Experimental Example 4. FIG. 1(c) and (d) are graphs showing the changes in survival rate of mice in each group in Experimental Example 4. [Diagram 5] 1 is a graph showing the results of analyzing the intestinal bacterial flora of each group of mice in Experimental Example 4. [Figure 6] 1 is a graph showing the results of quantifying the number of Enterococcus bacteria in the feces of mice in each group in Experimental Example 4. [Figure 7] 1(a) is a diagram illustrating the experimental schedule in Experimental Example 5. FIG. 1(b) is a graph showing the change in survival rate of mice in each group in Experimental Example 5. [Figure 8] 6(a) to 6(f) are graphs showing the results of quantifying Enterococcus bacteria in feces from mice in each group in Experimental Example 5. [Figure 9] 1 is a graph showing the percentage of bacteria of the genus Enterococcus isolated and identified in Experimental Example 6. [Figure 10] FIG. 13 is a diagram illustrating an experimental schedule in Experimental Example 7. [Figure 11]Graph (a) shows the results of measuring the survival rate after switching feed for mice administered with Enterococcus faecalis in Experimental Example 7. Graph (b) shows the results of quantifying Enterococcus bacteria in the feces of a mouse (number 1) administered with Enterococcus faecalis in Experimental Example 7. Graph (b) shows the results of quantifying Enterococcus bacteria in the feces of a mouse (number 2) administered with Enterococcus faecalis in Experimental Example 7. [Figure 12] Graph (a) shows the results of measuring the survival rate after switching feed for mice administered with Enterococcus faecium in Experimental Example 7. Graph (b) shows the results of quantifying Enterococcus bacteria in the feces of a mouse (number 1) administered with Enterococcus faecium in Experimental Example 7. Graph (b) shows the results of quantifying Enterococcus bacteria in the feces of a mouse (number 2) administered with Enterococcus faecium in Experimental Example 7. [Figure 13] Graph (a) shows the results of measuring the survival rate after switching feed for mice administered with Enterococcus hirae in Experimental Example 7. Graph (b) shows the results of quantifying Enterococcus bacteria in the feces of a mouse (number 1) administered with Enterococcus hirae in Experimental Example 7. Graph (b) shows the results of quantifying Enterococcus bacteria in the feces of a mouse (number 2) administered with Enterococcus hirae in Experimental Example 7. [Figure 14] Graph (a) shows the results of measuring the survival rate after switching the feed of mice administered with Enterococcus ratti in Experimental Example 7. Graph (b) shows the results of quantifying Enterococcus bacteria in the feces of a mouse (number 1) administered with Enterococcus ratti in Experimental Example 7. Graph (c) shows the results of quantifying Enterococcus bacteria in the feces of a mouse (number 2) administered with Enterococcus ratti in Experimental Example 7. [Figure 15]FIG. 1(a) is a diagram illustrating an experimental schedule in Experimental Example 8. FIG. 1(b) is a graph showing the quantitative results of phosphatidylcholine hydroperoxide (PC-OOH) in Experimental Example 8. FIG. 1(c) is a graph showing the quantitative results of phosphatidylcholine (PC) in Experimental Example 8. [Figure 16] FIG. 1(a) is a diagram illustrating the experimental schedule in Experimental Example 9. FIG. 1(b) is a graph showing the results of measuring the number of days until death (number of days of survival) for mice in each group in Experimental Example 9 after switching the feed. [Figure 17] 1 is a graph showing the results of quantifying the number of Enterococcus bacteria in the feces of mice in each group in Experimental Example 10. [Figure 18] 13 is a graph showing the results of a treadmill running test in Experimental Example 10. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] [Preventive or therapeutic pharmaceutical composition] In one embodiment, the present invention provides a pharmaceutical composition for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, comprising Enterococcus cells or a culture thereof and vitamin E or a derivative thereof as active ingredients. The pharmaceutical composition for prevention or treatment of the present embodiment may be a preventive agent for a disease associated with phospholipid hydroperoxide-dependent cell death, or may be a therapeutic agent for a disease associated with phospholipid hydroperoxide-dependent cell death.

[0011] As described later in the Examples, the inventors have demonstrated that by orally administering the preventive or therapeutic agent of this embodiment to a mouse model in which sudden death due to heart failure occurs, sudden death due to heart failure can be suppressed, and cardiac load during limit running (cardiac fatigue due to lipid oxidation in the heart) can be suppressed.

[0012] Therefore, diseases related to phospholipid hydroperoxide-dependent cell death include heart failure and cardiac fatigue caused by lipid oxidation in the heart.Heart failure, in particular, heart failure caused by lipid oxidation in the heart.More specifically, for example, heart failure caused by marathon running, heart failure in athletes such as Olympic athletes and soccer players.

[0013] In this specification, the term "disease" includes organ dysfunction. That is, the disease may be a condition that requires treatment with a drug or the like, or a condition that does not require treatment with a drug or the like but is preferably improved.

[0014] More specifically, the disease associated with phospholipid hydroperoxide-dependent cell death includes a decrease in cardiac function associated with phospholipid hydroperoxide-dependent cell death. The decrease in cardiac function includes, for example, a decrease in motor function, a delay in recovery from fatigue, etc. In the present specification, "treatment" includes improvement of symptoms.

[0015] In the preventive or therapeutic agent of this embodiment, examples of the Enterococcus bacteria include Enterococcus faecium, Enterococcus hirae, Enterococcus gallinarum, Enterococcus durans, Enterococcus raffinosus, Enterococcus pseudoavium, Enterococcus hermanniensis, Enterococcus rivorum, Enterococcus thailandicus, Enterococcus asini, Enterococcus avium, Enterococcus faecium, Enterococcus hirae, Enterococcus gallinarum, Enterococcus durans, Enterococcus raffinosus, Enterococcus pseudoavium, Enterococcus hermanniensis, Enterococcus rivorum, Enterococcus thailandicus, Enterococcus avium ... avium, Enterococcus villorum, Enterococcus lactis, Enterococcus ratti, Enterococcus feacalis, etc. Among them, Enterococcus feacalis, Enterococcus faecium, Enterococcus hirae, and Enterococcus ratti are preferred.

