JP2013190420A - Screening method for substance having curative properties against bacterial infection-induced ovarian dysfunction and therapeutic agent for bacterial infection-induced ovarian dysfunction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an evaluation system for bacterial infection-induced ovarian dysfunction and substances having curative properties against the same.SOLUTION: A screening method for substances having curative properties against bacterial infection-induced ovarian dysfunction includes the steps of subjecting an animal to an ovarian hyperstimulation treatment, dosing the animal with lipo-polysaccaride and/or lipoteichoic acid, dosing the animal with a test substance, and measuring the number of ovulation after these steps. In this screening method, an animal model having bacterial infection-induced ovarian dysfunction, which is prepared by the ovarian hyperstimulation treatment and dosing of lipo-polysaccaride and/or lipoteichoic acid, is used to inspect whether the number of ovulation is varied due to dosing of the test substance, so that curative properties against bacterial infection-induced ovarian dysfunction of the test substance can be easily evaluated.

Description

  The present invention relates to a method for screening a substance having an action of improving ovarian dysfunction caused by bacterial infection.

  Breeding of livestock and poultry caused by bacterial infections causes a decrease in meat production due to a decrease in the number of pups in pigs and cattle, and a decrease in milk production due to the inability to milk, and a decrease in the number of eggs in chickens. Therefore, it is a big problem in production efficiency. Bacterial infections that cause reproductive disorders include endometriosis and mastitis. Ovarian dysfunction due to these bacterial infections is presumed to be the cause of reproductive disorders.

  In order to prevent ovarian dysfunction caused by bacterial infection, antibiotics are administered to livestock. However, shipment of meat and raw milk is restricted during the antibiotic administration period or for a certain period after administration. Therefore, there is a need for a method for improving ovarian dysfunction using natural product-derived components regardless of antibiotics.

  Also in humans, pelvic inflammatory disease caused by bacteria transferred from the vagina during labor or sexual activity is known as the cause of infertility associated with ovarian dysfunction. Repeated recurrence of pelvic inflammatory disease is said to increase the risk of infertility.

  Improving ovarian dysfunction due to bacterial infection and promoting normal ovulation is extremely important for improving production efficiency in industrial animals and treating infertility in animals and humans.

  In relation to the present invention, Patent Document 1 discloses an ovarian function improving agent containing lactoferrin as an active ingredient. Patent Document 2 discloses an infertility treatment agent containing one or more selected from oleuropein, oleuropein derivatives or hydroxytyrosol as an active ingredient. The ovarian function improving agent disclosed in Patent Document 1 is for ovarian dysfunction caused by anticancer chemotherapy, and the infertility therapeutic agent disclosed in Patent Document 2 is used for ovarian dysfunction caused by heat stress or oxidative stress. Applicable.

International Publication No. 2009/101789 JP 2009-191012 A

  In order to improve ovarian dysfunction due to bacterial infection, it is necessary to develop an evaluation system that can appropriately reproduce the pathological condition and can be used for screening substances having an improving action.

  Therefore, the main object of the present invention is to provide a system for evaluating bacterial infection-induced ovarian dysfunction and substances having an ameliorating action thereof.

In order to solve the above problems, the present invention includes a procedure for superovulation treatment to an animal, a procedure for administering lipopolysaccharide and / or lipoteichoic acid to the animal, a procedure for administering a test substance to the animal, And a method for measuring the number of ovulations of the animal after the above procedure.
In this screening method, a bacterial infection-induced ovarian dysfunction animal model produced by superovulation treatment and administration of lipopolysaccharide and / or lipoteichoic acid is used to examine changes in the number of ovulations with or without test substance administration. Thus, the effect of the test substance on improving ovarian dysfunction induced by bacterial infection can be easily evaluated.

The present invention also provides a method for producing an animal model of bacterial infection-induced ovarian dysfunction, comprising a procedure for superovulation treatment to an animal and a procedure for administering lipopolysaccharide and / or lipoteichoic acid to the animal. To do.
Furthermore, the present invention provides a model animal of bacterial infection-induced ovarian dysfunction produced by superovulation treatment and administration of lipopolysaccharide and / or lipoteichoic acid.

  The present invention includes a procedure for superovulation treatment to an animal, a procedure for administering lipopolysaccharide and / or lipoteichoic acid to the animal, a procedure for administering a test substance to the animal, and after these procedures, And an agent for improving bacterial infection-induced ovarian dysfunction obtained by a method for screening a substance having an effect of improving bacterial infection-induced ovarian dysfunction. The agent for improving bacterial infection-induced ovarian dysfunction may contain hydroxytyrosol, may contain anthocyanins, or may contain a Hascup extract.

