Application of parabacteroides johnsonii in ovarian senescence

Abstract

The invention relates to the technical field of biology, in particular to application of parabacteroides johnsonii in ovarian senescence. The application comprises the following steps: taking low-abundance parabacteroides johnsonii as a biomarker of ovarian aging; according to the application, the parabacteroides johnsonii is applied to the medicament for delaying ovarian aging; the application also comprises the step of applying the fermentation product of the parabacteroides johnsonii to a medicament for delaying ovarian senescence. Experiments show that the parabacteroides johnsonii has significant difference in mice with different ovarian aging degrees, and subsequent experiments prove that the parabacteroides johnsonii has the effects of significantly increasing the number of oocytes, up-regulating anti-mullerian hormone, up-regulating estradiol level and down-regulating follicle-stimulating hormone level. Therefore, the parabacteroides johnsonii can be used as a medicament for delaying ovarian senescence.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to the application of Parabacteroides johnsonii in ovarian senescence. Background Art

[0002] Female reproductive senescence refers to the decline of ovarian reproductive function. The decline of female ovarian function generally begins at the age of 30 and accelerates after the age of 35. The reserve function in ovarian function is crucial for reproduction. Since the reserve of germ cells and follicles at birth determines the reproductive lifespan and menopause time of mammals, the accelerated reduction of ovarian reserve function will lead to premature senescence or insufficiency of the ovaries. At the same time, the ovarian reserve function will be depleted rapidly with age, mainly manifested as more follicular atresia and apoptosis. In addition to the reduction of follicle reserve, the quality of oocytes will also decline with the increase of female age. Currently, the quality of oocytes generally begins to deteriorate gradually after the age of 30, which coincides with the time of the reduction of oocyte number, and this also partly explains the phenomenon that female fertility declines before the onset of menopause and that reproductive senescent women show endocrine abnormalities. The senescence of oocytes is mainly manifested as characteristics such as lectin dysfunction, low cross-maturation efficiency, chromosome misalignment, damaged meiotic spindle, telomere wear, DNA damage, and mitochondrial dysfunction. And with age, the abnormal development of oocytes caused by the decline in oocyte quality has become one of the main reasons for female reproductive disorders.

[0003] In addition to reproductive function, the ovaries can also maintain female hormone secretion, which is also very important in maintaining female health. At present, ovarian senescence can cause disorders in the regulation of hypothalamic-pituitary-ovarian axis hormone secretion, resulting in an increase in the level of follicle-stimulating hormone (FSH) and a decrease in the level of anti-Müllerian hormone (AMH). Excessive FSH in the serum will directly act on hippocampal and cortical neurons, accelerating the deposition of amyloid-β and tau, damaging cognitive ability and at the same time making the body show the characteristics of Alzheimer's disease. At the same time, the depletion of follicles in the ovaries will cause a decrease in the level of estrogen secretion in the body until menopause. Estrogen is a steroid hormone with a wide range of important physiological functions and has obvious benefits for other systems of the body such as endocrine, bone, nerve, and cardiovascular. In addition, relevant studies have confirmed that estrogen also has potential therapeutic value for a variety of lung diseases such as asthma, pulmonary hypertension, and pulmonary fibrosis, and even has a certain protective effect on respiratory virus infections. In summary, ovarian senescence is a complex process involving multiple factors. Therefore, a deeper understanding of ovarian senescence will play an important role in the process of healthy aging of humans and bring more extensive social benefits.

[0004] The significant feature of ovarian aging is the decline in the quantity and quality of oocytes. The aging process is often accompanied by changes in the composition of gut microbiota, and gut microbiota also has an impact on aging. In recent years, studies have found that there may be an interaction between ovarian aging and gut microbiota, and the gut microbiota may play a role in the process of ovarian aging. Additionally, in a cohort study including women with premature ovarian insufficiency (POI) and healthy women, researchers found that compared with healthy women, the gut microbiota of POI women had changed, and these changes were closely related to the changes in hormone levels associated with ovarian aging. Therefore, these all suggest that the process of ovarian aging may be related to gut microbiota.

