Application of Lactobacillus plantarum AR501 in the manufacture of drugs for the prevention and treatment of premature ovarian failure

Abstract

To provide a novel concept for the prevention and treatment of premature ovarian insufficiency (POI).SOLUTION: Disclosed is an application of Lactobacillus plantarum AR501 (AR501) in the manufacture of a drug for the prevention and treatment of premature ovarian insufficiency, which belongs to the biopharmaceutical field. The reserve number of AR501 is CGMCC No. 13910. This strain prevents POI by alleviating estrous cycle disorders, serum hormone disorders, or depressive symptoms. AR501 is used to intervene into POI mice, significantly restoring the estrous cycle of the POI mice and improving serum hormone disorders such as estradiol, gonadotropin-releasing hormone, and anti-Mullerian duct hormone. Intervention with AR501 significantly improves the depressive state of the POI mice and increases their activity. AR501 can be used to manufacture a drug that improves POI, providing a novel concept to the prevention and treatment of POI.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, and in particular to the application of Lactobacillus plantarum AR501 in the manufacture of drugs for preventing or treating premature ovarian failure. [Background technology]

[0002] Premature ovarian insufficiency (POI) refers to the decline or failure of ovarian function in women before the age of 40. It is characterized by elevated serum follicle-stimulating hormone (FSH) and decreased estradiol levels. POI can cause infertility in women, leading to insufficient estrogen levels and accelerating the onset of menopause, severely impacting women's physical and mental health. Patients experience a range of symptoms, including irregular menstrual cycles, menopause, hot flashes, irritability, decreased libido, and osteoporosis, and some patients experience reduced fertility and infertility. The incidence of POI is approximately 1%-5% and severely impacts women's fertility and quality of life. The etiology of POI includes genetic, iatrogenic, immunological, and environmental factors. Because this condition is typically heterogeneous, it is primarily treated with hormone replacement therapy (HRT). However, clinically, there is no clearly defined and effective treatment for restoring or protecting ovarian function. HRT also has several limitations and side effects, including difficulty in controlling dosage, fluctuating hormone levels, and an increased risk of breast and endometrial cancer. Therefore, it is an object of the present invention to find a safe, effective and natural method for alleviating the symptoms of POI.

[0003] In recent years, the probiotic functions of probiotics have attracted public attention. Probiotics are a collective term for microorganisms that colonize the human intestine and reproductive system, primarily providing beneficial health benefits, improving the balance of the host's microecosystem, and exerting beneficial effects. They are widely used in the fields of food, feed, medicine, and other fields. Probiotics can secrete many beneficial metabolites, such as lactic acid, acetic acid, bacteriocin, and hydrogen peroxide, which can inhibit the growth of harmful bacteria, maintain the balance of the intestinal microflora, enhance intestinal barrier function, and promote the absorption and utilization of nutrients. Probiotics have also been shown to have excellent anti-inflammatory and immune regulation properties. Currently, there is little research on the therapeutic effects of probiotics on POI. Therefore, the objective of the present invention is to provide probiotics and their application methods for alleviating the symptoms of POI, thereby providing a new preventive and therapeutic approach for alleviating POI. Summary of the Invention [Problem to be solved by the invention]

[0004] The objective of the present invention is to solve the above-mentioned problems of the prior art by providing the application of Lactobacillus plantarum AR501 in the manufacture of drugs for the prevention and treatment of POI. The present invention uses Lactobacillus plantarum AR501 to intervene in POI mice, significantly restoring the estrous cycle of POI mice and improving the disturbance of serum hormones such as estradiol, gonadotropin-releasing hormone, and anti-Mullerian hormone, as well as the depression state of POI mice, thereby providing a new concept for the prevention and treatment of POI. [Means for solving the problem]

[0005] In order to achieve the above object, the present invention provides the following scheme.

[0006] The present invention provides the use of Lactobacillus plantarum AR501 in the manufacture of a medicament for preventing and treating premature functional ovarian failure, the reserve number of which is CGMCC No. 13910.

[0007] Furthermore, the Lactobacillus plantarum AR501 prevents premature ovarian failure by improving the disturbance of the estrous cycle, the disturbance of serum hormones, or depressive symptoms of the body.