  Furthermore, the present invention includes a procedure for superovulation treatment to an animal, a procedure for administering lipopolysaccharide and / or lipoteichoic acid to the animal, a procedure for administering a test substance to the animal, and after these procedures. A method of measuring the number of ovulations of the animal, and a method of administering a bacterial infection-induced ovarian dysfunction improving agent obtained by a method for screening a substance having an effect of improving bacterial infection-induced ovarian dysfunction. Also provided is a method for ameliorating ovarian dysfunction in livestock or poultry caused by.

  The present invention provides a system for evaluating a substance having an ovarian dysfunction induced by bacterial infection and an ameliorating action thereof.

It is a figure explaining the experiment schedule for creating the animal model of bacterial infection induction ovarian dysfunction (Experimental example 1). It is a drawing substitute graph which shows the result of having measured the number of ovulations of the mouse | mouth which performed administration of a lipopolysaccharide or peptidoglycan and superovulation treatment (Experimental example 1). It is a figure explaining the schedule of administration of a lipopolysaccharide and a superovulation treatment (Experimental example 1). It is a drawing substitute graph which shows the result of having measured the number of ovulations of the mouse | mouth which performed administration of a lipopolysaccharide and superovulation treatment (Experimental example 1). It is a drawing substitute photograph which shows the result of having confirmed the expression of the LPS receptor in the ovary of the mouse | mouth of each day 21-25 days old by RT-PCR (Experimental example 1). It is a drawing substitute graph which shows the result of having measured the number of ovulations of the mouse | mouth which administered lipoteichoic acid and superovulation treatment (Experimental example 2). It is a figure explaining the schedule of administration of a lipopolysaccharide and a superovulation treatment (Experimental example 3). It is a drawing substitute graph which shows the result of having measured the number of ovulations of the mouse | mouth which performed administration of a lipopolysaccharide and superovulation (Experimental example 3). It is a figure explaining the experiment schedule of the screening of the substance which has an improvement effect of bacterial infection induction ovarian dysfunction (Experimental example 4). It is a drawing substitute graph which shows the result of having evaluated the improvement effect of the bacterial infection induction ovarian dysfunction of hydroxytyrosol, oleuropein, and glycyrrhizin (Experimental example 4). It is a figure explaining the experiment schedule of the screening of the substance which has an improvement effect of bacterial infection induction ovarian dysfunction (Experimental example 5). It is a drawing substitute graph which shows the result of having evaluated the improvement effect of the bacterial infection induction ovarian dysfunction of anthocyanin (Experimental example 5). It is a figure explaining the experiment schedule of the screening of the substance which has an improvement effect of bacterial infection induction ovarian dysfunction (Experimental example 6). It is a drawing substitute graph which shows the result of having evaluated the improvement effect of the bacterial infection induction ovarian dysfunction of a Hascup extract (Experimental example 6).

1. Screening method (1) Creation of animal model of bacterial infection-induced ovarian dysfunction The present inventor has developed a “superovulatory treatment / bacterial component-administered animal model”, which is a combination of superovulation treatment and bacterial component administration. It has been found for the first time that it can be an appropriate pathological model of induced ovarian dysfunction (see Experimental Example 1 and Experimental Example 2). In the animal model of superovulation treatment / bacterial component administration, the number of eggs ovulated from the ovary stimulated by the superovulation treatment is reduced by administration of the bacterial component. Since this decrease in the number of ovulations is thought to be caused by ovarian dysfunction due to bacterial constituents, the animal model may reflect the pathology of ovarian dysfunction due to bacterial infection.

  A bacterial infection-induced ovarian dysfunction animal model can be created by combining superovulation treatment according to a standard method and administration of bacterial components. Superovulation treatment is usually performed by administering PMSG (pregnant mare serum gonadotropin) and hCG (human chorionic gonadotropin) to animals. Examples of the component of bacteria include LPS (lipopolysaccharide) which is a component of gram-negative bacteria and is called endotoxin, and LTA (lipotycoic acid) which is a component of gram-positive bacteria. These bacterial components are administered intraperitoneally.

  The model animal may be a mouse, rat, rabbit, pig, cow, monkey or the like, and is not particularly limited as long as it has an ovarian function (except for humans). For example, when a mouse is used as a model animal, superovulation can be induced after another 24 hours by administering PSG to the abdominal cavity and hCG after 48 hours. The dosage of PMSG and hCG in the mouse is 0.1-100 IU (international unit), preferably 1-10 IU. LPS and LTA are administered intraperitoneally before or after the administration of PMSG and / or hCG. The dose of LPS in mice is about 0.01 to 10 mg / kg body weight, preferably about 0.04 to 4 mg / kg body weight. The dose of LTA in the mouse is 0.4 mg / kg body weight or more, preferably 4.1 mg / kg body weight or more, more preferably 20 mg / kg body weight or more. However, the administration timing and dose of PMSG, hCG, LPS, and LTA can be adjusted as appropriate according to the model animal to be used.