[0005] Current exploration of the related mechanisms of small molecules in delaying ovarian aging suggests that microorganisms may be involved in the regulatory process of drugs in delaying ovarian aging. For example, some studies have found that resveratrol can significantly reduce the effects of tBHP-induced oxidative stress on the spawning rate and the number of follicles, and activate the SIRT1 / FoxO1 and Nrf2 pathways, thereby enhancing the expression of antioxidant genes. At the same time, some studies have found that the gut microbiota plays a crucial role in ovarian oxidative stress by maintaining tryptophan metabolism through the kynurenine pathway. However, there is currently no relevant report on Parabacteroides johnsonii in the gut microbiota in ovarian aging. Summary of the Invention

[0006] This application provides an application of Parabacteroides johnsonii in ovarian aging to fill the gap in the association between Parabacteroides johnsonii in gut microbiota and ovarian aging in the existing technology.

[0007] In the first aspect, this application provides an application of Parabacteroides johnsonii in ovarian aging biomarkers, and the application includes:

[0008] Using Parabacteroides johnsonii with low abundance as a biomarker for ovarian aging.

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

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

[0011] Optionally, when the abundance of Parabacteroides johnsonii is lower than 0.77 times the normal abundance of Parabacteroides johnsonii, it is determined that the ovary is in a state of aging at this time.

[0012] In the second aspect, this application provides a reagent for screening or assisting in screening ovarian aging, and the reagent includes an agent for detecting the relative abundance of Parabacteroides johnsonii.

[0013] Thirdly, the present application provides a reagent for evaluating or assisting in evaluating the prognosis of ovarian aging, and the reagent includes an agent for detecting the relative abundance of Parabacteroides johnsonii.

[0014] Fourthly, the present application provides an application of Parabacteroides johnsonii in delaying ovarian aging, and the application includes using Parabacteroides johnsonii in a medicament for delaying ovarian aging.

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

[0016] Increasing the number of oocytes, upregulating anti-Müllerian hormone, upregulating estradiol level, and downregulating follicle-stimulating hormone level.

[0017] Optionally, the application further includes using the fermentation product of Parabacteroides johnsonii in a medicament for delaying ovarian aging.

[0018] Optionally, the fermentation product of Parabacteroides johnsonii includes the supernatant of culturing Parabacteroides johnsonii.

[0019] The above technical solutions provided by the embodiments of the present application have the following advantages compared with the prior art:

[0020] In the application of Parabacteroides johnsonii in ovarian aging provided by the embodiments of the present application, through a large number of experiments, it is found that Parabacteroides johnsonii shows a high abundance in younger mice and a significantly reduced abundance in older mice. Based on this difference, it can be determined that Parabacteroides johnsonii has the potential to be used as a biomarker for ovarian aging. Subsequently, through verification experiments, it is confirmed that Parabacteroides johnsonii can significantly increase the number of oocytes, upregulate anti-Müllerian hormone, upregulate estradiol level, and downregulate follicle-stimulating hormone level. Therefore, Parabacteroides johnsonii can also be used as a medicament for delaying ovarian aging, thus filling the gap in the research of Parabacteroides johnsonii in the field of ovarian aging among gut microbiota. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings herein are incorporated into the specification and form a part of the specification, showing embodiments consistent with the present application, and are used together with the specification to explain the principles of the present application.