[0016] The Enterococcus bacteria may be killed or live. When live bacteria are used, some dead bacteria may be mixed in. Furthermore, Enterococcus bacteria may be used in a state where the shape of the bacteria remains, such as in the form of a disrupted product or extract.

[0017] The culture of bacteria of the genus Enterococcus is a composition containing a substance produced by bacteria of the genus Enterococcus. The culture of bacteria of the genus Enterococcus may be a culture supernatant of bacteria of the genus Enterococcus, a crude product of the culture supernatant, or the like.

[0018] The preventive or therapeutic pharmaceutical composition of the present embodiment is preferably administered orally. When administering live Enterococcus bacteria, the dosage is preferably an amount that allows the bacteria to colonize the large intestine to a certain extent, for example, 3×10 7 CFU (colony forming units) / kg body weight of Enterococcus spp. The number of administrations can be adjusted appropriately depending on the symptoms, and may be, for example, once every one to several days. One to several days may be, for example, 1 to 10 days.

[0019] Furthermore, when killed Enterococcus bacteria or a culture of Enterococcus bacteria is administered, for example, an amount of the killed bacteria or culture corresponding to the amount of the live bacteria may be administered once or multiple times.

[0020] The Enterococcus bacteria used in this embodiment can be prepared by a known method. The Enterococcus bacteria may be cultured by a known method, for example, by inoculating the Enterococcus bacteria into a medium and pre-culturing it for a predetermined period of time. The medium is not particularly limited as long as it can culture the Enterococcus bacteria, and examples of the medium include MRS liquid medium.

[0021] Alternatively, the Enterococcus bacteria may be used as a powder obtained by drying the bacteria. Methods for drying the bacteria include freeze-drying and spray-drying. For example, a liquid medium in which Enterococcus bacteria have been cultured can be concentrated by centrifugation or the like, and then freeze-drying the concentrated liquid to obtain a powder of the bacteria.

[0022] The preventive or therapeutic pharmaceutical composition of this embodiment may be a composition containing Enterococcus bacteria or a culture thereof and a pharma- ceutical acceptable carrier. The composition is preferably administered orally, and is preferably formulated in a dosage form for oral use. Examples of the dosage form for oral use include tablets and capsules.

[0023] Examples of pharma- ceutically acceptable carriers include binders such as gelatin, corn starch, gum tragacanth, and gum arabic; excipients such as starch and crystalline cellulose; and bulking agents such as alginic acid.

[0024] The composition may contain additives, such as lubricants such as calcium stearate and magnesium stearate, sweeteners such as sucrose, lactose, saccharin and maltitol, flavors such as peppermint and saffron oil, stabilizers such as benzyl alcohol and phenol, buffers such as phosphates and sodium acetate, antioxidants, and preservatives.

[0025] The compositions can be formulated by combining and mixing the Enterococcus cells or cultures, carriers and additives as appropriate, in a unit dosage form required for generally accepted pharmaceutical practice.

[0026] The preventive or therapeutic pharmaceutical composition of this embodiment further contains vitamin E or a derivative thereof. As described later in the Examples, the inventors have revealed that vitamin E or a derivative thereof is necessary for suppressing heart failure caused by administration of Enterococcus bacteria.

[0027] Examples of vitamin E derivatives include trolox (2,5,7,8-tetramethyl-6-hydroxychroman-2-carboxylic acid) and troglitazone (5-[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]-phenyl]methyl]-2,4-thiazolidinedione).

[0028] The dosage of vitamin E or a derivative thereof is, for example, 0.01 to 100 mg / kg body weight, for example, 0.6 to 14 mg / kg body weight of vitamin E or a derivative thereof per administration. The number of administrations can be appropriately adjusted according to the symptoms, for example, once every one to several days. For example, one to several days is 1 to 10 days.

[0029] The preventive or therapeutic pharmaceutical composition of this embodiment may be a mixture of Enterococcus bacteria or a culture thereof and vitamin E or a derivative thereof, or may be a kit in which the Enterococcus bacteria or a culture thereof and vitamin E or a derivative thereof are enclosed in separate containers and administered in combination.

[0030] [Food composition] In one embodiment, the present invention provides a food composition for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, comprising Enterococcus bacteria or a culture thereof and vitamin E or a derivative thereof as active ingredients. The Enterococcus bacteria are the same as those described above. The terms "disease" and "treatment" are the same as those described above. That is, the term "disease" includes a decrease in organ function, and includes a state in which treatment with a medicine or the like is required, and a state in which treatment with a medicine or the like is not required but is preferable to improve. The term "treatment" includes improvement of symptoms. It can be said that the food composition of this embodiment is useful for inhibiting lipid oxidation in the heart, inhibiting cardiac fatigue, and enhancing cardiac function.

[0031] The food composition of the present embodiment may be in the form of, for example, a supplement, yogurt, a drink, a gel food, any cooked food, etc. Examples of the shape of the supplement include capsules.

[0032] The Enterococcus bacteria and cultures are the same as those described above. The food composition of the present embodiment can be administered in an amount of, for example, 3×107 It is preferable to use live Enterococcus bacteria in an amount of 3×10 CFU / kg body weight or more, or a corresponding amount of killed bacteria or cultures of the live bacteria. The timing of ingestion may be before, after, or between meals. 7 "CFU / kg body weight or more" varies depending on the form of the food composition, but refers to the recommended daily intake amount indicated on the label, or the amount contained in one bottle of a drink that is usually consumed in one sitting.

[0033] The food composition of this embodiment further contains vitamin E or a derivative thereof. The vitamin E derivative is the same as that described above. The food composition of this embodiment is preferably used to allow the intake of, for example, 0.01 to 100 mg / kg body weight, for example, 0.6 to 14 mg / kg body weight of vitamin E or a derivative thereof per day. The intake may be before, after, or between meals. Note that "0.6 to 14 mg / kg body weight per day" refers to the recommended daily intake amount indicated on the label, or the amount contained in one bottle of a drink that is usually consumed in one sitting, although this varies depending on the form of the food composition.