  In order to reliably induce ovarian dysfunction, LPS and / or LTA are desirably administered twice or more as shown in Experimental Example 3 described later. When the ovarian dysfunction is reliably induced in the model animal, the accuracy of the evaluation of the screening result is increased when screening a substance having an effect of improving the bacterial infection-induced ovarian dysfunction described later in the model animal.

  For example, when a mouse is used as a model animal, the first administration of LPS and / or LTA is performed simultaneously with the PMSG administration, and the second administration of LPS and / or LTA is conducted 24 hours after the administration of PMSG. However, the number of administrations of PMSG, hCG, LPS, and LTA can be appropriately set according to the administration timing and dose. For example, in mice, ovarian dysfunction can be induced by a single administration of LPS 24 hours after administration of PMSG.

  As a model animal, it is preferable to use an immature mature animal. This is because in sexually mature animals, there is an endogenous ovulation cycle, which affects superovulation treatment. When a mouse is used as a model animal, it is appropriate to use an individual 27 to 28 days old. Further, in screening for a substance having an effect of improving bacterial infection-induced ovarian dysfunction described later, an individual with a smaller age may be used depending on the administration period of the test substance to the mouse.

  Bacterial infection-induced ovarian dysfunction animal model can easily evaluate the degree of ovarian dysfunction due to bacterial constituents using the number of ovulation as an indicator. Useful for method development etc.

  Furthermore, a test substance can be administered to an animal model of bacterial infection-induced ovarian dysfunction, and a substance having an effect of improving bacterial infection-induced ovarian dysfunction can be screened by searching for a substance capable of suppressing the decrease in the number of ovulations. Is possible. Next, this screening method will be described.

(2) Screening method for substances having an effect of improving bacterial infection-induced ovarian dysfunction The screening method according to the present invention is compared to the case where a test substance is administered to a bacterial infection-induced ovarian dysfunction animal model and no test substance is administered. By examining the change in the number of ovulations, the presence or absence of a test substance to improve ovarian dysfunction induced by bacterial infection is determined.

  Specifically, the screening method includes a procedure for superovulation treatment to an animal, a procedure for administering LPS and / or LTA to the animal, a procedure for administering a test substance to the animal, and a procedure for these procedures. And later, measuring the number of ovulations of the animal. Of these, both the superovulation treatment and the administration of LPS and / or LTA can be performed in the same manner as in the production of the animal model of ovarian dysfunction induced by bacterial infection described above.

  The test substance administration procedure is performed by administering the test substance to the animal before or after superovulation treatment and LPS and / or LTA administration. The test substance may be administered by any administration method such as intraperitoneal administration, oral administration, and subcutaneous administration.

  In the procedure for measuring the number of ovulations, the animal is opened and the number of ovulation eggs is counted. In the screening method according to the present invention, when the test substance is administered, the test substance has a bacterial infection when the number of ovulations measured in this procedure is larger than the number of ovulations measured when the test substance is not administered. It can be determined that it has an effect of improving induced ovarian dysfunction. On the other hand, when the test substance is administered, the number of ovulations measured in this procedure is equal to or less than the number of ovulations measured without administration, the test substance improves bacterial infection-induced ovarian dysfunction It is determined that there is no effect.

  The test substance in the screening method according to the present invention may be a natural compound or a synthetic compound, but is particularly preferably a natural product-derived component. If a natural product-derived component having an effect of improving bacterial infection-induced ovarian dysfunction is found, it becomes possible to treat or prevent bacterial infection-induced ovarian dysfunction regardless of antibiotics.

2. Bacterial infection-induced ovarian dysfunction-improving agent (1) Bacterial infection-induced ovarian dysfunction-improving agent The inventor may exhibit an ameliorating effect on ovarian dysfunction caused by heat stress or oxidative stress using the screening method according to the present invention. We investigated the effect of known oleuropein and hydroxytyrosol in improving bacterial infection-induced ovarian dysfunction. Hydroxytyrosol (3,4-dihydroxyphenylethanol) is a component contained in olives and is a kind of polyphenol. Hydroxytyrosol is a degradation product of oleuropein, also contained in olives.

  As a result, the present inventor confirmed the improvement effect only with hydroxytyrosol. As shown in Experimental Example 4, hydroxytyrosol significantly suppressed the decrease in the number of ovulations by LPS administration in a bacterial infection-induced ovarian dysfunction animal model. Accordingly, hydroxytyrosol can be an agent for improving bacterial infection-induced ovarian dysfunction. That is, the effectiveness of the bacterial infection-induced ovarian dysfunction improving agent containing hydroxytyrosol was shown.

  In addition, the inventor examined the improvement effect of bacterial infection-induced ovarian dysfunction on anthocyanins and lotus cups (Lonicera caerulea L.) extracts using the screening method according to the present invention. . Anthocyanins are glycosides in which anthocyanidins are combined with sugars as aglycones, and are water-soluble pigments contained in plants such as fruits. Examples of anthocyanins include delphinidin, cyanidin, malvidin, pelargonidin, peonidin, petunidin and the like.