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 It is a schematic diagram of the transcriptome sequencing results of 3-month-old and 10-month-old mice provided by the embodiments of the present application, whereFigure 1 A is the PCA result graph of two groups of mice, Figure 1 B is the schematic diagram of the differential expression gene analysis results of two groups of mice, Figure 1 C is the KEGG function enrichment result graph of two groups of mice;

[0024] Figure 2 It is the schematic diagram of the fecal microbiota transplantation results of SPF young mice and ovarian senescent mice provided in the embodiments of the present application. Among them, Figure 2 A is the schematic diagram of the fecal microbiota transplantation process of SPF young mice and ovarian senescent mice, Figure 2 B is the schematic diagram of the ovarian HE staining results of two groups of mice, Figure 2 C is the graph of the follicle count results of two groups of mice at all levels, Figure 2 D is the schematic diagram of the staining results of Ki-67 cell proliferation, Figure 2 E is the statistical result graph of the positive Ki-67 cell proliferation, Figure 2 F is the schematic diagram of the staining results of TUNEL cell apoptosis, Figure 2 G is the statistical result schematic diagram of the positive TUNEL cell apoptosis;

[0025] Figure 3 It is the schematic diagram of the metagenomic detection results of 3-month-old and 10-month-old C57BL / 6 mice provided in the embodiments of the present application. Among them, Figure 3 A is the graph of the α-diversity analysis results of two groups of mice, Figure 3 B is the graph of the β-diversity analysis results of two groups of mice, Figure 3 C is the graph of the species abundance of two groups of mice;

[0026] Figure 4 It is the result graph of the influence of Parabacteroides johnsonii and its culture medium supernatant on ovarian senescence provided in the embodiments of the present application. Among them, Figure 4 A is the result graph of the influence of Parabacteroides johnsonii and its culture medium supernatant on the number of oocytes, Figure 4 B is the result graph of the influence of Parabacteroides johnsonii and its culture medium supernatant on anti-Müllerian hormone, Figure 4 C is the result graph of the influence of Parabacteroides johnsonii and its culture medium supernatant on estradiol, Figure 4 D is the result graph of the influence of Parabacteroides johnsonii and its culture medium supernatant on follicle-stimulating hormone. Detailed implementation manners

[0027] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the following will clearly and completely describe the technical solutions in the embodiments of this application with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are some, but not all, of the embodiments of this application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts fall within the scope of protection of this application.

[0028] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, equipment, etc. used in this application can be obtained through market purchases or can be prepared by existing methods.

[0029] The creative thinking of this application is as follows:

[0030] A significant feature of ovarian aging is the decline in the quantity and quality of oocytes. The aging process is often accompanied by changes in the composition of the gut microbiota, and the gut microbiota also affects ovarian aging. In recent years, studies have found that there may also be an interaction between ovarian aging and the gut microbiota. For example, vaginal atrophy was observed in ovariectomized mice, accompanied by changes in the gut microbiota. When the gut microbiota of female mice with intact ovaries was transplanted into ovariectomized female mice, a significant alleviation of vaginal epithelial atrophy was found. This result indicates that normal ovarian function helps regulate the gut microbiota of mice, and the gut microbiota may have a regulatory effect on 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 the bodies of ovarian aging mice (42-week-old), it was found that the "young" gut microbiota could reshape the gut microbiota of ovarian aging mice through fecal microbiota transplantation, and slow down the loss of ovarian function in ovarian aging mice by reducing follicular atresia and apoptosis, increasing granulosa cell proliferation, and improving the ovarian immune microenvironment. In addition, in a cohort study involving women with premature ovarian insufficiency (POI) and healthy women, the researchers found that compared with healthy women, the gut microbiota of POI women had changed, and these changes were closely related to the changes in hormone levels associated with ovarian aging. These two studies both imply that the process of ovarian aging may be related to the gut microbiota.

[0032] Current research on the mechanisms of small molecules in delaying ovarian aging suggests that microorganisms may be involved in the regulatory process of drugs in delaying ovarian aging. For example, studies have found that resveratrol can significantly reduce the effects of tBHP-induced oxidative stress on oviposition rate and follicle number, and activate the SIRT1 / FoxO1 and Nrf2 pathways, thereby enhancing the expression of antioxidant genes. At the same time, studies have found that the gut microbiota plays a crucial role in ovarian oxidative stress by maintaining tryptophan metabolism through the kynurenine pathway. However, there is currently no report on the role of Parabacteroides johnsonii in ovarian aging among gut microorganisms.