[0034] The food composition of the present embodiment may be a mixture of Enterococcus bacteria or a culture thereof and vitamin E or a derivative thereof, or may be a kit in which the Enterococcus bacteria or a culture thereof and vitamin E or a derivative thereof are enclosed in separate containers and administered in combination.

[0035] The food composition of the present embodiment may be a functional food. "Functional food" refers to a food that has been notified to the Consumer Affairs Agency as a food that displays functionality on the product package based on scientific evidence. Examples of such display include, but are not limited to, "functionality to recover from cardiac fatigue." In addition, even if a food does not have a functional display, it is possible to manufacture and sell the food by advertising the functionality in leaflets, emails, or verbally.

[0036] The food composition of the present embodiment may be a food for special dietary use. A food for special dietary use means a food that is approved by the government and labeled with a special purpose such as being suitable for the growth and maintenance / recovery of health of infants, small children, pregnant women, sick people, etc.

[0037] The food composition of the present embodiment may be a food for specified health uses among foods for special dietary uses. A food for specified health uses is a food that is recognized based on scientific evidence as being useful for maintaining and promoting health, and is permitted to display its effects. The effects and safety of the foods are examined by the government, and approval is given for each food by the Commissioner of the Consumer Affairs Agency.

[0038] [Preventive or therapeutic methods] In one embodiment, the present invention provides a method for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death (excluding medical procedures on humans), comprising the step of administering to a subject a bacterial cell or culture of Enterococcus spp., and vitamin E or a derivative thereof.

[0039] In the preventive or therapeutic method of this embodiment, Enterococcus bacteria or a culture thereof and vitamin E or a derivative thereof may be administered simultaneously, or Enterococcus bacteria or a culture thereof and vitamin E or a derivative thereof may be administered separately.

[0040] In the prevention or treatment method of this embodiment, the terms "disease" and "treatment" are the same as those described above. The subject may be a human or a non-human animal. The prevention or treatment method of this embodiment excludes medical procedures for humans and does not include procedures performed by doctors. Specific examples of the prevention or treatment method of this embodiment include providing a food composition containing Enterococcus bacteria or a culture thereof and vitamin E or a derivative thereof at a restaurant or the like, or providing a food composition containing Enterococcus bacteria or a culture thereof and vitamin E or a derivative thereof as a supplement for preventing fatigue.

[0041] The Enterococcus bacteria, culture, vitamin E and derivatives thereof are the same as those described above. The Enterococcus bacteria or culture may be in the form of a formulation as described above, or in the form of a food composition. The dosage of the formulation or food composition is the same as that described above. The Enterococcus bacteria or culture is preferably administered orally.

[0042] Vitamin E or a derivative thereof may be administered simultaneously with Enterococcus cells or a culture, or may be administered separately from Enterococcus cells or a culture.

[0043] [Other embodiments] In one embodiment, the present invention provides a method for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, comprising administering an effective amount of Enterococcus cells or culture to a patient in need of treatment.

[0044] In one embodiment, the present invention provides a composition for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, the composition comprising a cell or culture of a bacterium of the genus Enterococcus.

[0045] In one embodiment, the present invention provides use of a cell or culture of Enterococcus for the production of an agent for the prevention or treatment of a disease associated with phospholipid hydroperoxide-dependent cell death.

[0046] In each of these embodiments, the Enterococcus bacteria, culture, dosage, etc. are the same as those described above. EXAMPLES

[0047] The present invention will now be described in more detail with reference to experimental examples, but the present invention is not limited to the following experimental examples.

[0048] [Mouse model] The inventors previously reported that GPx4 gene knockout mice (GPx4 - / - ) was produced. GPx4 - / - The mice became embryonic lethal at day 7.5 of development. Therefore, we transfected mice with the mouse GPx4 genomic gene flanked by loxP sequences (loxP-GPx4) and generated Tg(loxP-GPx4) mice. + / - :GPx4 + / + Mice were generated.

[0049] Next, Tg(loxP-GPx4) + / - :GPx4 + / + Mouse and GPx4 + / - The mice were crossed with Tg(loxP-GPx4) + / - :GPx4 + / - By breeding these mice, the endogenous GPx4 genomic gene was knocked out (KO), and the embryonic lethality was rescued by the introduced loxP-GPx4Tg gene. + / + :GPx4 - / - Mice were generated.

[0050] Also, GPx4 + / - The mice were then compared with the control mice, which had the Cre gene downstream of the muscle creatine kinase promoter, a cardiac-specific promoter. + / + ) and Cre + / + GPx4 + / - Got a mouse.

[0051] Next, Cre + / + GPx4 + / - Mice and the Tg(loxP-GPx4) + / + :GPx4 - / - By crossing with mice, + / - :Tg(loxP-GPx4) + / - :GPx4 - / - We obtained mice that lack GPx4 specifically in the heart. + / -:Tg(loxP-GPx4) + / - :GPx4 - / - The mice are referred to as "cardiac-specific GPx4-deficient mice."

[0052] When the cardiac-specific GPx4-deficient mice obtained in this way were observed, they grew normally up to day 16.5 of development, but on day 17.5, cardiomyocytes suddenly died, and on day 18.5, they developed edema and died.

[0053] However, when mother mice were fed a diet high in vitamin E every day, cell death in the cardiac tissue of the cardiac-specific GPx4-deficient mice on day 17.5 of development was completely suppressed, and cardiac-specific GPx4-deficient mice were born normally. The vitamin E-high diet used was a diet containing 100 mg of vitamin E acetate (dl-α-tocopherol acetate) per 100 g of normal diet CE-2 (manufactured by CLEA Japan).

[0054] The inventors have further previously demonstrated that when cardiac-specific GPx4-deficient mice are switched from a high-vitamin E diet to a normal diet (CE-2) after weaning (at approximately 3 weeks of age), they suffer sudden death due to heart failure within approximately 15 days.