  As a result, the present inventor confirmed the improvement effect for the anthocyanins and the lotus cup extract. As shown in Experimental Example 5 and Experimental Example 6, the anthocyanins and the Hascup extract significantly suppressed the decrease in the number of ovulations by LPS administration in an animal model of bacterial infection-induced ovarian dysfunction. Therefore, anthocyanins and lotus cup extracts can be agents for improving bacterial infection-induced ovarian dysfunction. That is, the effectiveness of the bacterial infection-induced ovarian dysfunction-improving agent containing anthocyanins and the bacterial infection-induced ovarian dysfunction-improving agent containing the Hascup extract was shown.

  Treat or prevent ovarian dysfunction caused by bacterial infection such as endometritis and mastitis, if hydroxytyrosol, anthocyanin or lotus cup extract is administered to livestock or poultry as an agent to improve ovarian dysfunction induced by bacterial infection It is possible to improve the production efficiency by preventing the number of pups and the number of eggs laid. It is also possible to treat or prevent ovarian dysfunction and infertility caused by pelvic inflammatory disease by applying a bacterial infection-induced ovarian dysfunction improving agent to humans.

(2) Pharmaceutical composition The above-mentioned agent for improving bacterial infection-induced ovarian dysfunction can be applied as a pharmaceutical composition containing this as an active ingredient.

  Since this pharmaceutical composition contains, as an active ingredient, an extract derived from hydroxytyrosol, anthocyanin, which is a natural product-derived component, or a fruit cup, it has a high possibility of being continuously applicable over a long period of time and has no side effects. Less likely.

  The pharmaceutical composition according to the present invention includes, for example, tablets, capsules, elixirs, microcapsules, etc., which are sugar-coated as needed, or water or other pharmaceutically acceptable liquids. It can be used parenterally in the form of an injection such as a sterile solution or suspension. The pharmaceutical composition comprises a generally recognized formulation of the agent for improving bacterial infection-induced ovarian dysfunction according to the present invention together with physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders and the like. Manufactured by blending in the unit dosage form required for practice. Additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth, gum arabic, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Leavening agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavorings such as peppermint, red oil and cherry.

  The dosage of the pharmaceutical composition is determined in consideration of the type of dosage form, the administration method, the age and weight of the administration subject (including animals), symptoms, and the like.

(3) Feed etc. Moreover, the said bacterial infection induction ovarian dysfunction improving agent is applicable also as a feed or a food composition which contains this as an active ingredient.

  If this feed or food composition is fed to industrial animals, there is a possibility that bacterial infection-induced ovarian dysfunction can be prevented. The food composition may be applied to humans as a health supplement (functional food).

  The feed according to the present invention comprises the above-mentioned agent for improving ovarian dysfunction induced by bacterial infection, raw grass and hay, green-cutting forage crops (green-cutting corn, etc.), straw, fresh grass and hay, green-cutting forage crops (blue-cutting corn, etc.), straw It can be obtained by mixing with the like.

  Generally, livestock feed is roughly divided into three types: roughage, concentrated feed, and special feed. Among these, roughage refers to feed that has a relatively high content of coarse fibers and a small amount of digestible nutrients for a large volume. Raw grass, hay, green-cutting forage crops (blue-cutting corn, etc.), root vegetables, straws, etc. are used for roughage. Concentrated feed refers to feed having a relatively high nutrient content and low moisture and coarse fiber content. Concentrated feed includes grains such as corn, milo, barley, oats, rice, millet, millet, millet, sorghum, and grain by-products such as rice bran, bran, peanut ginger, cottonseed meal, sunflower meal, Oil varieties such as rapeseed meal, sesame meal, and flaxseed meal are used.

  The feed according to the present invention can also be produced by adding olive fruits, flowers, stems, roots, preferably leaves, to these feeds in a raw state or as a dried product. Olive leaves and the like can be added as they are or pulverized into powder. It is also possible to add pomace generated during olive oil extraction.

  Moreover, fruits including anthocyanins and fruit of a lotus cup can also be produced by adding them to the above feed in a raw state or as a dried product. Fruits and the like containing anthocyanins and fruit of a lotus cup can be added as they are or after being pulverized into powder.

  In addition, the food composition includes the above-mentioned agent for improving bacterial infection-induced ovarian dysfunction, glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside, corn syrup, lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid. , L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester, propylene glycol fatty acid ester, gum arabic, carrageenan, casein , Gelatin, pectin, agar, vitamin B, nicotinamide, calcium pantothenate, amino acids, calcium salts, pigments, fragrances, preservatives, etc. It can be produced by Rukoto. Further, it may be mixed with rice, wheat, corn, potato, sweet potato, soybean, kelp, wakame, tengusa and the like which are generally used as food materials.

  Examples of the food composition include health foods, functional foods, foods for specified health use, nutritional supplements, enteral nutritional foods, and the like. These food compositions can also be fed to animals.

