Use of bacteroides xylanisolvens in preparation of medicine for preventing or treating polycystic ovary syndrome

Abstract

The application belongs to the field of microbiology and medical technology, and relates to application of Bacteroides xylanisolvens in preparation of a medicine for preventing or treating polycystic ovary syndrome. Bacteroides xylanisolvens In vitro experiments show that the Bacteroides xylanisolvens can degrade androstenedione in vitro. Animal experiments prove that the Bacteroides xylanisolvens can reduce the androgen level of a polycystic ovary syndrome model animal, restore the ovary function, and effectively improve the polycystic ovary syndrome symptoms. The application provides a safe and efficient new treatment scheme for preventing and treating the polycystic ovary syndrome in clinic. Therefore, the application has important clinical significance and wide market prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and pharmaceutical technology, specifically relating to Bacteroides xylanoplasmosis (… Bacteroides xylanisolvens The application of *Bacteroides xylana* in the preparation of drugs for the prevention or treatment of polycystic ovary syndrome, particularly in the preparation of drugs for the treatment of polycystic ovary syndrome-related hyperandrogenemia and its complications. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder characterized by hyperandrogenemia, ovulation disorders, and polycystic ovarian morphology, often accompanied by complications such as hirsutism and acne. The global prevalence of PCOS among women of reproductive age is approximately 6%-20%, with 70%-80% of patients experiencing infertility, severely impacting their fertility needs, mental and physical health, and quality of life.

[0003] Current treatments for PCOS mainly include the following categories: 1. Anti-androgen therapy, such as spironolactone, which works by competitively binding to androgen receptors; 2. Oral short-acting contraceptive therapy, such as ethinylestradiol and cyproterone acetate, used to regulate the menstrual cycle and lower androgen levels; 3. Ovulation induction therapy, such as letrozole, suitable for patients who wish to conceive; 4. Metabolic regulation therapy, such as metformin, used to improve metabolic disorders; 5. Topical symptomatic treatment, such as topical retinoic acid to improve acne and laser hair removal to improve hirsutism. However, all of the above treatments have significant limitations: anti-androgen drugs are prone to causing adverse reactions such as hyperkalemia, oral short-acting contraceptives pose a risk of thrombosis, and ovulation induction drugs may lead to complications such as ovarian hyperstimulation syndrome. Moreover, none of these drugs can fundamentally improve the pathophysiological disorders of PCOS. Therefore, developing novel, highly effective, safe, and targeted drugs for the treatment of PCOS has significant clinical value and application prospects.

[0004] The gut microbiota, often called "human's second genome," consists of approximately 100 trillion microorganisms that colonize the human digestive system, forming a symbiotic relationship of interdependence and mutual regulation with the host. The gut microbiota plays a crucial role in physiological processes such as digestion and absorption, immune regulation, drug metabolism, and hormone transformation. Recent studies have confirmed that gut microbiota imbalance is closely related to the pathogenesis of PCOS: compared to healthy women, PCOS patients have significantly reduced gut microbiota diversity, with a higher prevalence of Bacteroides commonis (Bacteroides vulgaris). Bacteroides vulgatus Abnormal changes in the abundance of specific gut microbiota, such as bile acid synthesis disorders, glucose metabolism disturbances, lipid metabolism imbalances, and sex hormone imbalances, can ultimately induce or aggravate PCOS symptoms. Therefore, intervention strategies targeting the gut microbiota-ovarian axis have become a new direction and research hotspot in the treatment of PCOS.

[0005] xylanbacterium ( Bacteroides xylanisolvens *Bacteroides xylanoplastinus* is a Gram-negative anaerobic bacterium widely found in the human gut. It possesses a highly efficient ability to degrade xylan, and the various bioactive substances produced during its metabolism can exert beneficial effects on the host through pathways such as regulating immune balance, improving metabolic function, and protecting neurological health. However, currently, no studies have reported any ameliorative or therapeutic effects of *Bacteroides xylanoplastinus* on PCOS and its hyperandrogenemia, nor has its application in the preparation of drugs for the prevention or treatment of PCOS been disclosed, indicating a significant technological gap. Summary of the Invention

[0006] To fill the gap in the application of Bacteroides xylanin in the treatment of PCOS and overcome the shortcomings of existing drugs for the treatment of hyperandrogenemia, such as large side effects and limited efficacy, this invention provides the application of Bacteroides xylaninin in the preparation of drugs for the prevention or treatment of PCOS, and at the same time provides a new, safe and efficient technical solution for the clinical treatment of hyperandrogenemia.

