Use of parabacteroides johnsonii in ovarian aging

Abstract

The application relates to the field of biotechnology, and particularly relates to an application of Bacteroides ovatus in ovarian aging; the application comprises: taking low-abundance Bacteroides ovatus as a biomarker of ovarian aging; the application comprises using Bacteroides ovatus in a medicine for delaying ovarian aging; the application further comprises using a fermentation product of Bacteroides ovatus in the medicine for delaying ovarian aging; it is found through experiments that there is a significant difference in Bacteroides ovatus in mice with different degrees of ovarian aging, and it is found through subsequent experiments that Bacteroides ovatus has the effects of significantly increasing the number of oocytes, up-regulating anti-Mullerian hormone, up-regulating the level of estradiol and down-regulating the level of follicle-stimulating hormone, which indicates that Bacteroides ovatus can be used in the medicine for delaying ovarian aging.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to the application of *Pseudomonas johnsonii* in ovarian aging. Background Technology

[0002] Female reproductive aging refers to the decline in ovarian fertility, which generally begins around age 30 and accelerates after age 35. Ovarian reserve is crucial for fertility; since the reserve of germ cells and follicles at birth determines the reproductive lifespan and menopause time in mammals, a rapid decrease in ovarian reserve leads to premature ovarian aging or dysfunction. Simultaneously, ovarian reserve is depleted more rapidly with age, primarily manifested by increased follicle atresia and apoptosis. In addition to reduced follicle reserve, oocyte quality also declines with age. Currently, oocyte quality generally begins to deteriorate gradually after age 30, which coincides with the time of oocyte reduction. This also partially explains why some women experience decreased fertility before menopause and endocrine abnormalities in women undergoing reproductive aging. Oocyte aging is characterized by lectin dysfunction, low cross-maturation efficiency, chromosomal misalignment, meiotic spindle damage, telomere wear, DNA damage, and mitochondrial dysfunction. Furthermore, with increasing age, abnormal oocyte development due to declining oocyte quality becomes one of the main causes of female reproductive disorders.

[0003] Besides reproductive function, the ovaries also maintain female hormone secretion, which is crucial for women's health. Ovarian aging can disrupt the regulation of hormone secretion along the hypothalamus-pituitary-ovarian axis, leading to elevated levels of follicle-stimulating hormone (FSH) and decreased levels of anti-Müllerian hormone (AMH). Excessive FSH in the serum directly affects hippocampal and cortical neurons, accelerating the deposition of amyloid-β and tau proteins, impairing cognitive function and contributing to Alzheimer's disease. Simultaneously, follicle depletion in the ovaries leads to a decrease in estrogen levels, eventually resulting in menopause. Estrogen is a steroid hormone with broad and important physiological functions, significantly benefiting other systems such as the endocrine, skeletal, nervous, and cardiovascular systems. Furthermore, research has confirmed that estrogen has potential therapeutic value for various lung diseases, such as asthma, pulmonary hypertension, and pulmonary fibrosis, and even offers some protection against respiratory viral infections. In conclusion, ovarian aging is a complex process involving multiple factors. Therefore, a deeper understanding of ovarian aging will play an important role in the healthy aging process of humankind and bring broader social benefits.

[0004] A significant characteristic of ovarian aging is the decline in both the number and quality of oocytes. The aging process is often accompanied by changes in the composition of the gut microbiota, which in turn influences aging. Recent studies have revealed a potential interaction between ovarian aging and the gut microbiota, suggesting that the gut microbiota may play a role in the process of ovarian aging. Furthermore, in a cohort study including women with early-onset ovarian insufficiency (POI) and healthy women, researchers found that the gut microbiota of POI women differed from that of healthy women, and these changes were closely related to changes in hormone levels associated with ovarian aging. Therefore, these findings suggest that the process of ovarian aging may be related to the gut microbiota.