  In the above-described agent for improving bacterial infection-induced ovarian dysfunction and a pharmaceutical composition comprising this as an active ingredient, hydroxytyrosol and anthocyanin can be derived from a natural product extract component and a purified component of the extract. It is of course possible to use chemically synthesized hydroxytyrosol and anthocyanins.

  When hydroxytyrosol is obtained as a natural product extraction component, extraction from olive (Olea europaea) is particularly preferable. Olive is an evergreen tree native to the Mediterranean Sea, and olive oil obtained from its fruits has been edible for a long time.

  Hydroxytyrosol is already known to be contained in a large amount in olives, and can be extracted from olives with water, organic solvents such as alcohol, oils, and the like. As the organic solvent for extraction, methanol, ethanol, propanol, butanol, propylene glycol, 1,3-butylene glycol, glycerin, acetone, methyl ethyl ketone, ethyl acetate, ethers, chloroform, dichloromethane and the like can be used. Furthermore, if the obtained extract is concentrated by a known method, hydroxytyrosol can be obtained as a purified component.

  For olives, any part of its fruit, leaves, flowers, stems, and roots can be used, and these may be used in a raw state or in a dried product. Among these, since oleuropein and the like are particularly found in olive leaves, hydroxytyrosol can be extracted by effectively utilizing pruned and cut olive leaves.

  Anthocyanins are known to be contained in many plants such as fruits such as blueberries and grapes and vegetables such as sweet potatoes. When anthocyanins are obtained as natural product extraction components, extraction from the fruits or flowers of these plants is preferred. About the extraction method of anthocyanin, it can select suitably from a well-known extraction method.

  The feed and food composition according to the present invention displays that it is used for improving ovarian dysfunction due to bacterial infection, if necessary.

<Experimental example 1>
1. Creation of an animal model of bacterial infection-induced ovarian dysfunction 1
In this experimental example, mice were used to try to produce pathological conditions of bacterial infection-induced ovarian dysfunction by combining superovulation treatment according to a standard method with administration of bacterial components.

The experimental schedule is shown in FIG.
In this experimental example, lipopolysaccharide (LPS) and peptidoglycan (PGN) were used as bacterial constituents.
On the first day of the experiment, LPS or PGN and pregnant mare serum gonadotropin (PMSG), 5 IU were administered into the abdominal cavity of 27-28 day old immature mature mice.
On the second day of the experiment, LPS or PGN was administered intraperitoneally again.
On the third day of the experiment, human chorionic gonadotropin (hCG) and 5 IU were administered intraperitoneally.
On the 4th day of the experiment, the abdomen was opened, and the number of ovulations was counted.

  The results are shown in FIG. FIG. 2A shows the number of ovulations of mice administered with different concentrations of LPS, and FIG. 2B shows the number of ovulations of mice administered with different concentrations of PGN. In mice administered with LPS, the number of ovulations decreased in a concentration-dependent manner compared to the control group (0 mg / kg body weight (mg / kg B.W)), and the maximum concentration (4.0 mg / kg B.W.) was observed. W) The number of ovulations was halved in the administration group. On the other hand, in mice administered with PGN, no significant change in the number of ovulations was observed compared to the control group.

  The relationship between the LPS administration schedule and the number of ovulations was also examined. The administration schedule is shown in FIG. In FIG. 3, black arrows represent LPS administration (2.0 mg / kg BW). The results are shown in FIG. In the case where a single administration is performed only on the first day of the experiment and the second LPS administration is not performed on the second day of the experiment (see FIGS. 3 and 4, once administration (first half)), the LPS is not administered Compared with, the number of ovulations decreased by several. On the other hand, when the LPS administration was not performed on the first day of the experiment and the single administration was performed only on the second day of the experiment (see FIG. 3 and FIG. 4, the number of administration once (the latter half)), In comparison, the number of ovulations decreased to about half.

  These results indicate that the function of the ovary stimulated by superovulation treatment is impaired by LPS administration called endotoxin, which is one of the components of bacteria, resulting in a decrease in the number of ovulations. . Therefore, it has been found that the animal model of superovulation treatment / bacterial component administration can reflect the pathology of bacterial infection-induced ovarian dysfunction. Ovarian dysfunction caused by administration of LPS (endotoxin), which is one of the components of bacteria, could be confirmed in both non-fasted mice and fasted mice.

  Prior to this experimental example, FIG. 5 shows the results of RT-PCR confirming the expression of LPS receptor in the ovaries of mice aged 21 to 25 days. It was confirmed that TLR4 (Toll-like receptor 4), which functions as an LPS receptor, was expressed in the ovary of mice after 24 days of age. In addition, expression of MD-2 and CD14 that form a complex with TLR4 and function in cooperation after 24 days of age was also confirmed. In addition, the expression of TLR2, NOD-1, and NOD-2, which are PGN receptors, was also confirmed in the ovaries of 27-28 day old mice subjected to the experiment.