[0033] An embodiment of the present application provides an application of Parabacteroides johnsonii in ovarian aging biomarkers, and the application includes:

[0034] Using low-abundance Parabacteroides johnsonii as a biomarker for ovarian aging.

[0035] In some alternative embodiments, the abundance of Parabacteroides johnsonii is positively correlated with the degree of ovarian aging.

[0036] In the embodiment of the present application, defining a positive correlation between the abundance of Parabacteroides johnsonii and the degree of ovarian aging can clarify the specific abundance of Parabacteroides johnsonii, thereby clarifying the accuracy of Parabacteroides johnsonii as a biomarker for ovarian aging.

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

[0038] In some alternative embodiments, when the abundance of Parabacteroides johnsonii is less than 0.77 times the normal abundance of Parabacteroides johnsonii, it is determined that the ovary is in a state of aging at this time.

[0039] In the embodiment of the present application, by defining the specific multiple of the abundance of Parabacteroides johnsonii higher than the normal abundance of Parabacteroides johnsonii, the minimum abundance of Parabacteroides johnsonii can be clarified, thereby improving the specificity and sensitivity of Parabacteroides johnsonii as a biomarker.

[0040] Based on a general inventive concept, an embodiment of the present application provides a reagent for screening or assisting in screening ovarian aging, and the reagent includes a medicament for detecting the relative abundance of Parabacteroides johnsonii.

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

[0042] Based on a general inventive concept, an embodiment of the present application provides a reagent for evaluating or assisting in evaluating the prognosis of ovarian aging, and the reagent includes an agent for detecting the relative abundance of Parabacteroides johnsonii.

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

[0044] Based on a general inventive concept, an embodiment of the present application provides an application of Parabacteroides johnsonii in delaying ovarian aging, and the application includes using Parabacteroides johnsonii in a medicament for delaying ovarian aging.

[0045] The application of Parabacteroides johnsonii in delaying ovarian aging is implemented based on the above application of Parabacteroides johnsonii in ovarian aging biomarkers. For the specific principle of the application of Parabacteroides johnsonii in ovarian aging biomarkers, reference can be made to the above embodiments. Since this reagent adopts some or all of the technical solutions of the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments, and will not be elaborated one by one here.

[0046] In some alternative embodiments, the delaying of ovarian aging includes at least one of the following:

[0047] Increasing the number of oocytes, upregulating anti-Müllerian hormone, upregulating estradiol level, and downregulating follicle-stimulating hormone level.

[0048] In the embodiments of the present application, by defining the specific ways of slowing down ovarian aging, it can be clarified that Parabacteroides johnsonii can affect the number of oocytes, anti-Müllerian hormone, estradiol level, and follicle-stimulating hormone level, so as to clarify the specific action mode of Parabacteroides johnsonii in delaying ovarian aging.

[0049] In some alternative embodiments, the application further includes using the fermentation product of Parabacteroides johnsonii in a medicament for delaying ovarian aging.

[0050] In some alternative embodiments, the fermentation product of Parabacteroides johnsonii includes the supernatant of culturing Parabacteroides johnsonii.

[0051] In the embodiments of the present application, it is clarified that the supernatant of Parabacteroides johnsonii can also delay ovarian aging by affecting the number of oocytes, anti-Müllerian hormone, estradiol level, and follicle-stimulating hormone level, so as to clarify that Parabacteroides johnsonii and its fermentation product can be used as medicaments for delaying ovarian aging.

[0052] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions noted in the following embodiments, they are generally determined according to industry standards. If there is no corresponding industry standard, they are carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0053] Example 1

[0054] I. Experimental materials and methods:

[0055] 1. Test materials:

[0056] (1) Test animals: C57BL / 6J wild-type mice of different weeks of age were purchased from the Animal Experiment Center of Huazhong Agricultural University, and all animal operations followed the relevant regulations of Huazhong Agricultural University on animal research.