[0008] Additionally, the serum hormones include estradiol, gonadotropin-releasing hormone, anti-Mullerian hormone, and luteinizing hormone.

[0009] Furthermore, the effective dose of Lactobacillus plantarum AR501 is 1×10 9 CFU / mL.

[0010] Furthermore, the POI includes 4-vinylcyclohexene dioxide-induced premature ovarian insufficiency.

[0011] The present invention also provides a drug for preventing or treating POI, comprising an effective dose of Lactobacillus plantarum AR501 and pharmaceutically acceptable auxiliary materials, and the storage number of said Lactobacillus plantarum AR501 is CGMCC No. 13910.

[0012] Furthermore, the effective dose of Lactobacillus plantarum AR501 is 1×10 9 CFU / mL.

[0013] Furthermore, the dosage form of the drug includes tablets, pills, granules, capsules, oral preparations or sprays. [Effects of the Invention]

[0014] The present invention discloses the following technical effects.

[0015] The present invention demonstrates that intervention with Lactobacillus plantarum AR501 in POI mice significantly restores the estrous cycle and improves serum hormone disturbances, such as estradiol, gonadotropin-releasing hormone, and anti-Mullerian hormone. Furthermore, intervention with Lactobacillus plantarum AR501 significantly improves the depressive state of POI mice and enhances their activity. The Lactobacillus plantarum AR501 provided by the present invention can be used to prepare drugs that improve POI, providing a new approach to the prevention and treatment of POI. [Brief explanation of the drawings]

[0016] In the following, in order to more clearly explain the embodiments of the present invention or the technical scheme in the prior art, the drawings necessary for the embodiments will be briefly described. However, the drawings in the following description are only some embodiments of the present invention, and those skilled in the art can obtain other drawings from these drawings without creative work. [Figure 1] Effect of Lactobacillus plantarum AR501 on the estrous cycle of POI mice. [Figure 2] Effect of Lactobacillus plantarum AR501 on serum estradiol in POI mice. [Figure 3] Effect of Lactobacillus plantarum AR501 on serum gonadotropin-releasing hormone in POI mice. [Figure 4] Effect of Lactobacillus plantarum AR501 on serum anti-Müllerian hormone in POI mice. [Figure 5] Effect of Lactobacillus plantarum AR501 on serum luteinizing hormone in POI mice. [Figure 6] Effect of Lactobacillus plantarum AR501 on the movement distance in open field experiments in POI mice. DETAILED DESCRIPTION OF THE INVENTION

[0017] Various exemplary embodiments of the present invention will now be described in detail, but this detailed description should not be considered a limitation of the present invention, but rather should be understood as a more detailed description of some aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the invention. Numerical ranges in the present invention should also be understood to specifically disclose every intermediate value between the upper and lower limits of the range. Any stated value or intermediate value within a stated range, as well as any other stated value or intermediate value within said range, are also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the range.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention describes. Although the present invention describes only preferred methods and materials, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials related to the publication. In the event of a conflict with any incorporated publication, the contents of this document will control.

[0020] It will be apparent to those skilled in the art that many modifications and variations can be made to the specific embodiments of the present specification without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art upon learning the present specification. The present specification and examples are merely illustrative.

[0021] As used herein, the terms "containing," "including," "having," "having" and the like are open-ended terms and mean including but not limited to.

[0022] Example 1: Creation of a mouse model of premature ovarian insufficiency (POI) and experimental grouping

[0023] (1) Creation of POI mouse model

[0024] Preparation of solutions:

[0025] Lactobacillus plantarum AR501 (this strain is preserved in the General Microbiology Center of the China Committee for the Preservation and Management of Microorganisms. Its preservation number is CGMCC No. 13910 and is described in the patent document "CN109593688A Lactobacillus plantarum for mitigating the hazards of frying oil and its application") bacterial suspension: AR501 bacterial cells collected by centrifugation were washed with saline twice. Then, the bacterial cell concentration was adjusted to 1 × 10 using saline. 9 Adjust to CFU / mL.