<Experimental example 2>
2. Creation of animal model of bacterial infection-induced ovarian dysfunction 2
In this experimental example, mice were used to attempt pathogenesis of bacterial infection-induced ovarian dysfunction by combining administration of lipoteichoic acid (LTA) with superovulation treatment according to a standard method.

The experiment schedule was the same as in Experiment Example 1 (see FIG. 1).
On the first day of the experiment, PSGSG, 5IU, and lipoteichoic acid (LTA) were added to the abdominal cavity of 27-28 day old immature mice at 0.4, 2.0, 4.0, 20 mg / kg body weight (mg / Kg B.W) at any concentration. In addition, LTA was not administered to 27-28 day-old immature mice, and only PMSG and 5IU were administered to serve as a control group.
On the second day of the experiment, the same amount of LTA as that of the first day of experiment was again administered intraperitoneally to the mice.
On the third day of the experiment, hCG and 5IU were intraperitoneally administered to the above mice.
On the 4th day of the experiment, the above mice were opened and the number of ovulations was counted.

  The results are shown in FIG. FIG. 6 shows the number of ovulations in mice administered with different concentrations of LTA. LTA is 0.4-4.0 mg / kg. In the mice administered in the range of W, the number of ovulations was reduced as compared with the control group (0 mg / kg B.W). Furthermore, in the mice administered with LTA at the maximum concentration (20 mg / kg BW), the number of ovulations was reduced to about half compared with the control group.

  These results indicate that the function of the ovary stimulated by the superovulation treatment is impaired by the administration of LTA, resulting in a decrease in the number of ovulations. Therefore, it has been found that an animal model that combines superovulation treatment and LTA administration can reflect the pathology of bacterial infection-induced ovarian dysfunction. In mice, there was no decrease in food intake due to LTA administration.

<Experimental example 3>
3. Examination of the frequency of administration of bacterial constituents In this experimental example, the frequency of administration of bacterial constituents necessary to create a pathology of bacterial infection-induced ovarian dysfunction in mice was examined.

The experimental schedule is shown in FIG.
In this Experimental Example, PMSG (5 IU) and hCG (5 IU) were administered to 27-28 day-old immature mature mice as superovulation treatment as in Experimental Examples 1 and 2. PMSG was administered to mice on the first day of experiment, and hCG was administered to mice on the third day of experiment.
In addition, in this experimental example, in order to create a pathology of bacterial infection-induced ovarian dysfunction in mice as in Experimental Example 1, LPS (2.0 mg / kg body weight (mg / kg B.B. W))) was administered.

As shown in FIG. 7, for mice with one LPS administration, LPS was administered at the above concentration only on the first day of the experiment (see FIG. 7, arrow (black)). For mice administered twice, LPS at the above concentrations was administered on the first and second days of the experiment. About the mouse | mouth of 3 times of administration frequency, LPS of the said density | concentration was administered every time on the 1st day of experiment, the 2nd day, and the 3rd day.
These mice were laparotomized on the fourth day of the experiment, and the number of ovulations was counted. In addition, a control group (only LPS administration 0 times) in which only superovulation treatment was performed and LPS administration was not performed was also prepared.

  The results are shown in FIG. In mice administered with LPS once, the number of ovulations decreased compared to the control group (LPS administration frequency 0). Moreover, in the mouse | mouth which administered LPS twice, the number of ovulations decreased to about half compared with the control group. Furthermore, in mice administered with LPS three times, the number of ovulations decreased to about 40% compared to the control group.

  From the results of this experimental example, even if a single component of bacterial components is administered, a decrease in the number of ovulations is observed in mice treated with superovulation, but in order to create a pathology of bacterial infection-induced ovarian dysfunction in mice. It was shown that the number of administration of the bacterial component is preferably 2 times or more. In addition, as shown in the present experimental example and the experimental example 1 described above, the number of ovulations can be reduced even if the number of LPS administration is one, but at least one LPS administration should be performed 24 hours after the administration of PMSG. It has been shown to be preferred.

<Experimental example 4>
4). Screening for substances that have the effect of improving ovarian dysfunction induced by bacterial infection using a superovulatory treatment / bacterial component-administered animal model 1
In this experimental example, the animal model of bacterial infection-induced ovarian dysfunction established in Experimental Example 1 was used to screen for natural product-derived components having an effect of improving the disorder.

The experimental schedule is shown in FIG.
On the first day of the experiment, LPS (4.0 mg / kg body weight (mg / kg B.W)) and PMSG (5 IU) were intraperitoneally administered to 27-28 day old immature mice, and the test substance was simultaneously administered. Orally administered. As test substances, olive tyrosol and oleuropein, which are components of olive leaves, and glycyrrhizin, which is a component of licorice root, were used.
On the second day of the experiment, LPS was intraperitoneally administered and the test substance was orally administered again.
On the third day of the experiment, hCG (5 IU) was intraperitoneally administered.
On the 4th day of the experiment, the abdomen was opened, and the number of ovulations was counted.