[0055] [Experimental Example 1] (Administration of antibiotics to cardiac-specific GPx4-deficient mice) Antibiotics were administered to cardiac-specific GPx4-deficient mice, and the survival rate was examined. Figure 1(a) is a diagram explaining the experimental schedule. First, the diet of cardiac-specific GPx4-deficient mice (3 weeks old) was switched from high vitamin E diet to normal diet (CE-2). Antibiotics were also administered to the mice. Cefoperazone (CPZ) was used as the antibiotic. The chemical formula of cefoperazone is shown in the following formula (1). In addition, oral administration via drinking water and intraperitoneal administration were examined as the administration method. 0.4 g / L of cefoperazone was added to the drinking water. In addition, for intraperitoneal administration, 0.2 mg / g body weight of cefoperazone was administered every other day. This amount is the same as the amount of cefoperazone that mice ingest from drinking water in a day. A control group was also prepared in which no antibiotics were administered and tap water was given.

[0056] [ka]

[0057] Figure 1(b) is a graph showing the change in survival rate of mice in each group. As a result, it was revealed that oral administration of antibiotics to cardiac-specific GPx4-deficient mice suppressed the decrease in survival rate. In contrast, intraperitoneal administration of antibiotics to cardiac-specific GPx4-deficient mice did not suppress the decrease in survival rate.

[0058] [Experimental Example 2] (Administration of antibiotics to cardiac-specific GPx4-deficient germ-free mice) Antibiotics were administered to cardiac-specific GPx4-deficient germ-free mice, and the survival rate was examined. First, mice were born and raised in a completely germ-free environment in an isolator to generate cardiac-specific GPx4-deficient germ-free mice that had no intestinal bacteria. Figure 2(a) is a diagram explaining the experimental schedule. First, the diet of cardiac-specific GPx4-deficient germ-free mice (6 weeks old) was switched from high vitamin E diet to normal diet. As the normal diet, AIN93G (containing 0.3 mg / 100 g of vitamin E (d-α-tocopherol, hereinafter sometimes referred to as "d-α-Toc")) was used. Antibiotics were orally administered to the mice via drinking water. Cefoperazone (CPZ) was used as the antibiotic. 0.4 g / L of cefoperazone was added to the drinking water. A control group was also prepared in which tap water was given without antibiotics.

[0059] Figure 2(b) is a graph showing the change in survival rate of mice in each group. As a result, it was revealed that oral administration of antibiotics did not suppress the decrease in survival rate in germ-free mice. This result indicates that intestinal bacteria are necessary for suppressing the decrease in survival rate caused by oral administration of cefoperazone. Furthermore, since lethality was induced by vitamin E reduction even in the absence of antibiotics, it was revealed that intestinal bacteria are not necessary for the induction of lipid oxidation-dependent heart failure. In other words, it was shown that intestinal bacteria that survived the administration of cefoperazone in drinking water are involved in the suppression of lipid oxidation-dependent heart failure.

[0060] [Experimental Example 3] (Analysis of mouse intestinal microbiota using next-generation sequencing) The intestinal microbiota of mice administered antibiotics was analyzed. Figure 3(a) is a diagram explaining the experimental schedule. First, the feed of cardiac-specific GPx4-deficient mice (3 weeks old) was switched from high-vitamin E-supplemented feed to normal feed (CE-2). Antibiotics were also administered to the mice. Cefoperazone (CPZ) was used as the antibiotic. The antibiotic was administered orally via drinking water. 0.4 g / L of cefoperazone was added to the drinking water. A control group was also prepared in which no antibiotics were administered and tap water was provided.

[0061] Feces were collected 0, 6, and 12 days after the diet change. After 12 days, mice were dissected and the cecum was collected. The bacterial flora in the feces and cecum were then analyzed using a next-generation sequencer.

[0062] Figure 3(b) is a graph showing the types and percentages of bacteria detected. As a result, it was revealed that Enterococcus bacteria significantly increased in the feces and cecum of mice administered cefoperazone.

[0063] [Experimental Example 4] (Administration of antibiotics to cardiac-specific GPx4-deficient pseudo-germ-free mice) We administered antibiotics to pseudo-germ-free mice lacking cardiac-specific GPx4, and analyzed their survival rate and intestinal microbiota. Figures 4(a) and (b) are diagrams explaining the experimental schedule.

[0064] As shown in Figure 4(b), in order to pseudo-eliminate the gut bacteria of cardiac-specific GPx4-deficient mice, four types of antibiotics were orally administered for two weeks from the age of three weeks to make them pseudo-germ-free. The mice were fed a diet containing high levels of vitamin E. The four antibiotics were neomycin (1 g / L), ampicillin (1 g / L), metronidazole (1 g / L), and vancomycin (0.5 g / L), and were administered in drinking water. For comparison, cardiac-specific GPx4-deficient mice that were not pseudo-germ-free were also prepared, as shown in Figure 4(a).

[0065] Next, at 5 weeks of age, the diet was switched from high vitamin E to normal diet AIN93G (with vitamin E 0.3mg / 100g). Additionally, the mice were orally administered cefoperazone (CPZ) via drinking water. 0.4g / L of cefoperazone was added to the drinking water. A control group was also prepared in which no antibiotics were administered and tap water was provided.

[0066] In addition, after switching the diet to AIN93G (supplemented with vitamin E 0.3 mg / 100 g), feces were collected on days 0, 6, and 12, and the intestinal flora was analyzed using a next-generation sequencer.

[0067] Figures 4(c) and (d) are graphs showing the change in survival rate of mice in each group. Figure 4(c) shows the results for cardiac-specific GPx4-deficient mice, and Figure 4(d) shows the results for cardiac-specific GPx4-deficient pseudo-germ-free mice.

[0068] As a result, as shown in FIG. 4(c), cardiac-specific GPx4-deficient mice died in about 20 days when they were switched from a high-vitamin E diet to AIN93G (with vitamin E 0.3 mg / 100 g) at 5 weeks of age. In contrast, the deaths were suppressed by administering cefoperazone in drinking water. On the other hand, as shown in FIG. 4(d), in pseudo-germ-free mice administered four types of antibiotics, when they were switched from a high-vitamin E diet to AIN93G (with vitamin E 0.3 mg / 100 g), the deaths were not suppressed even when cefoperazone was administered in drinking water. This result was similar to that of cardiac-specific GPx4-deficient germ-free mice in Experimental Example 2.