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

[0058] Table 1 Instruments and equipment

[0059]

[0060]

[0061] (3) Main reagents and reagent kits are shown in Table 2.

[0062] Table 2 Main reagents and reagent kits

[0063] Names of Main Reagents Manufacturer or Company Chloroform Equipment Section, Huazhong Agricultural University Isopropanol Equipment Section, Huazhong Agricultural University Absolute Ethanol Equipment Section, Huazhong Agricultural University Paraffin Equipment Section, Huazhong Agricultural University 10% Formalin Solution Equipment Section, Huazhong Agricultural University Fetal Bovine Serum Gibco Hematoxylin Staining Solution Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Eosin Solution Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Ki-67 Monoclonal Antibody Abcam, USA ZO-1 Monoclonal Antibody Abcam, USA Fecal DNA Kit OMEGA, USA Trizol Reagent Applied Biosystems, USA Taq DNA Polymerase Promega, USA Taq DNA Buffer Promega, USA dNTP Mix HyTest Ltd, Finland Primer Wuhan Qingke Innovation Biotechnology Co., Ltd. DNA Marker TaKaRa Reverse Transcription Kit Thermo Scientific Neutral Resin Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. DAB Chromogenic Kit Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. AxyPrep DNA Gel Extraction Kit OMEGA, USA Transfer Membrane Buffer Gibco

[0064] 2. Experimental methods:

[0065] (1) Mouse feeding:

[0066] Mice in each group were adaptively fed with standard diet and drinking water for one week. At the same time, the feed for feeding the mice was strictly irradiated and sterilized, and the water bottles, breeding cages and bedding were all used after being sterilized by high-pressure steam.

[0067] The breeding environment was of SPF level, the environmental temperature was set at 24°C ± 1°C, and the automatic lighting device was adjusted according to the day and night time. 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) Pick paraffin sections with intact cut surfaces and bake them in an oven at 65°C for 50 min;

[0070] 2) Dewax the sections to water: Xylene I: 15 min, Xylene II: 15 min, 100% Alcohol I: 5 min, 100% Alcohol II: 5 min, 95% Alcohol: 5 min, 90% Alcohol: 5 min, 80% Alcohol: 5 min, 70% Alcohol: 5 min, Distilled Water: 5 min;

[0071] 3) H&E staining: Hematoxylin: 8 min, Rinse with tap water: 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) Clearing: Xylene I: 5 min, Xylene II: 5 min;

[0074] 6) Mounting: Mount with neutral balsam, cover with a coverslip, and air dry at room temperature.

[0075] (3) Tissue immunofluorescence

[0076] 1) Pick paraffin sections with intact cut surfaces and bake the sections in an oven at 65 °C for 50 min;

[0077] 2) Dewax the paraffin sections to water: Xylene I: 20 min, Xylene II: 20 min, 100% Alcohol I: 5 min, 100% Alcohol II: 5 min, 95% Alcohol: 5 min, 90% Alcohol: 5 min, 80% Alcohol: 5 min, 70% Alcohol: 5 min, Distilled Water: 5 min;

[0078] 3) Antigen retrieval: Place the tissue sections in a retrieval box filled with 0.01 mol / L sodium citrate buffer (pH 7.0) and perform antigen retrieval in a microwave oven. After heating to boiling on medium power, turn off the power and let it stand for 10 min, then use medium-low power until boiling again. During this process, prevent the buffer from evaporating excessively and do not let the sections dry; after natural cooling, place the slides in PBS (pH 7.4) and shake on a shaker for 3 washes, 5 min each;

[0079] 4) BSA blocking: After draining the sections, use a histochemical pen to draw a circle around the tissue (to prevent the antibody from flowing away), and add 3% BSA in the circle to cover the tissue, and block at room temperature for 30 min;