[0026] Step 1: Modeling premature ovarian failure: Forty 8-week-old C57 female mice weighing 28-30g were used in the experiment. They were allowed to adapt to the environment for 7 days. After adaptation was complete, 30 mice were randomly selected for modeling and administered 4-vinylcyclohexene dioxide (VCD) (160mg / kg / day) intraperitoneally for 20 consecutive days. Ten mice were selected as normal controls and administered an equal volume of sesame oil intraperitoneally.

[0027] Step 2, treatment period: After successful modeling, the mice entered a treatment cycle. The treatment cycle lasted for 8 weeks, and all mice were orally administered with steroids at 9:00 a.m. every day. The 30 successfully modeled mice were randomly divided into three groups: 10 mice in the model group were injected with sterile water into the stomach; 10 mice in the positive control group were orally administered 0.1 mg / kg estradiol valerate; and 10 mice in the treatment group were administered with Lactobacillus plantarum AR501 (1 × 10 9 CFU / mL) were orally administered.

[0028] Example 2: Effect of Lactobacillus plantarum AR501 on the estrous cycle of POI mice

[0029] After the treatment cycle of Example 1 was completed, the estrous cycle of the mice was measured.

[0030] The estrous cycle of mice is measured as follows: The mouse is anesthetized with an anesthetic, secured to the operating table, and the vaginal opening is swabbed with a disinfectant cotton ball. A thin cotton swab is gently inserted into the vagina, rotated once, and then removed. The vaginal contents on the swab are applied to a glass plate to prepare a smear. The smear is fixed in 95% alcohol for 10 minutes, then rinsed with water and dried for storage. The smear is stained with an alkaline methylene blue solution for 5 minutes, then rinsed with water and dried for storage. The smear is then observed under a microscope, and the stage of the mouse's estrous cycle is determined based on the type and number of vaginal cells.

[0031] As shown in Figure 1, the disruption of the estrous cycle in the model group was significantly increased compared with the control group, while the disruption of the estrous cycle in the mice after oral administration of Lactobacillus plantarum AR501 was significantly reduced.

[0032] Example 3: Effect of Lactobacillus plantarum AR501 on serum hormones in POI mice

[0033] 1. Effect of Lactobacillus plantarum AR501 on serum estradiol in POI mice

[0034] After the treatment period in Example 1 was completed, mice in each group were fasted for 12 hours. Blood was collected from the anesthetized mice by eyeball blood collection, placed in sterile blood collection tubes, and centrifuged at 3,000 rpm for 10 minutes to collect the supernatant, which was then temporarily stored in a refrigerator at 4°C. Mouse serum estradiol content was measured using the E2 detection reagent Mouse Estradiol ELISA Kit EY-01H2013 (purchased from Shanghai Yiken Biotechnology Co., Ltd.). As shown in Figure 2, the serum estradiol content of mice in the model group was significantly lower than that of the control group. However, after oral administration of Lactobacillus plantarum AR501, the serum estradiol content of mice significantly increased and returned to normal levels.

[0035] 2. Effect of Lactobacillus plantarum AR501 on serum gonadotropin-releasing hormone in POI mice

[0036] After the treatment period in Example 1 was completed, mice in each group were fasted for 12 hours. Blood was collected from anesthetized mice by eyeball blood collection, placed in sterile blood collection tubes, and centrifuged at 3000 rpm for 10 minutes to collect the supernatant, which was then temporarily stored in a refrigerator at 4°C. The gonadotropin-releasing hormone (GnRH) ELISA kit (purchased from Shanghai Huabang Biotechnology Co., Ltd., product number: HB-P9S660X) was used to measure the content of mouse serum gonadotropin-releasing hormone. The results, as shown in Figure 3, showed that the content of mouse serum gonadotropin-releasing hormone in the model group was significantly higher than that in the control group, while the content of mouse serum gonadotropin-releasing hormone significantly decreased after oral administration of Lactobacillus plantarum AR501.

[0037] 3. Effect of Lactobacillus plantarum AR501 on serum anti-Mullerian hormone in POI mice

[0038] After the treatment period in Example 1 was completed, the mice in each group were fasted for 12 hours. Blood was collected from the anesthetized mice by eyeball blood collection, placed in sterile blood collection tubes, and centrifuged at 3000 rpm for 10 minutes to obtain the supernatant, which was then temporarily stored in a refrigerator at 4°C. The mouse anti-Müllerian hormone (AMH) serum anti-Müllerian hormone content was measured using a mouse anti-Müllerian hormone (AMH) ELISA test kit (purchased from Shanghai Zhenke Biotechnology Co., Ltd.). As shown in Figure 4, the mouse serum anti-Müllerian hormone content in the model group was significantly reduced compared to the control group, but after oral administration of Lactobacillus plantarum AR501, the mouse serum anti-Müllerian hormone content was significantly increased.

