JP7681851B2 - Pharmaceutical composition for preventing or treating diseases associated with phospholipid hydroperoxide-dependent cell death - Google Patents

Description

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.

Sudden death is defined as death that occurs within 24 hours of onset. 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 behind sudden death due to idiopathic cardiomyopathy has not been elucidated, and currently there are no methods of prevention or treatment.

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

The inventors also 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).

Patent No. 6652761 Patent No. 6253013

The present invention aims to provide a technology for preventing or treating diseases associated with phospholipid hydroperoxide-dependent cell death.

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 a 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 Enterococcus genus bacteria is selected from the group consisting of Enterococcus faecium, Enterococcus hirae, Enterococcus gallinarum, Enterococcus durans, Enterococcus raffinosus, Enterococcus pseudoavium, and Enterococcus hermaniensis. hermanniensis, Enterococcus rivorum, Enterococcus thailandicus, Enterococcus asini, Enterococcus avium, Enterococcus villorum, Enterococcus lactis, Enterococcus ratti or Enterococcus faecalis The pharmaceutical composition according to [1] or [2], wherein the
[4] A food 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.
[5] A method for preventing or treating a disease associated with phospholipid hydroperoxide-dependent cell death, comprising the step of administering to a subject a bacterial cell or culture of Enterococcus, and vitamin E or a derivative thereof (excluding medical procedures performed on humans).

The present invention provides a technology for preventing or treating diseases associated with phospholipid hydroperoxide-dependent cell death.

1A is a diagram illustrating the experimental schedule in Experimental Example 1. FIG. 1B is a graph showing the change in survival rate of mice in each group in Experimental Example 1. 1A is a diagram illustrating the experimental schedule in Experimental Example 2. FIG. 1B is a graph showing the change in survival rate of mice in each group in Experimental Example 2. 1A is a diagram illustrating an experimental schedule in Experimental Example 3. FIG. 1B is a graph showing the types and proportions of bacteria detected in Experimental Example 3. 1A and 1B are diagrams illustrating the experimental schedule in Experimental Example 4. FIG. 1C and 1D are graphs showing the change in survival rate of mice in each group in Experimental Example 4. 1 is a graph showing the results of analyzing the intestinal bacterial flora of each group of mice in Experimental Example 4. 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. 1A is a diagram illustrating the experimental schedule in Experimental Example 5. FIG. 1B is a graph showing the change in survival rate of mice in each group in Experimental Example 5. 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. 1 is a graph showing the percentage of bacteria of the genus Enterococcus isolated and identified in Experimental Example 6. FIG. 13 is a diagram illustrating an experimental schedule in Experimental Example 7. 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 feces of mice (number 1) administered with Enterococcus faecalis in Experimental Example 7. Graph (b) shows the results of quantifying Enterococcus bacteria in feces of mice (number 2) administered with Enterococcus faecalis in Experimental Example 7. 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 feces of a mouse (number 1) administered with Enterococcus faecium in Experimental Example 7. Graph (b) shows the results of quantifying Enterococcus bacteria in feces of a mouse (number 2) administered with Enterococcus faecium in Experimental Example 7. 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 feces of mice (number 1) administered with Enterococcus hirae in Experimental Example 7. Graph (b) shows the results of quantifying Enterococcus bacteria in feces of mice (number 2) administered with Enterococcus hirae in Experimental Example 7. Graph (a) shows the results of measuring the survival rate after switching feed for mice administered with Enterococcus ratti in Experimental Example 7. Graph (b) shows the results of quantifying Enterococcus bacteria in feces of mice (number 1) administered with Enterococcus ratti in Experimental Example 7. Graph (c) shows the results of quantifying Enterococcus bacteria in feces of mice (number 2) administered with Enterococcus ratti in Experimental Example 7. 1A is a diagram illustrating an experimental schedule in Experimental Example 8; FIG. 1B is a graph showing the quantitative results of phosphatidylcholine hydroperoxide (PC-OOH) in Experimental Example 8; and FIG. 1C is a graph showing the quantitative results of phosphatidylcholine (PC) in Experimental Example 8. 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 each group of mice in Experimental Example 9 after switching the feed. 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. 13 is a graph showing the results of a treadmill running test in Experimental Example 10.