  The results are shown in FIG. FIG. 10A shows the results for hydroxytyrosol, FIG. 10B shows the results for oleuropein, and FIG. 10C shows the results for glycyrrhizin. LPS with hydroxytyrosol 5.0 μg / kg In mice co-administered with W, the decrease in the number of ovulations was suppressed compared to the control group not administered hydroxytyrosol (0 μg / kg B.W), and the number of ovulations was comparable to that in the non-LPS administration group (experimental) (See Example 1). On the other hand, 0.5, 2.5, 5.0 μg / kg B.C. No effect of decreasing the number of ovulations was observed at any dose of W. Glycyrrhizin is also administered at a dose of 0.05 g / kg B.I. In W, the effect of suppressing the decrease in the number of ovulations was not observed.

  These results show that hydroxytyrosol suppresses the decrease in the number of ovulations caused by the administration of bacterial components such as endotoxin and has an effect of improving ovarian dysfunction induced by bacterial infection, and the treatment of superovulation and bacterial composition This shows that the component-administered animal model can be an effective evaluation system for screening for substances having the same improving action.

<Experimental example 5>
5. Screening for substances that have the effect of improving ovarian dysfunction induced by bacterial infection using animal models treated with superovulation and bacterial components 2
In this experimental example, in the same manner as in Experimental Example 4, the animal model of bacterial infection-induced ovarian dysfunction established in Experimental Example 1 was used to screen for natural product-derived components having an effect of improving the disorder.

The experimental schedule is shown in FIG.
From the first day to the fourth day of the experiment, an anthocyanin (Tokiwa Plant Chemistry Laboratory, Bilberon-25) as a test substance was orally administered to 23-24 day-old immature mice, and test group 1 and test group 2 It was. The amount of anthocyanin administered at a time was 4000 mg / kg body weight (mg / kg BW).
On the fifth day of the experiment, LPS (2.0 mg / kg B.W) and PMSG (5 IU) were intraperitoneally administered to the test group 2 mice, and the test substance was orally administered at the above concentration at the same time. About the mouse | mouth of the test group 1, only PMSG was intraperitoneally administered at the said density | concentration, and the test substance was orally administered at the said density | concentration simultaneously. In addition, PSGSG alone was administered intraperitoneally to a 27-28 day old immature mouse that had not been administered the test substance, and designated as control group 1. Furthermore, LPS and PMSG were intraperitoneally administered to 27- to 28-day-old immature mice that had not been administered the test substance at the above concentrations to obtain Control Group 2.
On the sixth day of the experiment, LPS was administered to the mice of Test Group 2 and Control Group 2 again at the above concentration. In addition, the test substance was orally administered to the mice of Test Group 1 and Test Group 2 again at the above concentrations.
On the 7th day of the experiment, hCG (5 IU) was intraperitoneally administered to each mouse of the test groups 1 and 2 and the control groups 1 and 2.
On the 8th day of the experiment, each mouse of test groups 1 and 2 and control groups 1 and 2 was laparotomized and the number of ovulations was counted.

  The results are shown in FIG. FIG. 12 shows the ratio (%) of the number of ovulations in control group 2, test group 1 and test group 2 when the number of ovulations in control group 1 is 100.

  In the test group 2 administered with LPS and anthocyanin, the decrease in the number of ovulations was suppressed as compared with the control group 2 not administered with anthocyanin, and recovered to the same level as the control group 1 not administered with LPS. On the other hand, in Test Group 1 in which LPS was not administered and only anthocyanin was administered, the number of ovulations was similar to that in Control Group 1. This indicates that ovulation is not affected even if anthocyanin at the above-mentioned concentration is administered in a state not infected with bacteria.

  The results of this experimental example show that anthocyanins suppress the decrease in the number of ovulations caused by the administration of bacterial constituents such as endotoxin and have an effect of improving bacterial infection-induced ovarian dysfunction. This shows that the component-administered animal model can be an effective evaluation system for screening for substances having the same improving action.

<Experimental example 6>
6). Screening for substances that have the effect of improving ovarian dysfunction induced by bacterial infection using an animal model of superovulation treatment and bacterial component administration 3
In this experimental example, in the same manner as in Experimental Examples 4 and 5, using the animal model of bacterial infection-induced ovarian dysfunction established in Experimental Example 1, screening for natural product-derived components having an effect of improving the disorder was performed.