[0005] Current research on the mechanisms by which small molecules delay ovarian aging suggests that microorganisms may participate in the regulatory process of drug-induced ovarian aging. For example, studies have found that resveratrol can significantly reduce the effects of tBHP-induced oxidative stress on oocyte production rate and follicle count, and activate the SIRT1 / FoxO1 and Nrf2 pathways, thereby enhancing the expression of antioxidant genes. Meanwhile, studies have found that gut microbiota plays a crucial role in maintaining tryptophan metabolism through the kynurenine pathway in ovarian oxidative stress. However, there are currently no reports on the role of *Parabacteroides johnsonii* in ovarian aging. Summary of the Invention

[0006] This application provides an application of *Pseudomonas johnsonii* in ovarian aging, filling the gap in the prior art regarding the association between *Pseudomonas johnsonii* in gut microbiota and ovarian aging.

[0007] In a first aspect, this application provides the application of *Pseudomonas johnsonii* in ovarian aging biomarkers, the application including:

[0008] Low abundance of *Pseudomonas johnsonii* was used as a biomarker for ovarian aging.

[0009] Optionally, the abundance of *Pseudomonas johnsonii* is positively correlated with the degree of ovarian aging.

[0010] Optionally, the abundance of *Pseudomonas johnsonii* is 0.77 times lower than that of normal *Pseudomonas johnsonii*.

[0011] Optionally, when the abundance of *Pseudomonas johnsonii* is less than 0.77 times the normal abundance of *Pseudomonas johnsonii*, the ovary is judged to be in a state of aging.

[0012] Secondly, this application provides a reagent for screening or assisting in the screening of ovarian aging, the reagent comprising an agent for detecting the relative abundance of *Parabacteroides johnsonii*.

[0013] Thirdly, this application provides a reagent for evaluating or assisting in the evaluation of the prognostic effects of ovarian aging, the reagent comprising an agent for detecting the relative abundance of *Pleurobacterium johnsonii*.

[0014] Fourthly, this application provides the use of *Pseudomonas johnsonii* in delaying ovarian aging, the use including the use of *Pseudomonas johnsonii* in pharmaceuticals for delaying ovarian aging.

[0015] Optionally, the delay in ovarian aging includes at least one of the following:

[0016] It increases the number of oocytes, upregulates anti-Müllerian hormone, upregulates estradiol levels, and downregulates follicle-stimulating hormone levels.

[0017] Optionally, the application also includes using the fermentation products of *Pseudomonas johnsonii* in pharmaceuticals that delay ovarian aging.

[0018] Optionally, the fermentation product of the *Pseudomonas johnsonii* includes the supernatant from the culture of *Pseudomonas johnsonii*.

[0019] The technical solutions provided in this application have the following advantages compared with the prior art:

[0020] This application provides an application of *Peribacterium johnsonii* in ovarian aging. Extensive experiments revealed that *Peribacterium johnsonii* is highly abundant in younger mice, while its abundance significantly decreases in older mice. This difference suggests that *Peribacterium johnsonii* may serve as a biomarker for ovarian aging. Subsequent verification experiments confirmed that *Peribacterium johnsonii* significantly increases oocyte count and can upregulate anti-Müllerian hormone, upregulate estradiol levels, and downregulate follicle-stimulating hormone levels. Therefore, *Peribacterium johnsonii* can also be used as a drug to delay ovarian aging, thus filling a gap in the understanding of *Peribacterium johnsonii* in gut microbiota regarding ovarian aging. Attached Figure Description

[0021] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of transcriptome sequencing results for 3-month-old and 10-month-old mice provided in the embodiments of this application, wherein, Figure 1 Figure A shows the PCA results of the two groups of mice. Figure 1 B is a schematic diagram showing the results of differentially expressed gene analysis between the two groups of mice. Figure 1 C shows the KEGG functional enrichment results for the two groups of mice;