[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.

As described later in the Examples, the inventors have demonstrated that oral administration of the preventive or therapeutic agent of this embodiment to a mouse model in which sudden death due to heart failure occurs can suppress sudden death due to heart failure and can suppress cardiac load during limit running (cardiac fatigue caused by lipid oxidation in the heart).

Therefore, diseases associated with phospholipid hydroperoxide-dependent cell death include heart failure and cardiac fatigue caused by lipid oxidation in the heart. Heart failure in particular includes heart failure caused by lipid oxidation in the heart. More specifically, examples of heart failure include marathon-related heart failure and heart failure in athletes such as Olympic athletes and soccer players.

In this specification, a disease includes a decrease in organ function. That is, a 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 preferable to improve.

More specifically, diseases associated with phospholipid hydroperoxide-dependent cell death include impaired cardiac function associated with phospholipid hydroperoxide-dependent cell death. Impaired cardiac function includes, for example, impaired motor function and delayed recovery from fatigue. In this specification, "treatment" includes improvement of symptoms.

In the preventive or therapeutic agent of this embodiment, the Enterococcus bacteria include Enterococcus faecium, Enterococcus hirae, Enterococcus gallinarum, Enterococcus durans, Enterococcus raffinosus, Enterococcus pseudoavium, Enterococcus hermaniensis, ... hermanniensis), Enterococcus rivorum, Enterococcus thailandicus, Enterococcus asini, Enterococcus avium, Enterococcus villorum, Enterococcus lactis, Enterococcus ratti, Enterococcus feacalis, and the like. Among these, Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, and Enterococcus ratti are preferred.

The Enterococcus bacteria may be dead or live. When live bacteria are used, some dead bacteria may be mixed in. Enterococcus bacteria may be disrupted, extracted, or otherwise used in a state where the shape of the bacteria remains.

The culture of Enterococcus is a composition containing a substance produced by Enterococcus. The culture of Enterococcus may be a culture supernatant of Enterococcus, a crude product of the culture supernatant, etc.

The preventive or therapeutic pharmaceutical composition of this 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 x ¹⁰⁷ CFU (colony forming units)/kg body weight of Enterococcus bacteria per administration. The number of administrations can be appropriately adjusted depending on the symptoms, for example, about once every one to several days. An example of one to several days is 1 to 10 days.

When administering killed Enterococcus bacteria or a culture of Enterococcus bacteria, for example, an amount of killed bacteria or culture corresponding to the amount of live bacteria may be administered once or multiple times.

The Enterococcus bacteria used in this embodiment can be prepared by a known method. The Enterococcus bacteria can 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.

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-dried to obtain a powder of the bacteria.

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 dosage forms for oral use include tablets and capsules.

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 leavening agents such as alginic acid.

The composition may contain additives. Examples of additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavorings such as peppermint and saffron oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; antioxidants; and preservatives.

The composition can be formulated by appropriately combining Enterococcus bacteria or cultures, carriers and additives, and mixing them in a unit dosage form required for generally accepted pharmaceutical practice.

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 demonstrated that vitamin E or a derivative thereof is necessary for suppressing heart failure caused by administration of Enterococcus bacteria.

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).

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

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. Alternatively, the Enterococcus bacteria or a culture thereof and vitamin E or a derivative thereof may be enclosed in separate containers to constitute a kit for administration in combination.

[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 described above. The terms "disease" and "treatment" are the same as described above. That is, the term "disease" includes a decline in organ function, and includes conditions requiring treatment with medicines or the like, and conditions not requiring treatment with medicines or the like but in which improvement is preferable. The term "treatment" includes improvement of symptoms. The food composition of this embodiment can be said to be useful for inhibiting lipid oxidation in the heart, inhibiting cardiac fatigue, enhancing cardiac function, and the like.

