Construction method and application of polycystic ovary syndrome animal model

Abstract

The application provides a construction method and application of a polycystic ovary syndrome animal model; through a way of intragastric administration, the mouse intestinal tract is transplanted with fusobacterium nucleatum, and through a certain frequency, the fusobacterium nucleatum can be stably planted, and a stable polycystic ovary syndrome symptom is successfully generated; the sex hormone level of the mouse is significantly different from the PCOS characteristic, is shown as the increase of the LH level, the LH / FSH ratio and the T level, and multiple vesicle structures are seen in the ovary, and no mature follicle and corpus luteum are seen, so that the typical symptom of the polycystic ovary syndrome is met, and a new model selection is provided for screening of a treatment drug or experimental research of the polycystic ovary syndrome.

Description

Technical Field

[0001] This invention relates to the field of animal model technology for diseases, specifically to the construction method and application of an animal model of polycystic ovary syndrome. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is the most common reproductive endocrine disorder in women of reproductive age, with a global incidence of 5-18%, and it is showing an increasing trend year by year. Its pathogenesis is not fully understood, but it is currently believed to be caused by the combined effects of genetic susceptibility, epigenetic regulation, and environmental factors.

[0003] In recent years, the role of the gut microbiota in the pathological process of PCOS has received widespread attention, providing new directions for mechanism exploration and treatment development. However, the gut microbiota dysbiosis presents in PCOS patients, and many key mechanisms by which it induces PCOS remain unknown.

[0004] Fusobacterium varium is a Gram-negative anaerobic bacterium that primarily colonizes the human oral cavity and gastrointestinal tract. Its pathogenicity has been confirmed in various diseases, including colorectal cancer, rheumatoid arthritis, and periodontal disease. In the context of gut microbiota dysbiosis, Fusobacterium varium can activate the TLR4 / NF-κB signaling pathway in intestinal epithelial cells through its adhesion factors, triggering a local inflammatory response and disrupting the intestinal barrier function. This leads to the entry of endotoxins such as lipopolysaccharide (LPS) into the circulatory system, inducing systemic chronic low-grade inflammation. This process further promotes insulin resistance and the formation of a hyperandrogenic state, and is one of the key mechanisms of PCOS metabolic disorders. Furthermore, Fusobacterium varium-induced inflammation may also inhibit the synthesis of sex hormone-binding globulin (SHBG) and enhance the activity of free androgens, thereby exacerbating phenotypes such as ovulation disorders, hirsutism, and acne.

[0005] In our previous study, we collected gut microbiota samples from PCOS patients and non-PCOS patients, and performed metagenomic sequencing and metabolomics analysis. We also collected peripheral blood and follicular fluid from both groups for metabolomics and proteomics analysis. The results showed that the abundance of *Fusobacterium varium* was significantly increased in the gut microbiota of PCOS patients.

[0006] A good animal model is crucial for studying the pathogenesis of PCOS. If a polycystic ovary syndrome (PCOS) model is constructed based on Fusobacterium nucleatum, it is expected to provide a new option for studying the pathogenesis of PCOS. Summary of the Invention

[0007] This invention proposes a method for constructing a polycystic ovary syndrome (PCOS) model to meet the need for animal models to study the pathogenesis of PCOS.

[0008] The technical solution of this invention is implemented as follows:

[0009] This invention proposes a method for constructing an animal model of polycystic ovary syndrome (PCOS). The method involves injecting a single suspension of Fusobacterium nucleatum into the intestines of mice via gavage at least once a day for several days until stable colonization is achieved, thereby obtaining an animal model of PCOS. Before receiving the gavage, the endogenous flora in the intestines of the mice is cleared.

[0010] Furthermore, the abundance of the stably established colonies satisfies a relative abundance of 1-5% and an absolute abundance of 1×10⁻⁶. 5 -1×10 7 Copy / g feces.

[0011] Furthermore, the coefficient of variation for the stable colonization is <30%.

[0012] Furthermore, the concentration of the single-cell suspension of *Fusobacterium nucleatum* was 1 × 10⁻⁶. 9 The dose was CFU / mL, and the gavage was administered once daily.

[0013] Furthermore, the gavage lasts for 14 days.

[0014] Furthermore, the elimination of the endogenous flora utilizes an antibiotic cocktail formulation.

[0015] Furthermore, the mice were fasted for 1-5 hours before gavage.

[0016] Furthermore, the mice are inbred strains, preferably C57BL / 6J mice.

[0017] The present invention also provides an animal model of polycystic ovary syndrome, which is constructed by the above-described construction method.