[0024] Figure 2 This is a schematic diagram illustrating the fecal microbiota transplantation results of SPF young mice and ovarian senescent mice provided in the embodiments of this application, wherein, Figure 2 A is a schematic diagram of the fecal microbiota transplantation process in SPF young mice and ovarian aging mice. Figure 2 B is a schematic diagram showing the HE staining results of the ovaries of the two groups of mice. Figure 2 C shows the follicle count results at each stage in the two groups of mice. Figure 2 D is a schematic diagram of the staining results for Ki-67 cell proliferation. Figure 2 E represents the statistical results of Ki-67 cell proliferation positivity. Figure 2 F is a schematic diagram of the colorimetric results of TUNEL cell apoptosis. Figure 2 G is a schematic diagram of the statistical results of TUNEL cell apoptosis positivity;

[0025] Figure 3 This is a schematic diagram illustrating the metagenomic detection results of 3-month-old and 10-month-old C57BL / 6 mice provided in the embodiments of this application, wherein... Figure 3 Figure A shows the results of α-diversity analysis for the two groups of mice. Figure 3 Figure B shows the results of β-diversity analysis for the two groups of mice. Figure 3 C shows the bacterial abundance of the two groups of mice;

[0026] Figure 4 The figure shows the effect of *Pseudomonas johnsonii* and its culture supernatant on ovarian aging, as provided in the embodiments of this application. Figure 4 Figure A shows the effect of *Pseudomonas johnsonii* and its culture supernatant on the number of oocytes. Figure 4 Figure B shows the effect of *Pseudomonas johnsonii* and its culture supernatant on the resistance of Müllerian hormones. Figure 4 Figure C shows the effect of *Pseudomonas johnsonii* and its culture supernatant on estradiol. Figure 4 Figure D shows the effect of *Pseudomonas johnsonii* and its culture supernatant on follicle-stimulating hormone (FSH). Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0028] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0029] The creative thinking behind this application is:

[0030] A significant characteristic of ovarian aging is the decline in the number and quality of oocytes. The aging process is often accompanied by changes in the composition of the gut microbiota, which in turn influences ovarian aging. Recent studies have revealed a potential interaction between ovarian aging and the gut microbiota. For example, studies have observed vaginal atrophy accompanied by alterations in the gut microbiota in ovariectomized mice. Transplanting the gut microbiota of intact ovarian female mice into ovariectomized female mice significantly alleviated vaginal epithelial atrophy. This suggests that normal ovarian function helps regulate the gut microbiota in mice, and the gut microbiota may play a regulatory role in other parts of the body.

[0031] The gut microbiota may play a role in the process of ovarian aging. For example, by transplanting the gut microbiota of young (5-week-old) female mice into ovarian-aged mice (42 weeks old), it was found that the "young" gut microbiota via fecal microbiota transplantation could reshape the gut microbiota of ovarian-aged mice and slow the loss of ovarian function by reducing follicular atresia and apoptosis, increasing granulosa cell proliferation, and improving the ovarian immune microenvironment. Furthermore, in a cohort study including women with early-onset ovarian insufficiency (POI) and healthy women, researchers found that the gut microbiota of POI women was altered compared to healthy women, and these changes were closely related to changes in hormone levels associated with ovarian aging. Both of these studies suggest that the process of ovarian aging may be related to the gut microbiota.

[0032] Current research into the mechanisms by which small molecules delay ovarian aging suggests that microorganisms may participate in the regulatory process of drug-induced ovarian aging. For example, studies have found that resveratrol can significantly reduce the effects of tBHP-induced oxidative stress on oocyte production rate and follicle count, and activate the SIRT1 / FoxO1 and Nrf2 pathways, thereby enhancing the expression of antioxidant genes. Simultaneously, studies have found that gut microbiota plays a crucial role in maintaining tryptophan metabolism through the kynurenine pathway in ovarian oxidative stress. However, there are currently no reports on the role of *Parabacteroides johnsonii* in ovarian aging.

[0033] This application provides an application of *Pseudomonas johnsonii* in ovarian aging biomarkers, the application including:

[0034] Low abundance of *Pseudomonas johnsonii* was used as a biomarker for ovarian aging.

[0035] In some alternative implementations, the abundance of *Pseudomonas johnsonii* is positively correlated with the degree of ovarian aging.