The food composition of this embodiment may be in the form of, for example, a supplement, yogurt, a drink, a gel food, any cooked food, or the like. Examples of the shape of the supplement include capsules.

The Enterococcus bacteria and culture are the same as those described above. The food composition of this embodiment is preferably used to ingest, for example, 3×10 ⁷ CFU/kg body weight or more of live Enterococcus bacteria per day, or a corresponding amount of killed bacteria or culture. The timing of ingestion may be before, after, or between meals. Note that "3×10 ⁷ CFU/kg body weight or more per day" 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.

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" 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.

The food composition of this embodiment may be a mixture of Enterococcus bacteria or a culture thereof and vitamin E or a derivative thereof. Alternatively, the Enterococcus bacteria or a culture thereof and vitamin E or a derivative thereof may be enclosed in separate containers to constitute a kit for administration in combination.

The food composition of this embodiment may be a food with functional claims. "Food with functional claims" refers to food that has been notified to the Consumer Affairs Agency as a food that displays functionality on the product packaging based on scientific evidence. Examples of such claims include, but are not limited to, "functionality to relieve cardiac fatigue." In addition, even if a food does not have a functional claim, it is conceivable that the food may be manufactured and sold by touting these functionalities in leaflets, emails, or verbally.

The food composition of this embodiment may be a special purpose food. Special purpose food means a food that, with national permission, is labeled with a special purpose, such as being suitable for the development and maintenance/recovery of the health of infants, young children, pregnant women, sick people, etc.

The food composition of this embodiment may be a food for specified health uses, which is a type of food for special dietary uses. A food for specified health uses is a food that has been recognized based on scientific evidence to be useful in maintaining and promoting health, and is permitted to display such 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.

[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 for humans), comprising the step of administering to a subject a bacterial cell or culture of Enterococcus spp., and vitamin E or a derivative thereof.

In the prevention or treatment method of this embodiment, Enterococcus bacteria or a culture and vitamin E or a derivative thereof may be administered simultaneously. Alternatively, Enterococcus bacteria or a culture and vitamin E or a derivative thereof may be administered separately.

In the preventive or therapeutic 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 preventive or therapeutic method of this embodiment excludes medical procedures for humans and does not include procedures performed by a doctor. Specific examples of the preventive or therapeutic 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.

The Enterococcus bacteria, culture, vitamin E and its derivatives 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.

Vitamin E or a derivative thereof may be administered simultaneously with Enterococcus cells or cultures, or may be administered separately from Enterococcus cells or cultures.

[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.

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 Enterococcus spp.

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

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

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.

[Mouse model]
The inventors previously generated GPx4 gene knockout mice (GPx4 ^-/- ) by standard homologous recombination using ES cells. GPx4 ^-/- mice were embryonic lethal at day 7.5 of development. Therefore, mouse GPx4 genomic gene flanked by loxP sequences (loxP-GPx4) was transfected (Tg) into mice to generate Tg(loxP-GPx4) ^+/- :GPx4 ^+/+ mice.

Subsequently, Tg(loxP-GPx4) ^+/- :GPx4 ^+/+ mice were crossed with GPx4 ^+/- mice to produce Tg(loxP-GPx4) ^+/- :GPx4 ^+/- mice, and these mice were crossed with each other to produce Tg(loxP-GPx4) ^+/+ :GPx4 ^-/- mice in which the endogenous GPx4 genomic gene was knocked out (KO) and embryonic lethality was rescued by the introduced loxP-GPx4Tg gene.

Furthermore, GPx4 ^+/- mice were repeatedly crossed with mice (Cre ^+/+ ) carrying the Cre gene downstream of the muscle creatine kinase promoter, which is a cardiac-specific promoter, to obtain Cre ^+/+ GPx4 ^+/- mice.

Next, Cre ^+/+ GPx4 ^+/- mice were crossed with the above-mentioned Tg(loxP-GPx4) ^+/+ :GPx4 ^-/- mice to obtain Cre ^+/- :Tg(loxP-GPx4) ^+/- :GPx4 ^-/- mice. These mice are cardiac-specifically deficient in GPx4. Hereinafter, Cre ^+/- :Tg(loxP-GPx4) ^+/- :GPx4 ^-/- mice are referred to as "cardiac-specific GPx4-deficient mice."