[0007] Specifically, the present invention is achieved through the following technical solutions: This invention provides the use of Bacteroides xylanisolvens in the preparation of medicaments for the prevention or treatment of polycystic ovary syndrome.

[0008] Preferably, the xylanobacterium used in this invention is a known strain, which has been reported in many publications and can be purchased commercially.

[0009] Specifically, when test animals develop PCOS-like symptoms (including but not limited to elevated levels of key androgens such as androstenedione, ovulation disorders, and polycystic ovarian changes) through subcutaneous injection of dehydroepiandrosterone (DHEA), the *Bacteroides xylana* can significantly reduce androgen levels and restore normal ovarian function in the test animals, thereby improving PCOS-like symptoms.

[0010] Alternatively, in the above applications, the *Bacteroides xylana* strain can be stably passaged in conventional anaerobic culture media and has in vitro proliferation activity and in vivo colonization ability.

[0011] As an optional method, in the above application, the culture method of Bacteroides xylana is as follows: under a strictly anaerobic environment, Bacteroides xylana is inoculated into a closed liquid anaerobic culture medium and cultured statically at 37°C for 24 hours; the liquid anaerobic culture medium is GAM medium, and after the culture medium is autoclaved, heme chloride is added to the medium at a final concentration of 5 μg / mL.

[0012] Alternatively, in the above applications, the drug uses Bacteroides xylana as the active ingredient and also contains a pharmaceutically acceptable carrier.

[0013] Specifically, the excipients mentioned above can be rationally selected according to the specific route of administration (such as oral, enteral, etc.) to ensure the stability, efficacy, and safety of the drug. The drug may further include auxiliary active ingredients, diluents, sweeteners, flavorings, or preservatives to optimize the dosage form, taste, or shelf life of the drug.

[0014] Alternatively, in the above applications, the drug is in oral dosage form.

[0015] Alternatively, in the above applications, the oral dosage form is selected from granules, capsules, tablets, powders, oral solutions, suspensions, or emulsions.

[0016] The above dosage forms can be prepared using conventional processes in the field of pharmaceutical formulation.

[0017] Alternatively, in the above applications, the drug is used to prevent or treat hyperandrogenemia, ovulation disorders, or polycystic ovarian changes associated with polycystic ovary syndrome.

[0018] In the pharmaceutical applications described herein, the timing, frequency, and duration of administration of Bacteroides xylana should be determined based on the specific diagnostic results of the condition, which is within the technical scope of those skilled in the art.

[0019] Applying treatment regimens used on test animals to humans allows for the conversion of the effective dose of all drugs in humans to the effective dose in test animals, which is easily achievable by those skilled in the art.

[0020] To better understand the essence of the present invention, the following detailed description uses pharmacodynamic experiments and their results to further illustrate the novel use of Bacteroides xylanoplastin in the pharmaceutical field.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The advantages of this invention are that in vitro experiments show that *Bacteroides xylana* can degrade androstenedione in vitro. Animal experiments have confirmed that *Bacteroides xylana* can reduce androgen levels and restore ovarian function in polycystic ovary syndrome (PCOS) models, effectively improving PCOS symptoms. This invention provides a safe and efficient novel treatment option for the clinical prevention and treatment of PCOS. Therefore, this invention has significant clinical value and broad market prospects.

[0022] It should be understood that, within the scope of this invention, the above-mentioned technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or better technical solutions, all of which fall within the protection scope of this invention. Detailed Implementation

[0023] The embodiments of the present invention are described in detail below. These embodiments are only used to explain the present invention and should not be construed as limiting the present invention. Those skilled in the art should understand that the experimental methods, reagents, instruments, etc. used in the following embodiments are all conventional technical means, and unless otherwise stated, they are all conventional products that can be obtained through formal channels.

[0024] The *Bacteroides xylanoplasmophilus* used in the following examples was isolated and cultured by the experimental team from fecal samples of healthy individuals: Fresh fecal samples from healthy volunteers were serially diluted and inoculated into a selective anaerobic medium containing xylan. The samples were cultured at 37°C under strictly anaerobic conditions for 24 hours. Single colonies were picked and purified multiple times. Morphological observation and 16S rRNA gene sequencing confirmed that the strain was *Bacteroides xylanoplasmophilus*. Bacteroides xylanisolvens They were inoculated into anaerobic broth medium for further culture and used in subsequent experiments.

[0025] However, the xylan-bacteroides used in this invention is a known strain, which has been reported in many publications and can also be purchased commercially.