[0036] In this embodiment of the application, the abundance of *Pseudomonas johnsonii* is defined as being positively correlated with the degree of ovarian aging, which clarifies the specific abundance of *Pseudomonas johnsonii* and thus clarifies the accuracy of *Pseudomonas johnsonii* as a biomarker of ovarian aging.

[0037] In some alternative embodiments, the abundance of *Pseudomonas johnsonii* is less than 0.77 times that of normal *Pseudomonas johnsonii*.

[0038] In some alternative implementations, when the abundance of *Pseudomonas johnsonii* is less than 0.77 times the normal abundance of *Pseudomonas johnsonii*, the ovary is determined to be in a state of aging.

[0039] In this embodiment of the application, by specifying the exact multiple by which the abundance of *Pseudomonas johnsonii* is higher than that of normal *Pseudomonas johnsonii*, the minimum abundance of *Pseudomonas johnsonii* can be determined, thereby improving the specificity and sensitivity of *Pseudomonas johnsonii* as a biomarker.

[0040] Based on a general inventive concept, embodiments of this application provide a reagent for screening or assisting in the screening of ovarian aging, the reagent comprising an agent for detecting the relative abundance of *Pseudomonas johnsonii*.

[0041] This reagent is based on the above application. The specific principle of this application can be referred to the above embodiments. Since this reagent adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0042] Based on a general inventive concept, embodiments of this application provide a reagent for evaluating or assisting in the evaluation of the prognostic effects of ovarian aging, the reagent comprising an agent for detecting the relative abundance of *Pleurobacterium johnsonii*.

[0043] This reagent is based on the above application. The specific principle of this application can be referred to the above embodiments. Since this reagent adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0044] Based on a general inventive concept, embodiments of this application provide the application of *Peribrobacter johnsonii* in delaying ovarian aging, the application including the use of *Peribrobacter johnsonii* in agents for delaying ovarian aging.

[0045] The application of *Pseudomonas johnsonii* in delaying ovarian aging is based on the application of *Pseudomonas johnsonii* in ovarian aging biomarkers. The specific principle of the application of *Pseudomonas johnsonii* in ovarian aging biomarkers can be referred to the above embodiments. Since the reagent adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0046] In some alternative implementations, the delay in ovarian aging includes at least one of the following:

[0047] It increases the number of oocytes, upregulates anti-Müllerian hormone, upregulates estradiol levels, and downregulates follicle-stimulating hormone levels.

[0048] In the embodiments of this application, the specific ways of slowing down ovarian aging are defined, which can clarify that *Pseudomonas johnsonii* can affect the number of oocytes, anti-Müllerian hormone, estradiol level and follicle-stimulating hormone level, thereby clarifying the specific mechanism by which *Pseudomonas johnsonii* slows down ovarian aging.

[0049] In some alternative embodiments, the application also includes using the fermentation products of *Pseudomonas johnsonii* in agents for delaying ovarian aging.

[0050] In some alternative embodiments, the fermentation product of the *Pseudomonas johnsonii* includes the supernatant from the culture of *Pseudomonas johnsonii*.

[0051] In the embodiments of this application, it is clear that the supernatant of *Pseudomonas johnsonii* can also delay ovarian aging by affecting the number of oocytes, anti-Müllerian hormone, estradiol levels and follicle-stimulating hormone levels, thereby clarifying that *Pseudomonas johnsonii* and its fermentation products can be used as agents to delay ovarian aging.

[0052] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to industry standards. If there is no corresponding industry standard, then common international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0053] Example 1

[0054] I. Experimental Materials and Methods:

[0055] 1. Experimental materials:

[0056] (1) Experimental animals: C57BL / 6J wild-type mice of different ages were purchased from the Animal Experiment Center of Huazhong Agricultural University. All animal operations were carried out in accordance with the relevant regulations of Huazhong Agricultural University for animal research.

[0057] (2) The main instruments and equipment are shown in Table 1.

[0058] Table 1 Instruments and Equipment

[0059]

[0060]

[0061] (3) The main reagents and kits are shown in Table 2.