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

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

The inventors have also 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.

[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. FIG. 1(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-added feed to normal feed (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). As the administration method, oral administration via drinking water and intraperitoneal administration were examined. 0.4 g/L of cefoperazone was added to the drinking water. In addition, in the 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 one day. A control group was also prepared in which no antibiotics were administered and tap water was given.

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, it was revealed that intraperitoneal administration of antibiotics to cardiac-specific GPx4-deficient mice did not suppress the decrease in survival rate.

[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 condition in an isolator to generate cardiac-specific GPx4-deficient germ-free mice that had no intestinal bacteria. FIG. 2(a) is a diagram explaining the experimental schedule. First, the feed of cardiac-specific GPx4-deficient germ-free mice (6 weeks old) was switched from a high vitamin E-added feed to a normal feed. As the normal feed, 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 administration of antibiotics.

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 death was induced by a decrease in vitamin E even when antibiotics were not administered, 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 suppressing lipid oxidation-dependent heart failure.

[Experimental Example 3]
(Analysis of mouse intestinal microbiota using next-generation sequencing)
The intestinal flora of mice administered antibiotics was analyzed. FIG. 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 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 tap water was given without antibiotics.

Feces were collected 0, 6, and 12 days after the diet change. After 12 days, the mice were dissected and the cecum was collected. Next, sequencing was performed using a next-generation sequencer to analyze the bacterial flora in the collected feces and cecum.

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

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

As shown in Figure 4(b), in order to simulate the elimination of intestinal bacteria in cardiac-specific GPx4-deficient mice, four types of antibiotics were orally administered for two weeks from the age of three weeks to make the mice pseudo-sterile. The mice were fed a diet containing high levels of vitamin E. The four types of 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-sterile were also prepared, as shown in Figure 4(a).

Next, at the age of 5 weeks, the high vitamin E diet was switched to normal diet AIN93G (containing 0.3 mg/100 g vitamin E). Additionally, the mice were orally administered cefoperazone (CPZ) 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.

Furthermore, after switching the diet to AIN93G (with added 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.

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.

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 (vitamin E 0.3 mg/100 g) at 5 weeks of age. In contrast, administration of cefoperazone in drinking water prevented mortality. On the other hand, as shown in FIG. 4(d), in pseudo-germ-free mice administered four types of antibiotics, administration of cefoperazone in drinking water did not prevent mortality when they were switched from a high-vitamin E diet to AIN93G (vitamin E 0.3 mg/100 g). This result was similar to that of cardiac-specific GPx4-deficient germ-free mice in Experimental Example 2.

Figure 5 is a graph showing the results of analyzing the intestinal flora 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.

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

Figure 6 is a graph showing the results of quantifying the number of Enterococcus bacteria in the feces of mice in each group using the plate method on the sixth day after the diet was switched to AIN93G (with vitamin E 0.3 mg/100 g added).

In Figure 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.

As a result, it was found that Enterococcus spp. increased in the group of non-pseudo-sterilized cardiac-specific GPx4-deficient mice that were administered cefoperazone. In contrast, no Enterococcus spp. were detected in the pseudo-sterilized cardiac-specific GPx4-deficient mice, even in the group that was administered cefoperazone. These results suggest that Enterococcus spp. is involved in the suppression of heart failure by administration of cefoperazone in drinking water.

[Experimental Example 5]
(Consideration of fecal transplants)
We performed fecal transplants of feces containing 99% Enterococcus bacteria into pseudo-germ-free mice lacking cardiac-specific GPx4, and examined the survival rate.

<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.

Next, the diet of the cardiac-specific GPx4-deficient pseudo-germ-free mice was switched from high vitamin E diet to normal AIN93G diet (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.