[0018] The present invention also provides applications of the above-described animal models of polycystic ovary syndrome, including screening drugs for the treatment of polycystic ovary syndrome or experimental studies of polycystic ovary syndrome.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention involves transplanting Fusobacterium nucleatum into the intestines of mice via gavage. Stable colonization of Fusobacterium nucleatum is achieved through repeated gavage, successfully generating stable symptoms of polycystic ovary syndrome (PCOS). The mice exhibit significant PCOS characteristics, including elevated LH levels, LH / FSH ratio, and T levels, and multiple follicular structures are observed in the ovaries, without mature follicles or corpora lutea, meeting the typical symptoms of PCOS. This provides a new model for screening therapeutic drugs or conducting experimental research on PCOS. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The results show the comparison of estrous cycles between mice with Fusobacterium nucleatum transplantation model in Example 2 and the control group.

[0023] Figure 2 The results show the detection of sex hormone levels in mice after Fusobacterium nucleatum transplantation modeling in Example 2.

[0024] Figure 3 The results are pathological examinations of mouse ovarian tissue after Fusobacterium nucleatum transplantation modeling in Example 2. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] In one embodiment, a method for constructing an animal model of polycystic ovary syndrome is proposed, wherein a single suspension of Fusobacterium nucleatum is transplanted into the intestine of mice by gavage at least once a day for several days until stable colonization is achieved to obtain an animal model of polycystic ovary syndrome; the endogenous flora of the mice is cleared before gavage.

[0027] In a preferred embodiment, the abundance of the stable colonization satisfies a relative abundance of 1-5% and an absolute abundance of 1×10⁻⁶. 5 -1×10 7 Copy / g feces; the coefficient of variation for stable colonization is <30%.

[0028] In a preferred embodiment, the concentration of the single-strain of *Fusobacterium nucleatum* is 1 × 10⁻⁶. 9 The concentration of CFU / mL was determined, and the gavage was administered once daily for 14 days.

[0029] In the above embodiments, Fusobacterium nucleatum was transplanted into the intestines of mice by gavage. Stable colonization of Fusobacterium nucleatum was achieved through a certain frequency, and stable polycystic ovary syndrome symptoms were successfully generated. The mice showed significant PCOS characteristics with bleeding in sex hormone levels, manifested as elevated LH levels, LH / FSH ratio and T levels, and multiple follicular structures were observed in the ovaries, but no mature follicles or corpus luteum were observed.

[0030] In a preferred embodiment, the *Fusobacterium nucleatum* has ≥99% homology with the 16S rRNA sequence of *Fusobacterium nucleatum* in the NCBI GenBank database; common commercially available strains, such as the *Fusobacterium nucleatum* standard strain ATCC 25586, can be selected.

[0031] In a preferred embodiment, the endogenous flora is eliminated using an antibiotic cocktail formulation: ampicillin 1 g / L, vancomycin 1 g / L, neomycin 1 g / L, and metronidazole 1 g / L, to eliminate the endogenous flora in SPF-grade mice.

[0032] In a preferred embodiment, to improve the colonization efficiency of the strain, an electrolyte solution was used as the sole water source the day before transplantation, allowing free drinking to physically flush the intestinal cavity and reduce subsequent immune rejection. Mice were fasted for 1-5 hours before gavage.

[0033] Example 1: Preparation of single-strain suspension of Fusobacterium nucleatum

[0034] 1) Culture medium preparation

[0035] The standard strain of *Fusobacterium nucleatum*, ATCC 25586, was selected. ATCC medium 1490 (modified minced meat broth medium) was used. After boiling to remove oxygen and cooling, 0.5 g / L cysteine ​​hydrochloride (S1505), 1 mg / L vitamin K1 (S5606), and 5 mg / L heme chloride (S5605) were added. The mixture was thoroughly mixed and dispensed into suitable sealed containers (with 1-2 cm of *S5604* minced beef granules placed in the sealed containers beforehand). The containers were then autoclaved at 121°C for 15 minutes and cooled for later use. This medium meets the growth requirements of *Fusobacterium nucleatum* (an anaerobic bacterium), and the added cysteine ​​hydrochloride, vitamin K1, and heme chloride promote the proliferation and maintain the activity of the strain.

[0036] 2) Recovery and amplification culture of the strain

[0037] Resuscitation: The frozen standard strain of Fusobacterium nucleatum was taken out of the -80℃ freezer and quickly thawed in a 37℃ water bath (1-2 min). Under aseptic conditions, 0.5 mL of bacterial culture was inoculated into 5 mL of freshly prepared modified minced meat broth medium and placed in an anaerobic incubator (anaerobic environment: 80% N2, 10% H2, 10% CO2) and incubated at 37℃ for 24 h to complete the strain resuscitation.

[0038] Amplification: Take 2 mL of the revived bacterial culture and inoculate it into 100 mL of modified minced meat broth medium for expansion culture. Incubate at 37°C for 48 hours under the same anaerobic conditions. Observe the bacterial culture daily during this period. When the bacterial culture shows uniform turbidity and OD... 600 When the value reaches 0.6-0.8 (in the logarithmic growth phase, when the strain activity is strongest), stop the culture and use it for subsequent preparation of single-strain suspensions.