[0069] Figure 5 is a graph showing the results of analyzing the intestinal microbiota of non-pseudo-sterilized cardiac-specific GPx4-deficient mice on days 0, 6, and 12 after switching the diet to AIN93G (with vitamin E 0.3 mg / 100 g). In Figure 5(a), "CPZ" indicates the results for the group that received cefoperazone in drinking water, and "water" indicates the results for the group that did not receive cefoperazone.

[0070] As a result, it was revealed that in the group that received cefoperazone in drinking water, Enterococcus spp. accounted for 99% of the intestinal flora 6 and 12 days after the feed was switched to AIN93G (containing 0.3 mg / 100 g vitamin E).

[0071] FIG. 6 is a graph showing the results of quantifying, by the plate method, the number of Enterococcus bacteria in the feces of mice in each group on the sixth day after the feed was switched to AIN93G (with vitamin E added at 0.3 mg / 100 g).

[0072] In FIG. 6, "Water" indicates the results of the group of non-pseudo-sterilized cardiac-specific GPx4-deficient mice that were not administered cefoperazone. "CPZ" indicates the results of the group of non-pseudo-sterilized cardiac-specific GPx4-deficient mice that were administered cefoperazone in drinking water. "Four drugs" indicates the results of the group of non-pseudo-sterilized cardiac-specific GPx4-deficient mice that were not administered cefoperazone. "Four drugs + CPZ" indicates the results of the group of non-pseudo-sterilized cardiac-specific GPx4-deficient mice that were administered cefoperazone.

[0073] As a result, it was revealed that Enterococcus spp. increased in the non-pseudo-sterilized cardiac-specific GPx4-deficient mice treated with cefoperazone. In contrast, no Enterococcus spp. were detected in the pseudo-sterilized cardiac-specific GPx4-deficient mice treated with cefoperazone. These results suggest that Enterococcus spp. are involved in the suppression of heart failure by cefoperazone in drinking water.

[0074] [Experimental Example 5] (Consideration of fecal transplants) We performed fecal transplants of feces containing 99% Enterococcus spp. into cardiac-specific GPx4-deficient pseudo-germ-free mice and examined their survival rates.

[0075] <Preparation of recipient mice> FIG. 7(a) is a diagram explaining the experimental schedule. First, cardiac-specific GPx4-deficient pseudo-germ-free mice were prepared. Specifically, cardiac-specific GPx4-deficient mice were orally administered four types of antibiotics in combination for two weeks from the age of three weeks to make them pseudo-germ-free. They were fed a diet containing high amounts of vitamin E. The four antibiotics were neomycin (1 g / L), ampicillin (1 g / L), metronidazole (1 g / L), and vancomycin (0.5 g / L), and were administered in drinking water.

[0076] Next, the diet of the cardiac-specific GPx4-deficient pseudo-germ-free mice was switched from high vitamin E diet to normal diet AIN93G (with vitamin E 0.3 mg / 100 g) at 5 weeks of age to obtain recipient mice. The recipient mice were orally administered the antibiotic cefoperazone in drinking water to prevent colonization of the intestinal bacteria other than Enterococcus spp.

[0077] <Preparing the donor mouse> The diet of cardiac-specific GPx4-deficient mice (3 weeks old) was switched from a high-vitamin E diet to a normal diet. AIN93G (with vitamin E 0.3 mg / 100 g added) was used as the normal diet. Antibiotics were also administered to the mice. Cefoperazone (CPZ) was used as the antibiotic and was administered orally via drinking water. 0.4 g / L of cefoperazone was added to the drinking water. A control group was also prepared in which no antibiotics were administered and tap water was given.

[0078] Next, feces from mice whose mortality was suppressed by administration of cefoperazone were collected on the 12th day after the diet change, and a transplant solution (hereafter, referred to as "CPZ-administered fecal transplant solution") was prepared. As a control, feces from mice fed tap water were collected on the 10th day after the diet change, and a transplant solution (hereafter, referred to as "water-administered fecal transplant solution") was prepared.

[0079] 《Fecal transplant》 Recipient mice were administered CPZ-administered fecal transplant fluid or water-administered fecal transplant fluid. Feces were collected before and after administration, and colonization of Enterococcus spp. was confirmed by the plate method.

[0080] Fecal transplantation was performed by oral gavage once a week using a tube. For comparison, a control group was not administered fecal transplantation and a group was administered phosphate-buffered saline (PBS).

[0081] Figure 7(b) is a graph showing the change in survival rate of mice in each group. As a result, it was revealed that the decrease in survival rate was suppressed when CPZ-administered fecal transplants were transplanted into cardiac-specific GPx4-deficient pseudo-germ-free mice.

[0082] Figures 8(a)-(f) are graphs showing the results of quantifying Enterococcus bacteria in the feces of mice in each group. Figure 8(a) shows the results for the group that did not receive fecal transplants, Figure 8(b) shows the results for the group that received PBS, Figure 8(c) shows the results for the group that received water-administered fecal transplants, Figure 8(d) shows the results for the mouse (number 1) that died after receiving CPZ-administered fecal transplants, Figure 8(e) shows the results for the mouse (number 2) that died after receiving CPZ-administered fecal transplants, and Figure 8(f) shows the results for the two mice that survived after receiving CPZ-administered fecal transplants.

[0083] As a result, it was found that, of the four mice that received CPZ-administered fecal transplants, no colonization of Enterococcus spp. was observed in the feces of the two mice that died. On the other hand, it was found that Enterococcus spp. colonized the intestines of the two mice that survived. This result indicates that colonization of the intestines by ingestion of Enterococcus spp. suppresses the decline in survival rate.

[0084] [Experimental Example 6] (Isolation of enterobacterial strains from feces) We isolated and identified gut bacteria from the feces of cardiac-specific GPx4-deficient mice treated with cefaperazone.