[0062] Table 2 Main Reagents and Kits

[0063] Main reagent names Manufacturer or unit Chloroform Equipment Department of Huazhong Agricultural University Isopropanol Equipment Department of Huazhong Agricultural University Anhydrous ethanol Equipment Department of Huazhong Agricultural University paraffin Equipment Department of Huazhong Agricultural University 10% formalin solution Equipment Department of Huazhong Agricultural University fetal bovine serum Gibco Hematoxylin staining solution Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Eosin liquid Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Ki-67 monoclonal antibody Abcam, Inc. (USA) ZO-1 monoclonal antibody Abcam, Inc. (USA) Fecal DNA Kit OMEGA Corporation, USA Trizol Reagent Applied Biosystems, Inc. (USA) Taq DNA polymerase Promega, Inc. (USA) Taq DNA Buffer Promega, Inc. (USA) dNTP mixture HyTest Ltd, Finland Primers Wuhan Qingke Innovation Biotechnology Co., Ltd. DNA marker TaKaRa Company Reverse transcription kit Thermo Scientific neutral resin Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. DAB colorimetric reagent kit Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. AxyPrep DNA Gel Recovery Kit OMEGA Corporation, USA Transfer buffer Gibco

[0064] 2. Experimental Methods:

[0065] (1) Mouse feeding:

[0066] Mice in each group were acclimatized to a standard diet and drinking water for one week. The feed for the mice was strictly sterilized by radiation, and the water bottles, cages and bedding were sterilized by high-pressure steam before use.

[0067] The rearing environment was SPF grade, with the ambient temperature set at 24℃±1℃, and the automatic lighting system adjusted according to the daytime. All procedures followed the guidelines approved by the Animal Experiment Ethics Committee of Huazhong Agricultural University.

[0068] (2) Hematoxylin-eosin (H&E) staining:

[0069] 1) Select paraffin sections with intact cut surfaces and bake them in an oven at 65℃ for 50 minutes;

[0070] 2) Dewaxing the sections to water: Xylene I: 15 min, Xylene II: 15 min, 100% ethanol I: 5 min, 100% ethanol II: 5 min, 95% ethanol: 5 min, 90% ethanol: 5 min, 80% ethanol: 5 min, 70% ethanol: 5 min, distilled water: 5 min;

[0071] 3) H&E staining: hematoxylin: 8 min, tap water rinse: 15 min, 70% alcohol: 4 min, eosin: 1 min;

[0072] 4) Dehydration: 95% alcohol I: 3 min, 95% alcohol II: 3 min, 100% alcohol I: 3 min, 100% alcohol II: 3 min;

[0073] 5) Transparent: Xylene I: 5 min, Xylene II: 5 min;

[0074] 6) Mounting: Mount the slide with neutral resin, cover with a coverslip, and let it air dry at room temperature.

[0075] (3) Tissue immunofluorescence

[0076] 1) Select paraffin sections with intact cut surfaces and bake them in an oven at 65℃ for 50 minutes;

[0077] 2) Dewaxing paraffin sections to water: Xylene I: 20 min, Xylene II: 20 min, 100% ethanol I: 5 min, 100% ethanol II: 5 min, 95% ethanol: 5 min, 90% ethanol: 5 min, 80% ethanol: 5 min, 70% ethanol: 5 min, distilled water: 5 min;

[0078] 3) Antigen retrieval: Place the tissue slides in a retrieval box filled with 0.01 mol / L sodium citrate buffer (pH 7.0) and perform antigen retrieval in a microwave oven. Bring to a boil on medium heat, then turn off the power and wait 10 minutes. Then bring to a boil again on medium-low heat. During this process, prevent excessive evaporation of the buffer and do not dry the slides. After natural cooling, place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker for 5 minutes each time.

[0079] 4) BSA blocking: After the sections are dried, draw a circle around the tissue with a histochemical pen (to prevent the antibody from flowing away), add 3% BSA to the circle to cover the tissue, and block at room temperature for 30 minutes.