[0080] 5) Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared by mixing with PBS in a certain proportion on the sections, place the sections flat in a wet box, and incubate overnight at 4 °C (add a small amount of water in the wet box to prevent the antibody from evaporating);

[0081] 6) Add secondary antibody: Place the slides in PBS (pH 7.4) and shake on a shaker for 3 washes, 5 min each. After the sections are air-dried, add the secondary antibody corresponding to the primary antibody within the circle to cover the tissue and incubate in the dark at room temperature for 50 min;

[0082] 7) DAPI counterstain for cell nuclei: Place the slides in PBS (pH 7.4) and wash 3 times within 5 min on a shaker. After adding DAPI and incubating for 10 min, wash 3 times with PBS (pH 7.4) within 5 min;

[0083] 8) Air-dry the slides, mount with anti-quencher, and take pictures.

[0084] (4) EdU staining:

[0085] The EdU staining kit was purchased from Guangzhou Ribobio Co., Ltd. The specific experimental steps were carried out according to the instructions of the kit (C10310).

[0086] (5) TUNEL staining:

[0087] Cell apoptosis was detected by the TUNEL method: Testes from wild-type and knockout mice were taken, fixed, dehydrated, cleared, and embedded in paraffin sections. TUNEL staining was performed according to the following steps:

[0088] 1) Bake the sections: Select paraffin sections with intact cut surfaces and bake in an oven at 65 °C for 50 min;

[0089] 2) Dewax 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) Wash: Rinse once with 0.85% NaCl for 5 min and once with PBS for 5 min;

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

[0092] 5) Wash: Wash 3 times with PBS, 5 min each;

[0093] 6) Permeabilize: Incubate with proteinase K (1:500 in volume, diluted with PBS) at room temperature for 10 min;

[0094] 7) Wash: Wash 3 times with PBS, 5 min each;

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

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

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

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

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

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

[0101] 14) Inactivation: Use H 2 O 2 with a concentration of 3% and incubate at room temperature for 5 min to inactivate endogenous peroxidase;

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

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

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

[0105] 18) Color development: Add DAB color development solution (water: substrate: diluent: H 2 O 2 = 17:1:1:1), immediately observe under the microscope to control the appropriate color development degree, and terminate immediately;

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

[0107] 20) Counterstaining: Add hematoxylin stain solution for 30 sec and rinse with tap water for 10 min;

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

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

[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) Clearing: Xylene I: 3 min, Xylene II: 3 min;

[0112] (25) Mounting the specimen: Mount the specimen with neutral resin and then observe and photograph it under a microscope.

[0113] (6) Superovulation and mating:

[0114] For the superovulation experiment in mice, 44 h after intraperitoneal injection of pregnant mare serum gonadotropin (PMSG) into female mice at 21 days old, hCG was injected 16 h later. The cumulus-oocyte complex (COC) cell mass was removed from the ampulla of the fallopian tube, and the cumulus cells were fully digested in hyaluronic acid (1:10 in M2). Female mice at 8 weeks old were naturally mated with male mice, and the vaginal plugs of female mice were checked the next morning. The presence of a vaginal plug was used as a marker of successful mating.

[0115] (7) Collection and culture of denuded oocytes

[0116] 1) Preparation and equilibration of microdrops: Microdrops (about 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) were prepared 30 min before culture. After covering the microdrops with paraffin oil, they were placed in an incubator for pH equilibration. At the same time, a small amount of M16 culture medium was added to a 1.5 mL centrifuge tube and placed in the incubator for equilibration to prepare for washing the oocytes.