[0085] First, the diet of cardiac-specific GPx4-deficient mice was switched from high-vitamin E diet to normal diet. AIN93G (with vitamin E 0.3 mg / 100 g) was used as the normal diet. In addition, the mice were administered antibiotics. Cefoperazone was used as the antibiotic and was orally administered via drinking water. 0.4 g / L of cefoperazone was added to the drinking water.

[0086] Next, 6 days after the diet change, feces from the mice were collected. The feces were then suspended in 1 mL of sterile PBS and stirred. The supernatant was then serially diluted 10-fold with sterile PBS and plated on EF agar medium (Enterococcus selective medium, catalog number "#05679", Nissui) at 100 μL each. The medium was then cultured at 37°C under aerobic conditions for 48 hours. Colonies were then picked and subcultured on a master plate. The medium was then cultured at 37°C for 48 hours and stored in a cold room.

[0087] Next, the same colonies as those that had been subcultured were suspended in a PCR reaction solution and amplified using a sense primer (primer name "27F", sequence number 1) and an antisense primer (primer name "1492R", sequence number 2), which are primers that amplify the full length of 16S rDNA. The amplified fragment was then purified, and the 16S rDNA V3 V4 region was sequenced to identify the isolated strains. The identified strains were grown in MRS liquid medium and glycerol stocks were prepared.

[0088] Fig. 9 is a graph showing the ratio of isolated and identified bacteria of the genus Enterococcus. Table 1 shows the types of isolated and identified bacteria of the genus Enterococcus. As a result, as shown in Fig. 9 and Table 1, a number of bacteria of the genus Enterococcus were isolated.

[0089] [Table 1]

[0090] [Experimental Example 7] (Study of transplantation of isolated intestinal bacteria) We transplanted isolated gut bacteria into cardiac-specific GPx4-deficient pseudo-germ-free mice and examined the number of days until death. The maximum observation period was 50 days.

[0091] <Preparation of recipient mice> FIG. 10 is a diagram explaining the experimental schedule. First, cardiac-specific GPx4-deficient pseudo-germ-free mice were prepared. Specifically, cardiac-specific GPx4-deficient mice were orally administered four types of antibiotics in combination for two weeks from the age of three weeks to make them pseudo-germ-free. They were fed a diet containing high amounts of vitamin E. The four antibiotics were neomycin (1 g / L), ampicillin (1 g / L), metronidazole (1 g / L), and vancomycin (0.5 g / L), and were administered in drinking water.

[0092] Next, the diet of the cardiac-specific GPx4-deficient pseudo-germ-free mice was switched from high vitamin E diet to normal diet AIN93G (with vitamin E 0.3 mg / 100 g) at 5 weeks of age to obtain recipient mice. The recipient mice were orally administered the antibiotic cefoperazone in drinking water to prevent colonization of the intestinal bacteria other than Enterococcus spp.

[0093] <Intestinal bacteria transplant> The isolated and identified Enterococcus bacteria were cultured under aerobic conditions using MRS medium until the absorbance at 600 nm reached 0.5. The concentration of the bacteria was then increased to 1 × 10 7 The culture was adjusted to CFU / mL. 1 mL of the culture was then transplanted into recipient mice. Bacterial transplantation was performed by oral gavage once a week using a tube. Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, and Enterococcus rattii were used. A control group was also prepared, to which phosphate-buffered saline (PBS) was administered.

[0094] Figure 11(a) is a graph showing the results of measuring the survival rate of mice administered with Enterococcus faecalis after switching the diet (n=2). As a result, one of the mice administered with Enterococcus faecalis (number 1) survived until the 48th day, and the other (number 2) survived for 50 days (the maximum observation period was set to 50 days).

[0095] FIG. 11(b) is a graph showing the results of quantifying Enterococcus bacteria in the feces of a mouse (number 1) administered with Enterococcus faecalis. FIG. 11(c) is a graph showing the results of quantifying Enterococcus bacteria in the feces of a mouse (number 2) administered with Enterococcus faecalis. As a result, it was revealed that Enterococcus bacteria were established in the intestines of both mice (number 1 and number 2).

[0096] Figure 12(a) is a graph showing the results of measuring the survival rate of mice administered with Enterococcus faecium after switching the diet (n=2). As a result, both mice administered with Enterococcus faecium survived until the 36th day.

[0097] Figure 12(b) is a graph showing the results of quantifying Enterococcus spp. in the feces of a mouse (number 1) administered with Enterococcus faecium. Figure 12(c) is a graph showing the results of quantifying Enterococcus spp. in the feces of a mouse (number 2) administered with Enterococcus faecium. As a result, in both mice (number 1 and number 2), the colonization rate of Enterococcus spp. in the intestines tended to be low up to 21 days after the feed was switched.

[0098] Figure 13(a) is a graph showing the results of measuring the survival rate of mice administered with Enterococcus hirae after switching their diet (n=2). As a result, one of the mice administered with Enterococcus hirae (number 1) survived until the 32nd day, and the other (number 2) survived for 50 days (the maximum observation period was set at 50 days).

[0099] Figure 13(b) is a graph showing the results of quantifying Enterococcus spp. in the feces of a mouse (number 1) administered with Enterococcus hirae. Figure 13(c) is a graph showing the results of quantifying Enterococcus spp. in the feces of a mouse (number 2) administered with Enterococcus hirae. As a result, it was observed that the number 1 mouse (survived until day 32) tended to have significantly lower colonization of Enterococcus spp. in the intestine compared to the mouse that survived for 50 days.

[0100] Figure 14(a) is a graph showing the results of measuring the survival rate of mice administered with Enterococcus ratti after switching the diet (n=2). As a result, one of the mice administered with Enterococcus ratti (number 1) survived until the 35th day, and the other (number 2) survived for 50 days (the maximum observation period was set to 50 days).