[0080] 5) Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in PBS at a certain ratio to the slide, place the slide flat in a humidified chamber, and incubate overnight at 4°C (add a small amount of water to the humidified chamber to prevent antibody evaporation);

[0081] 6) Add secondary antibody: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After the slide is dried, add the secondary antibody of the same species as the primary antibody to the circle to cover the tissue. Incubate at room temperature in the dark for 50 minutes.

[0082] 7) DAPI counterstaining of cell nuclei: Place the slide in PBS (pH 7.4) and wash it 3 times in 5 min on a decolorizing shaker. Add DAPI and incubate for 10 min. Then wash it 3 times in PBS (pH 7.4) in 5 min.

[0083] 8) Shake the slide dry, seal it with an anti-quenching agent, and take a picture.

[0084] (4) EdU staining:

[0085] The EdU staining kit was purchased from Guangzhou Ruibo Biotechnology Co., Ltd. Specific experimental procedures were performed according to the kit (C10310) instructions.

[0086] (5) TUNEL staining:

[0087] Apoptosis was detected using the TUNEL assay: Testes from wild-type and knockout mice were fixed, dehydrated, and cleared, then embedded to prepare paraffin sections, which were then stained with TUNEL according to the following steps:

[0088] 1) Baking: Select paraffin slices with intact cut surfaces and bake them in an oven at 65℃ for 50 minutes;

[0089] 2) Dewaxing the sections to water: Xylene I: 15 min, Xylene II: 15 min, 100% ethanol I: 5 min, 100% ethanol II: 5 min, 95% ethanol: 5 min, 90% ethanol: 5 min, 80% ethanol: 5 min, 70% ethanol: 5 min, distilled water: 5 min;

[0090] 3) Cleaning: Rinse once with 0.85% NaCl for 5 min, then rinse once with PBS for 5 min;

[0091] 4) Fixation: Fix with 4% paraformaldehyde for 15 min;

[0092] 5) Washing: Rinse three times with PBS, 5 minutes each time;

[0093] 6) Penetration: Incubate proteinase K (1:500 v / v, diluted with PBS) at room temperature for 10 min;

[0094] 7) Washing: Rinse three times with PBS, 5 minutes each time;

[0095] 8) Fixation: Fix with 4% paraformaldehyde for 5 minutes;

[0096] 9) Washing: Rinse three times with PBS, 5 minutes each time;

[0097] 10) Equilibrium: Add rTdT reaction equilibrium solution dropwise and incubate at room temperature for 10 min;

[0098] 11) Reaction: Wipe off the equilibration solution, add the pre-prepared TdT reaction solution (equilibration solution: rTdT enzyme: dUTP substrate = 98:1:1), and react at 37℃ for 1.5h;

[0099] 12) Termination: Incubate with 2×SSC for 15 min to terminate the reaction;

[0100] 13) Washing: Rinse three times with PBS, 5 minutes each time;

[0101] 14) Inactivation: Incubate with 3% H2O2 at room temperature for 5 min to inactivate endogenous peroxidase;

[0102] 15) Washing: Rinse three times with PBS, 5 minutes each time;

[0103] 16) Add HRP reactants (1:500, diluted with PBS, by volume) and incubate at room temperature for 30 min;

[0104] 17) Washing: Rinse three times with PBS, 5 minutes each time;

[0105] 18) Color development: Add DAB color development solution (water:substrate:diluent:H2O2 = 17:1:1:1), observe under a microscope immediately to determine the appropriate color development degree, and stop immediately;

[0106] 19) Termination: Rinse with distilled water to stop color development;

[0107] 20) Counterstaining: Add hematoxylin staining solution, 30 seconds, rinse with tap water for 10 minutes;

[0108] 21) Differentiation: Use 1% hydrochloric acid alcohol for 5 seconds;

[0109] 22) Rinse with distilled water for 5 minutes;

[0110] 23 Dehydration: 50% alcohol: 3 min, 70% alcohol: 3 min, 80% alcohol: 3 min, 90% alcohol: 3 min, 100% alcohol I: 3 min, 100% alcohol II: 3 min;

[0111] 24) Transparent: Xylene I: 3 min, Xylene II: 3 min;

[0112] 25) Mounting: After mounting with neutral resin, observe and photograph under a microscope.