[0117] 2) Culture of denuded oocytes: Five 3-week-old female mice were selected from each experimental group, quickly sacrificed by cervical dislocation, and both ovaries were removed. The ovaries were washed several times in PBS to remove residual blood and adipose tissue on the ovarian surface, and then the ovaries were placed in the center of a disposable culture dish (35 mm). The ovaries were chopped with a blade to release the oocytes from the follicles; an appropriate amount of denuded oocyte picking solution was added to suspend the ovarian tissue fragments, and the oocytes at the germinal vesicle (GV) stage were selected with a self-made mouth pipette under a stereomicroscope; subsequently, the washed oocytes were transferred into the pre-prepared microdrops for culture.

[0118] III. Experimental results:

[0119] 1. Select 3M and 10M SPF mice and use transcriptome to characterize the aging changes of ovarian tissue:

[0120] Three-month-old and 10-month-old SPF C57BL / 6 mice were selected to represent the young and ovarian aging states respectively, and the ovaries of the two groups of mice were subjected to transcriptome sequencing.

[0121] The results of PCA are as Figure 1 shown in A. The ovarian samples of 3-month-old and 10-month-old mice could be clearly distinguished on PC1, indicating that significant changes occurred in the expression profiles during ovarian aging.

[0122] The KEGG functional enrichment results are as follows Figure 1 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 responses associated with ovarian aging. And as Figure 1 shown in Figure B, pathways such as the cell cycle, oocyte meiosis, and PI3K-Akt signaling are downregulated with aging, reflecting a decline in ovarian function, cellular aging, and a reduction in follicle reserve. These results are consistent with previous studies.

[0123] 2. Exploring the effect of gut microbiota on ovarian aging through fecal microbiota transplantation experiments:

[0124] To explore whether gut microbiota affects ovarian aging, this application uses the fecal microbiota transplantation technique to simultaneously transplant the feces of SPF young mice and ovarian aging mice into germ-free mice (as Figure 2 shown in Figure A). After gavage, relevant phenotypes of ovarian aging were detected. The results showed that compared with the recipient mice transplanted with the feces of young mice (young group), the ovaries of the recipient mice transplanted with the feces of ovarian aging mice (aging group) were more senescent: by performing HE staining of the ovaries and counting follicles at all levels, it was found that the young group of mice had more antral follicles and fewer atretic follicles (as Figure 2 shown in Figures B and Figure 2 C); in addition, the Ki-67 cell proliferation experiment showed that the proliferation rate of ovarian granulosa cells in the young group of mice was significantly higher than that in the aging group (as Figure 2 shown in Figures D and Figure 2 E); the TUNEL cell apoptosis experiment showed that the apoptosis rate of ovarian granulosa cells in the young group of mice was significantly lower than that in the aging group (as Figure 2 shown in Figures F and Figure 2 G). The above results indicate that gut microbiota can affect the process of ovarian aging.

[0125] 3. Detecting changes in the gut microbiota composition during ovarian aging and screening for bacteria species related to ovarian aging:

[0126] Wild-type C57BL / 6 mice were selected as the research model, and feces from young (3-month-old) and ovarian aging (10-month-old) mice were collected for metagenomic detection. After processing through the standard procedure, α-diversity and β-diversity were evaluated respectively and the bacterial species abundances of the two groups of samples were compared. The results are as Figure 3 shown, indicating that there are no significant differences in the gut microbiota community structures between young and ovarian aging mice, but there are significant differences in the abundances of some bacterial species (as Figure 3 shown in Figure A, Figure 3 Figure B, and Figure 3As shown in C, Bacteroides vulgatus showed a high abundance at 10 months of age. Currently, some studies have shown that this bacterium may affect the normal function of the ovaries and induce polycystic ovary syndrome. At the same time, Akkermansia and Parabacteroides johnsonii showed high abundances at 3 months of age. Some studies have shown that these two bacterial species are depleted after menopause in women. At the same time, the abundance of Parabacteroides johnsonii was significantly reduced in the intestines of 10-month-old old mice.

[0127] The above results indicate that there are differences in the gut microbiota between young and ovarian senescent mice, and these differences may be related to ovarian function.