<Preparing the donor mouse>
The diet of cardiac-specific GPx4-deficient mice (3 weeks old) 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. Antibiotics were also administered to the mice. Cefoperazone (CPZ) was used as the antibiotic and 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 tap water was given without antibiotics.

Next, feces from mice whose mortality was suppressed by administration of cefoperazone were collected 12 days 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 that were given tap water were collected 10 days after the diet change, and a transplant solution (hereafter referred to as "water-administered fecal transplant solution") was prepared.

《Fecal transplant》
The recipient mice were administered with either CPZ-administered fecal transplant solution or water-administered fecal transplant solution. Feces were collected before and after administration, and colonization of Enterococcus spp. was confirmed by the plate method.

Fecal transplantation was performed by forced oral administration using a probe once a week. For comparison, a group that did not undergo fecal transplantation (Control) and a group that received phosphate-buffered saline (PBS) were also prepared.

Figure 7(b) is a graph showing the change in survival rate of mice in each group. As a result, it was revealed that transplantation of CPZ-administered fecal transplant fluid into cardiac-specific GPx4-deficient pseudo-germ-free mice suppressed the decline in survival rate.

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 undergo fecal transplantation, 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 transplant solution, Figure 8(d) shows the results for the mouse (number 1) that died after receiving CPZ-administered fecal transplant solution, Figure 8(e) shows the results for the mouse (number 2) that died after receiving CPZ-administered fecal transplant solution, and Figure 8(f) shows the results for the two mice that survived after receiving CPZ-administered fecal transplant solution.

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

[Experimental Example 6]
(Isolation of enterobacterial strains from feces)
We isolated and identified enterobacteria from the feces of cardiac-specific GPx4-deficient mice administered cefaperazone.

First, the diet of the cardiac-specific GPx4-deficient mice was switched from high-vitamin E diet to normal diet. AIN93G (with 0.3 mg/100 g vitamin E) was used as the normal diet. Antibiotics were also administered to the mice. 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.

Six days after the diet change, mouse feces 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.

Next, the same colonies as those that had been passaged 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.

Figure 9 is a graph showing the percentage of bacteria of the genus Enterococcus that were isolated and identified. Table 1 shows the types of bacteria of the genus Enterococcus that were isolated and identified. As a result, as shown in Figure 9 and Table 1, multiple bacteria of the genus Enterococcus were isolated.

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

<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. As feed, the mice were given a feed containing high amounts of vitamin E. The four types of 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.

Next, the diet of the cardiac-specific GPx4-deficient pseudo-germ-free mice was switched from high vitamin E diet to normal AIN93G diet (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.

<Intestinal bacteria transplant>
The isolated and identified Enterococcus bacteria were cultured under aerobic conditions using MRS medium until the absorbance at a wavelength of 600 nm reached 0.5. The concentration of the bacteria was then adjusted to 1×10 ⁷ CFU/mL. Then, 1 mL of the culture solution was transplanted into the recipient mice. The bacteria were transplanted once a week by forced oral administration using a sonde. Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, and Enterococcus rattii were used as Enterococcus bacteria. A control group (Control) administered with phosphate buffered saline (PBS) was also prepared.

Figure 11 (a) is a graph showing the results of measuring the survival rate of mice administered with Enterococcus faecalis after switching feed (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 at 50 days).

Figure 11(b) is a graph showing the results of quantifying Enterococcus bacteria in the feces of a mouse (number 1) administered Enterococcus faecalis. Also, Figure 11(c) is a graph showing the results of quantifying Enterococcus bacteria in the feces of a mouse (number 2) administered Enterococcus faecalis. As a result, it was revealed that Enterococcus bacteria had colonized in the intestines of both mice (number 1 and number 2).

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

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

Figure 13 (a) is a graph showing the results of measuring the survival rate of mice administered with Enterococcus hirae after switching feed (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).

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

Figure 14 (a) is a graph showing the results of measuring the survival rate of mice administered with Enterococcus ratti after switching feed (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 at 50 days).

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

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

[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. Fig. 15(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). 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.

On the 12th day after the diet change, mice from 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).