[0026] Example 1: Bacteroides xylanolyticus ( Bacteroides xylanisolvens To investigate the ability of androstenedione to degrade in vitro.

[0027] 1. Co-incubation culture of *Bacteroides xylanoplastin* and androstenedione. Elevated androstenedione levels are an important characteristic of hyperandrogenemia in PCOS patients. This experiment used GAM (5 mL) medium containing a final concentration of 50 μM androstenedione to incubate *Bacteroides xylana*. A control group and a *Bacteroides xylana* culture group were set up. Bx ) and heat-inactivated xylan-degrading Bacteroides culture group (HK- Bx The culture medium collected after 48 hours of co-incubation was used for subsequent testing.

[0028] 2. Detection of the ability of Bacteroides xylanolyticus to degrade androstenedione. Androstenedione in culture medium samples was quantitatively detected using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system. The system consisted of a Waters ultra-high performance liquid chromatography (UPLC) system (Waters Corporation, Milford, USA) coupled with an ABSCIEX 5500 triple quadrupole linear ion trap mass spectrometer (AB SCIEX Corporation, Framingham, Massachusetts, USA). Chromatographic separation was performed using an ACQUITY UPLC CSH C18 column (2.1 mm × 100 mm, 1.7 μm particle size, Waters Corporation), with a column temperature of 40 ℃, a flow rate of 0.25 mL / min, and an injection volume of 3 μL. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid. All analytes were detected using positive ion multiple reaction monitoring (MRM) mode.

[0029] Sample pretreatment: 50 μL of the sample to be tested was placed in a centrifuge tube, and 200 μL of pre-chilled chromatographic grade methanol was added. The tube was vortexed for 20 min and then incubated at -80℃ for 3 h to complete metabolite extraction. All samples were centrifuged at 4 ℃ and 14000 rpm for 20 min. The supernatant was filtered through a 0.22 μm microporous membrane and then analyzed by LC-MS / MS.

[0030] The chromatographic elution was performed using a linear gradient elution program. The gradient process was set as follows: 0–0.5 min, the volume fraction of mobile phase B was maintained at 50%; 0.5–3.5 min, the volume fraction of mobile phase B was linearly increased from 50% to 100%; 3.5–5 min, the volume fraction of mobile phase B was maintained at 100%; 5–6 min, the volume fraction of mobile phase B was linearly decreased from 100% to 50%.

[0031] The operation and control of the liquid chromatography-tandem mass spectrometry system were completed using Analyst 1.6.2 software, and the quantitative analysis of samples was carried out using MultiQuant 3.0.1 software.

[0032] The experimental data were analyzed, and the percentage of androstenedione remaining in the culture medium of different groups after 48 h of co-incubation was calculated. The results are shown in Table 1. It can be seen that Bacteroides xylanide has a significant ability to degrade androstenedione under in vitro conditions.

[0033] Table 1. Degradation capacity of xylan-degrading Bacteroides for androstenedione in in vitro culture systems Example 2: Bacteroides xylanolyticus ( Bacteroides xylanisolvens This study investigated the intervention effects of DHEA on DHEA-induced PCOS model mice (which exhibit hyperandrogenemia and ovulation dysfunction).

[0034] Specific detection indicators include the estrous cycle of mice, the number of cystic follicles and corpora lutea in the ovary, plasma and fecal androgen levels, and plasma luteinizing hormone (LH) levels.

[0035] 1. Materials and Methods 1.1 Experimental Animals: Three-week-old female pre-pubertal SPF-grade C57BL / 6J mice were selected and provided by the Department of Laboratory Animal Science, Peking University School of Medicine (Beijing, China). Mice were randomly divided into groups of three per cage and housed in a standard laboratory animal environment with controlled temperature (22±2℃), suitable humidity (50±5%), and a 12-hour light / 12-hour dark cycle. Mice had free access to standard feed and sterile drinking water. Experiments began after acclimatization.

[0036] 1.2 Experimental Grouping: Mice were randomly divided into 3 groups of 6 mice each: (1) negative control group (Control); (2) DHEA model group (DHEA); (3) Bacteroides xylanase intervention group (DHEA+). Bx ).

[0037] 1.3 PCOS Model Construction: DHEA was dissolved in sesame oil to prepare a suitable concentration, and model groups (DHEA group, DHEA+ group, etc.) were constructed. Bx The group of mice was injected subcutaneously with DHEA at a dose of 6 mg / 100 g body weight daily; the negative control group was injected subcutaneously with an equal volume of sesame oil daily for 21 consecutive days to establish a PCOS mouse model.