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

[0129] Parabacteroides johnsonii (experimental group) and normal saline (control group), as well as the supernatant of the medium cultured with Parabacteroides johnsonii (experimental group) and blank medium (control group) were respectively gavaged into 6-month-old C57 female mice for 12 consecutive weeks. Oocytes of each group of mice were collected for counting, and sera of each group of mice were collected to detect hormone levels in the blood. The results are as Figure 4 shown, indicating that Parabacteroides johnsonii and its medium supernatant can significantly increase the number of oocytes compared with the control group (as Figure 4 shown in A), and can up-regulate the levels of anti-Müllerian hormone and estradiol (as Figure 4 shown in B and Figure 4 C), and down-regulate the level of follicle-stimulating hormone (FSH) (as Figure 4 shown in D).

[0130] In summary, the application of Parabacteroides johnsonii provided in the embodiments of the present application in ovarian senescence biomarkers found through experiments that there are significant differences in Parabacteroides johnsonii in mice with different degrees of ovarian senescence, and through subsequent experiments, it was clarified that Parabacteroides johnsonii can significantly increase the number of oocytes, up-regulate anti-Müllerian hormone, up-regulate estradiol levels and down-regulate follicle-stimulating hormone levels, suggesting that Parabacteroides johnsonii can be used as an agent for delaying ovarian senescence.

[0131] The various embodiments of the present application may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be construed as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single 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 that range, such as 1, 2, 3, 4, 5, and 6, and this applies regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0132] In the present application, unless otherwise stated, the orientation terms such as "upper" and "lower" specifically refer to the drawing directions in the figures. Additionally, in the description of the specification of the present application, the terms "comprising", "including", etc. mean "including but not limited to". In this document, relational terms such as "first" and "second" are only used 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 associated relationship of associated objects and indicates that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist simultaneously, or B exists alone. Where A and B may be singular or plural. In this document, "at least one" means one or more, and "a plurality" means two or more. "At least one kind", "at least one of the following items (pieces)" or similar expressions refer to any combination of these items, including any combination of single items (pieces) or plural items (pieces). For example, "at least one of a, b, or c", or, "at least one of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0133] The above description is only the specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. Application of Parabacteroides johnsonii in ovarian senescence biomarkers, Characterized in that, The application includes: Using low-abundance Parabacteroides johnsonii as a biomarker for ovarian senescence.

2. The application according to claim 1, Characterized in that, The abundance of Parabacteroides johnsonii is positively correlated with the degree of ovarian senescence.

3. The application according to claim 1, Characterized in that, The abundance of Parabacteroides johnsonii is lower than 0.77 times the abundance of normal Parabacteroides johnsonii.

4. The application according to claim 1, Characterized in that, When the abundance of Parabacteroides johnsonii is lower than 0.77 times the abundance of normal Parabacteroides johnsonii, it is determined that the ovary is in a state of senescence at this time.

5. A reagent for screening or assisting in screening ovarian senescence, Characterized in that, The reagent includes a medicament for detecting the relative abundance of Parabacteroides johnsonii.

6. A reagent for evaluating or assisting in evaluating the prognosis of ovarian senescence, Characterized in that, The reagent includes a medicament for detecting the relative abundance of Parabacteroides johnsonii.

7. Application of Parabacteroides johnsonii in delaying ovarian senescence, Characterized in that, The application includes using Parabacteroides johnsonii in a medicament for delaying ovarian senescence.

8. The application according to claim 7, Characterized in that, The delaying of ovarian senescence includes at least one of the following: Increasing the number of oocytes, upregulating anti-Müllerian hormone, upregulating estradiol levels, and downregulating follicle-stimulating hormone levels.

9. The application according to claim 7, Characterized in that, The application further includes using the fermentation product of Parabacteroides johnsonii in a medicament for delaying ovarian senescence.

10. The application according to claim 9, Characterized in that, The fermentation product of Parabacteroides johnsonii includes the supernatant of culturing Parabacteroides johnsonii.

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