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

Figure 15(b) is a graph showing the quantitative results of phosphatidylcholine hydroperoxide (PC-OOH). Also, Figure 15(c) is a graph showing the quantitative results of phosphatidylcholine (PC).

As a result, in the control group of mice, accumulation of PC-OOH was observed in cardiac myocytes immediately before death from 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 numbers of Enterococcus bacteria due to cefoperazone administration suppress lipid oxidation in the heart.

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

Figure 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)). Groups were also 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 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.

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 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.

As a result, it was found that oral administration of antibiotics did not extend survival rates when the feed did not contain any vitamin E. Furthermore, when vitamin E was added to a vitamin E-deficient diet (AIN93G (0 mg vitamin E)) in an amount 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 (with 0.3 mg/100 g vitamin E added), lethality was suppressed up to 90 days after the feed was switched. These results indicate that vitamin E is necessary to suppress heart failure caused by Enterococcus bacteria, which increased with cefoperazone administration.

Table 2 below summarizes the amount of vitamin E required by type of food to suppress 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 the drinking water. Furthermore, "+" indicates that Enterococcus bacteria were administered or cefoperazone (CPZ) was administered in the drinking water. Furthermore, "x" indicates that heart failure could not be suppressed, and "o" indicates that heart failure was suppressed.

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 or administration of cefoperazone (CPZ) in drinking water was able to suppress heart failure at a vitamin E level of 7 mg/100 g. Furthermore, in the CE-2 diet, which contained 12 mg/100 g of vitamin E, heart failure was suppressed even without oral administration of Enterococcus.

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

[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.

<Administering bacteria to mice>
150 μL of bacterial solution (Enterococcus hirae:Enterococcus faecium = 1:1) suspended in PBS to 1×10 ⁹ CFU/mL was forcibly administered to 4-week-old Balb/c male mice using a probe. A group was administered the bacterial solution once a week until the age of 10 weeks, and a group was administered the solution every day until the age of 10 weeks. As a control, a group was administered 150 μL of PBS forcibly using a probe. PBS was given once a week until the age of 10 weeks.

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

The conditions for running habituation 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 it to become accustomed to the environment.
Tilt angle: 0 degrees (no tilt)
Running time: 20 minutes Initial speed: 9 m/min Acceleration: 3 m/min acceleration step every 4 minutes Number of times: 4 times (2 times/week)

The conditions for the main running test (repeated running test) were as follows. Using mice that had been acclimatized to running, the main 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.
Incline angle: 10 degrees Run time: until exhaustion Initial speed: 9 m/min Acceleration: 3 m/min acceleration step every 4 minutes Number of times: 2

When the mouse could no longer endure the run and could no longer escape from the electric stimulation rod at the back of the running track, it was judged to have become exhausted, and the running time was measured from the start of the run to when it became exhausted. The mouse was then made to run again after two hours. During this time, the mouse was allowed to drink water ad libitum.

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

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

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 for the group that was not administered bacteria, "entero-1" indicates the results for mice (number 1) that were administered Enterococcus bacteria once a week until the age of 10 weeks, and "entero-2" indicates the results for mice (number 2) that were administered Enterococcus bacteria daily until the age of 10 weeks. Additionally, "7w" indicates the results for feces collected at 7 weeks of age, and "10w" indicates the results for feces collected at 10 weeks of age.

Figure 18 is a graph showing the results of the treadmill running test. In Figure 18, "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, and "entero-2" indicates the results of mice (number 2) that were administered Enterococcus bacteria daily 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 conducted after a 2-hour break. "P<0.05" indicates that a significant difference exists at P<0.05.

As a result, colonization of Enterococcus was observed in the intestines of both mice number 1 and number 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).

The group receiving Enterococcus spp. once a week showed similar results to those receiving Enterococcus spp. daily. These results indicate that administering Enterococcus spp. once a week is sufficient and has the same effect as daily administration.

The present invention provides a technology for preventing or treating diseases associated with phospholipid hydroperoxide-dependent cell death.

Claims (3)

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 . 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 . The method includes 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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