[0038] 1.4 Bacterial intervention: From day 1 of model establishment, DHEA+ Bx Mice in the group were administered a bacterial suspension of *Bacteroides xylanopyramidus* via gavage every 3 days, with a bacterial concentration of 2 × 10⁻⁶. 8 CFU / mL, with each gavage volume being 0.2 mL; the negative control group and DHEA group were given the same dose of sterile anaerobic PBS by gavage. The intervention in each group was carried out continuously for 21 days, synchronized with the modeling cycle.

[0039] 1.5 Detection Indicators and Methods: (1) Detection of estrous cycle: Starting from the 10th day of the experiment, vaginal exfoliated cells of mice were collected at the same time every morning. After preparing smears and staining, the cell morphology was observed under a microscope to determine the estrous cycle of mice (divided into metestinal / diestrus: M / D; estrus: E; proestrus: P). The monitoring was carried out for 10 consecutive days, and the percentage of each estrous cycle of mice in each group was counted.

[0040] (2) Sample collection and index detection: After anesthetizing the mice, blood was collected from the orbital cavity, and plasma was separated by centrifugation. The levels of androstenedione (A4) and testosterone (T) in the plasma were measured by LC-MS / MS, and the level of luteinizing hormone (LH) was detected by ELISA. Fresh fecal samples of mice were collected, and the contents of A4 and T in the feces were measured by LC-MS / MS. Bilateral ovarian tissue was taken, fixed, sectioned, stained with H&E, and observed and counted under a microscope. The number of cystic follicles and corpus luteum was counted.

[0041] 1.6 Statistical Analysis: SPSS 27.0 software was used for data analysis. Quantitative data are expressed as mean ± standard error (mean ± SEM). The Shapiro-Wilk normality test was used to determine the sample distribution type. For normally distributed data, one-way ANOVA was used for inter-group comparisons, and subsequent multiple comparisons were performed: Tukey's test was used when variances were homogeneous, and Dunnett's T3 test was used when variances were unequal. For non-normally distributed data, the Kruskal-Wallis test was used. P <0.05 indicates a statistically significant difference.

[0042] 2. Experimental Results The experimental results are shown in Tables 2-6 below.

[0043] Table 2. Effects of Bacteroides xylaniae on the estrous cycle in a DHEA-induced PCOS mouse model Table 3. Effects of Bacteroides xylaniae on the estrous cycle in a DHEA-induced PCOS mouse model Table 4. Effects of Bacteroides xylanica on plasma androgen levels in a DHEA-induced PCOS mouse model Table 5. Effects of Bacteroides xylanate on fecal androgen levels in a DHEA-induced PCOS mouse model Table 6. Effects of Bacteroides xylanate on LH levels in a DHEA-induced PCOS mouse model Note: Compared to the Control group, P <0.05, P <0.01; compared with the DHEA group, # P <0.05,## P <0.01.

[0044] Experimental results showed that, compared with the negative control group, the model group mice exhibited a significant PCOS-like phenotype; compared with the model group, DHEA+ Bx The estrous cycle of mice was restored, polycystic ovarian changes were improved, and the levels of two androgens in feces and plasma were significantly reduced, as was the plasma LH level.

[0045] It should be understood that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of Bacteroides xylanisolvens in the preparation of drugs for the prevention or treatment of polycystic ovary syndrome.

2. The application as described in claim 1, characterized in that, The *Bacteroides xylana* strain can be stably passaged in conventional anaerobic culture media and exhibits both in vitro proliferation activity and in vivo colonization ability.

3. The application as described in claim 1, characterized in that, The culture method of the *Bacteroides xylana* is as follows: Under a strictly anaerobic environment, the *Bacteroides xylana* is inoculated into a closed liquid anaerobic culture medium and cultured statically at 37°C for 24 hours; the liquid anaerobic culture medium is GAM medium, and after the culture medium is autoclaved, heme chloride is added to an additional concentration of 5 μg / mL.

4. The application as described in claim 1, characterized in that, The drug contains Bacteroides xylana as its active ingredient and also includes a pharmaceutically acceptable carrier.

5. The application as described in claim 4, characterized in that, The drug is an oral dosage form.

6. The application as described in claim 5, characterized in that, The oral dosage form is selected from granules, capsules, tablets, powders, oral solutions, suspensions, or emulsions.

7. The application as described in claim 1, characterized in that, The drug is used to prevent or treat hyperandrogenemia, ovulation disorders, or polycystic ovarian changes associated with polycystic ovary syndrome.

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