[0101] Figure 14(b) is a graph showing the results of quantifying Enterococcus spp. in the feces of a mouse (number 1) administered with Enterococcus ratti. Figure 14(c) is a graph showing the results of quantifying Enterococcus spp. in the feces of a mouse (number 2) administered with Enterococcus ratti. As a result, it was observed that the number 1 mouse (survived until the 35th day) tended to have a significantly lower colonization of Enterococcus spp. in the intestine compared to the mouse that survived for 50 days.

[0102] These results indicate that Enterococcus has an inhibitory effect on lipid oxidation-dependent heart failure.

[0103] [Experimental Example 8] (Study of oxidized phospholipids in the heart) Antibiotics were administered to cardiac-specific GPx4-deficient mice, and the amount of oxidized phospholipids in the heart was examined. Figure 15(a) is a diagram explaining the experimental schedule.

[0104] First, the diet of cardiac-specific GPx4-deficient mice (3 weeks old) was switched from a high-vitamin E diet to a normal diet (CE-2). The mice were also administered antibiotics. The antibiotic cefoperazone (CPZ) was orally administered via drinking water. 0.4 g / L of cefoperazone was added to the drinking water. A control group was also prepared in which no antibiotics were administered and tap water was given.

[0105] On the 12th day after the diet change, the mice in each group were euthanized and their hearts were collected. The collected hearts were then homogenized in methanol, and lipids, including oxidized lipids, were extracted using a standard method (Bligh & Dyer method).

[0106] Next, the molecular species of phosphatidylcholine (PC), the major lipid in the heart, and its peroxidized form, phosphatidylcholine hydroperoxide (PC-OOH), were quantitatively analyzed using LC-ESI-MS / MS.

[0107] Fig. 15(b) is a graph showing the quantitative analysis results of phosphatidylcholine hydroperoxide (PC-OOH), and Fig. 15(c) is a graph showing the quantitative analysis results of phosphatidylcholine (PC).

[0108] As a result, in the control group of mice, accumulation of PC-OOH was observed in cardiomyocytes just before death due to heart failure caused by vitamin E deficiency. In contrast, it was revealed that accumulation of PC-OOH was significantly suppressed in the cefoperazone-treated group of mice. Since cefoperazone itself does not have antioxidant properties, this result indicates that the increased number of Enterococcus bacteria due to cefoperazone treatment suppresses lipid oxidation in the heart.

[0109] [Experimental Example 9] (Requirement of small amounts of vitamin E in diet to prevent lethality caused by cefoperazone administration) We switched the diet of cardiac-specific GPx4-deficient mice from high-vitamin E diet to vitamin E-deficient diet (AIN93G (vitamin E 0 mg)), administered antibiotics, and examined the number of days until death. At this time, we added a small amount of vitamin E (d-α-tocopherol) to the vitamin E-deficient diet (AIN93G (vitamin E 0 mg)) and examined the effect.

[0110] FIG. 16(a) is a diagram explaining the experimental schedule. The diet of cardiac-specific GPx4-deficient mice (3 weeks old) was switched from a high vitamin E diet to a vitamin E-deficient diet (AIN93G (vitamin E 0 mg)). In addition, groups were prepared in which vitamin E (d-α-tocopherol) was added to the vitamin E-deficient diet (AIN93G (vitamin E 0 mg)) at 0.15 mg / 100 g, 0.2 mg / 100 g, and 0.3 mg / 100 g). Antibiotics were also administered to the mice. The antibiotic cefoperazone (CPZ) was orally administered via drinking water. 0.4 g / L cefoperazone was added to the drinking water. A control group was also prepared in which no antibiotics were administered and tap water was given.

[0111] Figure 16(b) is a graph showing the results of measuring the number of days until death (survival days) for mice in each group after switching the feed. In Figure 16(b), "CPZ" shows the results for the group that received cefoperazone in drinking water, and "water" shows the results for the group that did not receive cefoperazone.

[0112] As a result, it was found that oral administration of antibiotics did not extend survival rates when the diet did not contain any vitamin E. When vitamin E was added to a vitamin E-deficient diet (AIN93G (vitamin E 0 mg)) at a level that did not suppress lethality when administered in drinking water, cefoperazone-induced lethality was suppressed in a vitamin E concentration-dependent manner, and in AIN93G (vitamin E 0.3 mg / 100 g added), lethality was suppressed up to 90 days after the diet was switched. These results indicate that vitamin E is necessary for suppressing heart failure caused by Enterococcus spp., which increased with cefoperazone administration.

[0113] Table 2 below summarizes the amount of vitamin E required by type of food in suppressing heart failure caused by Enterococcus bacteria, based on the results of Experimental Examples 1 to 9. In Table 2, "-" indicates that Enterococcus bacteria were not administered, and cefoperazone (CPZ) was not administered in drinking water. Furthermore, "+" indicates that Enterococcus bacteria were administered or cefoperazone (CPZ) was administered in drinking water. Furthermore, "x" indicates that heart failure could not be suppressed, and "o" indicates that heart failure was suppressed.

[0114] As a result, in the CE-2 diet, the amount of vitamin E contained in the feed (7 mg / 100 g) did not suppress heart failure. In contrast, oral administration of Enterococcus spp. or administration of cefoperazone (CPZ) in drinking water suppressed heart failure at a vitamin E content of 7 mg / 100 g. In addition, in the CE-2 diet, which contains 12 mg / 100 g of vitamin E, heart failure was suppressed even without oral administration of Enterococcus spp.

[0115] On the other hand, in the AIN93G diet, the amount of vitamin E contained in the diet (0.3 mg / 100 g) did not suppress heart failure. In contrast, oral administration of Enterococcus spp. or administration of cefoperazone (CPZ) in drinking water suppressed heart failure with a vitamin E amount of 0.3 mg / 100 g. In addition, the AIN93G diet, which contains 1.5 mg / 100 g of vitamin E, suppressed heart failure without oral administration of Enterococcus spp. The reason why the AIN93G diet is effective even though it contains less vitamin E than the CE-2 diet is thought to be because the amount of vitamin E absorbed into the body is 8 to 10 times higher in the AIN93G diet than in the CE-2 diet.