[0113] (6) Superovulation and mating:

[0114] For the mouse superovulation experiment, 21-day-old female mice were intraperitoneally injected with pregnant mare serum gonadotropin (PMSG) for 44 hours, followed by hCG injection for 16 hours. The COC complex cell clusters were then extracted from the ampulla of the fallopian tube and thoroughly digested in hyaluronic acid (1:10 in M2). Eight-week-old female mice were then naturally mated with male mice. The vaginal plug in the female mice was examined the following morning; the presence of the plug was considered a sign of successful mating.

[0115] (7) Collection and culture of naked eggs

[0116] 1) Droplet preparation and equilibration: Prepare droplets 30 minutes before culture (approximately 100 μL each of M16 culture medium, 1 ng / mL rh-AMH M16 culture medium, 10 ng / mL rh-AMH M16 culture medium, and 100 ng / mL rh-AMH M16 culture medium). After covering the droplets with paraffin oil, place them in an incubator for pH equilibration. Simultaneously, add a small amount of M16 culture medium to a 1.5 mL centrifuge tube and place it in an incubator for equilibration in preparation for washing oocytes.

[0117] 2) Culture of naked oocytes: Five 3-week-old female mice in each experimental group were quickly euthanized by cervical dislocation, and both ovaries were removed. The ovaries were washed several times in PBS to remove residual blood and fatty tissue from the surface of the ovaries. Then, the ovaries were placed in the center of a disposable culture dish (35 mm), and the ovaries were chopped with a blade to release the oocytes from the follicles. An appropriate amount of naked oocyte collection fluid was added to suspend the ovarian tissue fragments. Oocytes in the GV stage were selected under a stereomicroscope using a homemade mouth pipette. Subsequently, the washed oocytes were transferred into pre-prepared microdroplets for culture.

[0118] III. Experimental Results:

[0119] 1. Using 3M and 10M SPF mice, transcriptome analysis was used to characterize aging changes in ovarian tissue:

[0120] SPF-grade C57BL / 6 mice aged 3 months and 10 months were selected to represent young and ovarian senescence, respectively, and the ovaries of the two groups of mice were sequenced.

[0121] PCA results are as follows Figure 1 As shown in Figure A, ovarian samples from 3-month-old and 10-month-old mice can be clearly distinguished by PC1, indicating that the expression profile of the ovary changes significantly during aging.

[0122] KEGG functional enrichment results are as follows: Figure 1As shown in Figure C, pathways such as B cell receptor signaling, NF-κB signaling, and MAPK signaling are upregulated with aging, suggesting an increase in inflammation and stress response associated with ovarian aging; while... Figure 1 As shown in B, pathways such as cell cycle, oocyte meiosis, and PI3K-Akt signaling are downregulated with aging, reflecting a decline in ovarian function, cell aging, and reduced follicular reserve; these results are consistent with previous research findings.

[0123] 2. Investigating the effects of gut microbiota on ovarian aging through fecal microbiota transplantation:

[0124] To investigate whether gut microbiota affects ovarian aging, this application employed fecal microbiota transplantation (FMT) technology, simultaneously transplanting fecal microbiota from SPF young mice and ovarian-aged mice into germ-free mice (e.g., SPF young mice and mice with ovarian aging mice). Figure 2 A) After gavage, phenotypes related to ovarian aging were examined. Results showed that, compared to recipient mice transplanted with fecal microbiota from young mice (young group), recipient mice transplanted with fecal microbiota from ovarian-aged mice (aged group) had more aged ovaries: Ovarian HE staining and follicle counting at each stage revealed that the young group mice had more cavitary follicles and fewer atretic follicles (e.g., ...). Figure 2 B and Figure 2 (as shown in C); In addition, Ki-67 cell proliferation experiments showed that the proliferation rate of ovarian granulosa cells in young mice was significantly higher than that in the aging group (as shown in C). Figure 2 D and Figure 2 (as shown in E); TUNEL cell apoptosis experiments showed that the apoptosis rate of ovarian granulosa cells in young mice was significantly lower than that in the aging group (as shown in E); Figure 2 F and Figure 2 (As shown in G). The above results indicate that gut microbiota can influence the ovarian aging process.