[0039] 4. Effect of Lactobacillus plantarum AR501 on serum luteinizing hormone levels in POI mice

[0040] After the treatment period in Example 1 was completed, the mice in each group were fasted for 12 hours. Blood was collected from the anesthetized mice by eyeball blood collection, placed in sterile blood collection tubes, and centrifuged at 3000 rpm for 10 minutes to obtain the supernatant, which was then temporarily stored in a refrigerator at 4°C. Mouse serum luteinizing hormone (LH) ELISA kits (purchased from Shanghai Qi Biotechnology Co., Ltd.) were used to measure the serum luteinizing hormone (LH) content. The results, as shown in Figure 5, showed that compared with the control group, the serum luteinizing hormone content in the model group was significantly elevated, while the serum luteinizing hormone content in the model group was significantly reduced after oral administration of Lactobacillus plantarum AR501.

[0041] Example 4: Effect of Lactobacillus plantarum AR501 on depressive symptoms in POI mice

[0042] After the treatment period in Example 1 was completed, mice in each group were subjected to an open field experiment to measure their movement distance and evaluate the effect of POI on depressive symptoms. As shown in Figure 6, the movement distance of the mice in the model group was significantly reduced compared to the control group, but after oral administration of Lactobacillus plantarum AR501, the movement distance of the mice significantly increased, suggesting that the activity of the mice was enhanced and depressive symptoms were improved.

[0043] In summary, the present invention demonstrates that intervention with Lactobacillus plantarum AR501 in POI mice significantly restores the impaired ovarian estrous cycle and improves serum hormone disturbances, such as estradiol, gonadotropins, and anti-Müllerian hormone. Furthermore, intervention with Lactobacillus plantarum AR501 significantly improves the depressive state of POI mice and enhances their activity. Therefore, the Lactobacillus plantarum AR501 provided by the present invention provides a new approach to the prevention and treatment of POI.

[0044] The above-mentioned embodiments merely illustrate preferred embodiments of the present invention and do not limit the scope of the present invention. Various modifications and improvements made by those skilled in the art without departing from the design spirit of the present invention should also be included in the scope of protection defined by the claims of the present invention. [Accession number]

[0045] Depository institution: General Microbiology Center, China Committee for the Preservation of Microbial Species Accession number: CGMCC NO.13910 Date of acceptance: March 22, 2017 TIFF0007742674000001.tif192168

Claims

1. A method for using Lactobacillus plantarum AR501 in the manufacture of a medicament for preventing and treating premature ovarian failure, characterized in that the storage number of the Lactobacillus plantarum AR501 is CGMCC No. 13910.

2. The method of claim 1, wherein the Lactobacillus plantarum AR501 prevents premature ovarian insufficiency by improving the disturbance of the estrous cycle, the disturbance of serum hormones, or depressive symptoms of the body.

3. 3. The method of claim 2, wherein the serum hormones include estradiol, gonadotropin-releasing hormone, anti-Mullerian hormone, and luteinizing hormone.

4. The effective dose of the Lactobacillus plantarum AR501 is 1×10 ９ The method of claim 2, wherein the concentration is CFU / mL.

5. 2. The method according to claim 1, wherein the premature ovarian insufficiency includes 4-vinylcyclohexene dioxide-induced premature ovarian insufficiency.

6. A drug for preventing or treating premature ovarian failure, comprising an effective dose of Lactobacillus plantarum AR501 and pharmaceutically acceptable auxiliary materials, wherein the storage number of the Lactobacillus plantarum AR501 is CGMCC No. 13910.

7. The effective dose of the Lactobacillus plantarum AR501 is 1×10 ９ The drug according to claim 6, characterized in that it is CFU / mL.

8. The drug according to claim 6, wherein the dosage form of the drug includes tablets, pills, granules, capsules, oral preparations, or sprays.

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