[0116] [Table 2]

[0117] [Experimental Example 10] (Investigation of the effect of bacterial administration on cardiac fatigue in mice) We investigated the effect of administration of Enterococcus spp. on cardiac fatigue in mice.

[0118] <Administering bacteria to mice> 1 × 10 9 The mice were forced to orally administer 150μL of bacteria suspension (Enterococcus hirae:Enterococcus faecium = 1:1) in PBS at CFU / mL using a probe. One group was given the bacteria suspension once a week until the age of 10 weeks, and the other group was given the bacteria suspension every day until the age of 10 weeks. As a control group, the mice were forced to orally administer 150μL of PBS using a probe. PBS was given once a week until the age of 10 weeks.

[0119] <Treadmill running test> Between 8 and 9 weeks of age, the animals were acclimatized to running using a treadmill device (model "MK-680", Muromachi Kikai Co., Ltd.), and the main test was conducted at 10 weeks of age.

[0120] The conditions for running acclimation were as follows: Before starting running, the mouse was placed in the treadmill apparatus for 5 minutes while the apparatus was kept stationary to allow the mouse to become accustomed to the apparatus. Tilt angle: 0 degrees (no tilt) Journey time: 20 minutes Initial speed: 9m / min Acceleration: 3m / min acceleration steps every 4 minutes Frequency: 4 times (2 times / week)

[0121] The conditions for the actual running test (repeated running test) were as follows. Using mice that had been acclimatized to running, the actual running test was conducted the week after the acclimatization. To eliminate the effects of hunger and fullness, the mice were fasted for 3 hours before the start of the test. However, drinking water was allowed ad libitum. Before the start of the run, the mice were placed in the treadmill device for 5 minutes while the device was stationary to allow them to become acclimatized. Tilt angle: 10 degrees Running time: Until exhaustion Initial speed: 9m / min Acceleration: 3m / min acceleration steps every 4 minutes Number of times: 2

[0122] The mice were judged to be exhausted when they could no longer run and could no longer escape from the electric stimulation rod at the back of the running track, and the running time was measured from the start of the run to exhaustion. They were then made to run again after two hours. During this time, they were allowed to drink water ad libitum.

[0123] Quantitative analysis of Enterococcus in feces Enterococcus spp. in feces was quantified, and colonization of Enterococcus spp. in the intestine was examined.

[0124] 0.01 g of feces collected from Balb / c mice at 7 weeks of age and at 10 weeks of age just before the race were suspended in 1 mL of sterile PBS and mixed using a vortex mixer. The supernatant was then serially diluted 10-fold with 1 mL of sterile PBS. The supernatant was then serially diluted 10-fold with EF agar medium (Enterococcus selective medium, catalog number "#05679", Nissui) at 10 1 ~10 6 100 μL of the diluted solution was inoculated and cultured under aerobic conditions at 36° C. for 12 hours. Then, CFU / mL was measured and the Enterococcus spp. was quantified.

[0125] Figure 17 is a graph showing the results of quantifying Enterococcus bacteria in the feces of mice in each group. In Figure 17, "PBS" indicates the results of the group that was not administered bacteria, "entero-1" indicates the results of mice (number 1) that were administered Enterococcus bacteria once a week until the age of 10 weeks, "entero-2" indicates the results of mice (number 2) that were administered Enterococcus bacteria every day until the age of 10 weeks. "7w" indicates the results of feces collected at 7 weeks of age, and "10w" indicates the results of feces collected at 10 weeks of age.

[0126] FIG. 18 is a graph showing the results of the treadmill running test. In FIG. 18, "PBS" indicates the results of the group that was not administered bacteria, "entero-1" indicates the results of the mice (number 1) that were administered Enterococcus bacteria once a week until the age of 10 weeks, and "entero-2" indicates the results of the mice (number 2) that were administered Enterococcus bacteria every day until the age of 10 weeks. "1st" indicates the results of the first actual running test, and "2nd" indicates the results of the second actual running test that was performed after a 2-hour break. "P<0.05" indicates that there is a significant difference at P<0.05.

[0127] As a result, colonization of Enterococcus was observed in the intestines of both mice numbered 1 and 2. Furthermore, a treadmill running test revealed that mice administered Enterococcus had reduced cardiac load during limit running (cardiac fatigue caused by lipid oxidation in the heart).

[0128] The group receiving Enterococcus spp. once a week showed similar results to those receiving Enterococcus spp. daily, indicating that once a week is sufficient and as effective as daily administration. [Industrial Applicability]

[0129] The present invention can provide a technique for preventing or treating diseases associated with phospholipid hydroperoxide-dependent cell death.

Claims

1. Contains Enterococcus bacteria and vitamin E or a derivative thereof as active ingredients, The Enterococcus bacterium is Enterococcus faecium, Enterococcus hirae, Enterococcus ratti, or Enterococcus faecalis; The vitamin E derivative is trolox (2,5,7,8-tetramethyl-6-hydroxychroman-2-carboxylic acid) or troglitazone (5-[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]-phenyl]methyl]-2,4-thiazolidinedione); A pharmaceutical composition for preventing or treating heart failure or cardiac fatigue.

2. Contains Enterococcus bacteria and vitamin E or a derivative thereof as active ingredients, The Enterococcus bacterium is Enterococcus faecium, Enterococcus hirae, Enterococcus ratti, or Enterococcus faecalis; The vitamin E derivative is trolox (2,5,7,8-tetramethyl-6-hydroxychroman-2-carboxylic acid) or troglitazone (5-[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]-phenyl]methyl]-2,4-thiazolidinedione); A food composition for preventing or treating heart failure or cardiac fatigue.

3. A method comprising the step of administering Enterococcus bacteria and vitamin E or a derivative thereof to a non-human animal, The Enterococcus bacterium is Enterococcus faecium, Enterococcus hirae, Enterococcus ratti, or Enterococcus faecalis; The vitamin E derivative is trolox (2,5,7,8-tetramethyl-6-hydroxychroman-2-carboxylic acid) or troglitazone (5-[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]-phenyl]methyl]-2,4-thiazolidinedione); A method for preventing or treating heart failure or cardiac fatigue.

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