[0125] 3. Detect changes in gut microbiota composition during ovarian aging and screen for bacterial strains associated with ovarian aging:

[0126] Wild-type C57BL / 6 mice were selected as the research model. Fecal samples from young (3 months old) and ovarian-senescent (10 months old) mice were collected for metagenomic analysis. After standard processing, α-diversity and β-diversity were assessed, and the abundance of bacterial species in the two groups was compared. The results are as follows: Figure 3 As shown, this indicates that there are no significant differences in gut microbiota structure between young and ovarian-aged mice, but there are significant differences in the abundance of some bacterial species (e.g., Figure 3 A, Figure 3 B and Figure 3As shown in C), Bacteroides commonis showed high abundance at 10 months of age, and current research suggests that this bacterium may affect normal ovarian function and induce polycystic ovary syndrome; meanwhile, Akkermania and Parabacteroides johnsonii showed high abundance at 3 months of age, and studies have shown that these two species are depleted after menopause in women; meanwhile, Parabacteroides johnsonii was significantly reduced in the intestines of 10-month-old aged mice.

[0127] These results indicate that there are differences in gut microbiota between young and ovarian-aged mice, and that these differences may be related to ovarian function.

[0128] 4. Parabacteroides johnsonii slows down ovarian aging in mice:

[0129] Parabacteroides johnsonii (experimental group) and physiological saline (control group), as well as the supernatant of Parabacteroides johnsonii culture medium (experimental group) and blank culture medium (control group), were administered to six-month-old female C57 mice by gavage for 12 consecutive weeks. Oocytes from each group were collected and counted. Serum from each group was collected to detect hormone levels in the blood. Results are as follows: Figure 4 As shown, this indicates that Parabacteroides johnsonii and its culture supernatant significantly increased the number of oocytes compared to the control group (e.g., Figure 4 As shown in A), and it can upregulate anti-Müllerian hormone and estradiol levels (e.g., Figure 4 B and Figure 4 As shown in C), downregulate follicle-stimulating hormone (FSH) levels (as shown in Figure C). Figure 4 (as shown in D).

[0130] In summary, the application of *Pseudomonas johnsonii* in ovarian aging biomarkers provided in this application has shown significant differences in *Pseudomonas johnsonii* among mice with different degrees of ovarian aging through experiments. Further experiments have clarified that *Pseudomonas johnsonii* significantly increases the number of oocytes and can upregulate anti-Müllerian hormone, upregulate estradiol levels, and downregulate follicle-stimulating hormone levels. This suggests that *Pseudomonas johnsonii* could be used as a drug to delay ovarian aging.

[0131] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0132] In this application, unless otherwise stated, directional terms such as "upper" and "lower" specifically refer to the drawing directions in the accompanying drawings. Furthermore, in the description of this application, terms such as "comprising" and "including" mean "including but not limited to." In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. In this document, "and / or" describes the relationship between related objects, indicating that three relationships can exist; for example, A and / or B can represent: A alone, A and B simultaneously, or B alone. A and B can be singular or plural. In this document, "at least one" means one or more, and "more than one" means two or more. "At least one," "at least one of the following," or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c" or "at least one of a, b, and c" can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be a single or multiple.

[0133] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Use of Parabacteroides johnsonii for the preparation of a medicament for delaying ovarian aging, characterized in that, The delaying of ovarian aging comprises at least one of: increasing the number of oocytes, upregulating anti-Mullerian hormone, upregulating estradiol levels, and downregulating follicle-stimulating hormone levels.

2. Use according to claim 1, characterized in that, The use also comprises the fermentation product of Parabacteroides johnsonii for use in a medicament for delaying ovarian aging.

3. Use according to claim 2, characterized in that, The fermentation product of Parabacteroides johnsonii comprises a supernatant of a culture of Parabacteroides johnsonii.

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