The experimental schedule is shown in FIG.
In this experimental example, a lotus cup extract was selected as a test substance, and a lotus juice obtained by squeezing a raw lotus cup excluding the peel was used as the liquid containing the lotus cup extract (the lotus cup extract).
From the first day to the fourth day of the experiment, the Hascup fruit juice was orally administered to 23-24 day-old immature mature mice to obtain Test Group 3 and Test Group 4. The amount of lotus juice to be administered at a time was 100 μl.
On the fifth day of the experiment, LPS (2.0 mg / kg body weight (mg / kg B.W)) and PMSG (5 IU) were intraperitoneally administered to the mice of Test Group 4, and at the same time, the same amount of Hascup juice was orally administered. Administered. About the mouse | mouth of the test group 3, only PMSG was intraperitoneally administered at the said density | concentration, and the same amount of Hascup fruit juice was orally administered simultaneously. Moreover, only the PMSG was intraperitoneally administered at the said density | concentration to the 27-28 day-old immature mature mouse which is not administering said Hascup fruit juice, and it was set as the control group 3. Furthermore, LPS and PMSG were administered intraperitoneally at the above concentrations to 27- to 28-day-old immature mice that had not been administered the above-mentioned lotus cup juice, and designated as control group 4.
On the sixth day of the experiment, LPS was administered to the mice of Test Group 4 and Control Group 4 again at the above concentrations. In addition, the same amount of lotus juice was orally administered to the mice of Test Group 3 and Test Group 4 again.
On the 7th day of the experiment, hCG (5 IU) was intraperitoneally administered to each mouse of the test groups 3 and 4 and the control groups 3 and 4.
On the 8th day of the experiment, each mouse of test groups 3 and 4 and control groups 3 and 4 was laparotomized and the number of ovulations was counted.

  The results are shown in FIG. FIG. 14 shows the ratio (%) of the number of ovulations in control group 4, test group 3 and test group 4 when the number of ovulations in control group 3 is 100.

  In test group 4 administered with LPS and lotus juice, the decrease in the number of ovulations was suppressed as compared to control group 4 not administered with lotus juice, and up to about 90% of control group 3 not administered with LPS. Recovered. On the other hand, the number of ovulations was similar to that in the control group 3 even in the test group 3 in which only LPS was not administered and only the Hascup juice was administered. This indicates that ovulation is not affected even when the above-mentioned volume of Hascup juice is administered without being infected with bacteria.

  These results show that the component contained in the Haskup juice, that is, the Haskup extract, suppresses the decrease in the number of ovulations due to the administration of bacterial components such as endotoxin, and has the effect of improving bacterial infection-induced ovarian dysfunction, In addition, it is shown that an animal model for superovulation treatment / bacterial component administration can be an effective evaluation system for screening a substance having the same improvement effect.

  The method for screening a substance having an effect of improving bacterial infection-induced ovarian dysfunction according to the present invention improves ovarian dysfunction caused by bacterial infection for the purpose of improving production efficiency in industrial animals and treating fertility in animals and humans. It can be used to find a substance having an action of promoting ovulation.

Claims (10)

Procedures for superovulation treatment of animals;
Administering lipopolysaccharide and / or lipoteichoic acid to the animal;
A procedure for administering a test substance to the animal;
After these procedures, a procedure for measuring the number of ovulations of the animal,
A screening method for a substance having an action of improving bacterial infection-induced ovarian dysfunction. Procedures for superovulation treatment of animals;
Administering to said animal lipopolysaccharide and / or lipoteichoic acid,
A method for producing an animal model of bacterial infection-induced ovarian dysfunction.   A model animal of bacterial infection-induced ovarian dysfunction produced by superovulation treatment and administration of lipopolysaccharide and / or lipoteichoic acid. A procedure for superovulation treatment to an animal; a procedure for administering lipopolysaccharide and / or lipoteichoic acid to the animal;
A procedure for administering a test substance to the animal;
After these procedures, a procedure for measuring the number of ovulations of the animal,
Bacterial infection-induced ovarian dysfunction improving agent obtained by a method for screening a substance having an action of improving bacterial infection-induced ovarian dysfunction.   The agent for improving ovarian dysfunction induced by bacterial infection according to claim 4, comprising hydroxytyrosol.   The agent for improving ovarian dysfunction induced by bacterial infection according to claim 4, comprising anthocyanin.   The agent for improving bacterial infection-induced ovarian dysfunction according to claim 4, comprising a lotus cup extract.   The pharmaceutical composition which contains the bacterial infection induction ovarian dysfunction improving agent as described in any one of Claim 4 to 7 as an active ingredient.   A feed comprising the bacterial infection-induced ovarian dysfunction improving agent according to any one of claims 4 to 7. Procedures for superovulation treatment of animals;
Administering lipopolysaccharide and / or lipoteichoic acid to the animal;
A procedure for administering a test substance to the animal;
After these procedures, a procedure for measuring the number of ovulations of the animal,
Including a step of administering a bacterial infection-induced ovarian dysfunction improving agent obtained by a method for screening a substance having an improving effect on bacterial infection-induced ovarian dysfunction,
A method for improving ovarian dysfunction in livestock or poultry caused by bacterial infection.

Images