[0039] 3) Strain identification: Morphological identification and 16S rRNA sequencing were combined to ensure that the cultured strain was the target Fusobacterium nucleatum.

[0040] Morphological identification: Take a smear of the cultured bacterial solution, stain it with Gram, and observe it under an optical microscope. Fusobacterium nucleatum is a Gram-negative bacterium, which is spindle-shaped or rod-shaped, without spores, and is arranged irregularly, which is consistent with the typical morphological characteristics.

[0041] 16S rRNA sequencing identification: Genomic DNA of the strain was extracted and PCR amplification was performed using universal primers for bacterial 16S rRNA (upstream primer: 27F 5'-AGAGTTTGATCCTGGCTCAG-3', downstream primer: 1492R 5'-GGTTACCTTGTTACGACTT-3'). After sequencing, the amplified products were compared with the 16S rRNA sequences of Fusobacterium nucleatum in the NCBI GenBank database for homology. The homology was ≥99%, confirming the identity of the strain.

[0042] 4) Preparation of single-strain suspension of Fusobacterium nucleatum

[0043] Amplification culture to logarithmic growth phase (OD) 600 Transfer the culture of Fusobacterium nucleatum with a pH of 0.6-0.8 to a sterile centrifuge tube, centrifuge at 4°C and 5000 r / min for 10 min, discard the supernatant and collect the bacterial precipitate.

[0044] Washing and purification: Add sterile, pre-reduced PBS (containing 0.05% cysteine ​​hydrochloride) to the bacterial pellet for resuspending, centrifuge again, gently pipette to resuspend, and centrifuge again at 4℃ and 5000r / min for 10min. Discard the supernatant. Repeat washing twice to remove culture medium residues and metabolites to obtain purified nucleated Fusobacterium pellet.

[0045] Suspension preparation: Sterile physiological saline was added to the purified bacterial precipitate, and the mixture was agitated to prepare a single-bacterial suspension. The suspension was then analyzed using a hemocytometer and CFU spot test to adjust the concentration of Fusobacterium nucleatum in the suspension to 1×10⁻⁶. 9 The concentration of CFU / mL was kept on ice for later use. Transplantation was to be completed within 2 hours after preparation to avoid a decrease in bacterial activity.

[0046] Negative control suspension preparation: Take an equal volume of sterile saline and prepare a negative control suspension according to the same procedure as above (centrifugation, washing, no bacterial precipitation, only saline treatment), which was used for transplantation in the negative control group mice.

[0047] Example 2: Construction of a PCOS Model by Transplantation of Bacterial Suspension into Mice

[0048] 1. Preparation stage

[0049] 1) Breed, sex, age, and rearing environment

[0050] Three-week-old female C57BL / 6J mice were housed in an SPF barrier system with free access to food and water, a 12-hour light-dark cycle (lights on at 07:00), a temperature of 22.0±2 ℃, and a humidity of 40–70%. Experiments began after one week of acclimatization.

[0051] 2) Antibiotics eliminate native flora

[0052] The endogenous flora of SPF mice was eliminated by an antibiotic cocktail formulation (ampicillin 1g / L, vancomycin 1g / L, neomycin 1g / L and metronidazole 1g / L) administered via drinking water for 7 days.

[0053] 3) Antibiotic cleaning period

[0054] The day before the transplant, compound polyethylene glycol electrolyte solution (PEG-ELS) was used as the sole water source, which was allowed to be drunk freely for 24 hours to physically flush the intestinal cavity and reduce subsequent immune rejection.

[0055] 2. Pre-transplant preparation

[0056] All mice were fasted for 4 hours before and after transplantation to reduce dilution of the transplanted bacterial culture by gastric contents and improve colonization efficiency. They were allowed free access to compound polyethylene glycol electrolyte solution (PEG-ELS). Sterile gavage syringes (1 mL, 0.8 mm needle diameter), sterile syringes, and other equipment were prepared and autoclaved before use. Mice were housed individually for 4 hours after each transplantation to prevent cross-contamination from licking.

[0057] 3. Fecal microbiota transplantation via gavage

[0058] Using a sterile syringe, draw up a prepared single-strain of *Fusobacterium nucleatum* (model group) or sterile saline (negative control group), connect it to a gavage needle, and slowly insert the needle into the mouse's mouth from one side, advancing it slowly along the esophagus into the stomach (insertion depth approximately 2-3 cm, avoiding insertion into the trachea). After gavage, slowly remove the gavage needle, return the mouse to its cage, resume normal feeding, and observe for 1 hour to ensure the mouse shows no abnormal reactions such as vomiting or respiratory distress.

[0059] The transplantation frequency was once daily for 14 consecutive days, establishing a stable colonization environment for Fusobacterium nucleatum in the mouse intestine, laying the foundation for the induction of PCOS pathological features.

[0060] Microbial colonization detection (microbial count at three consecutive sampling points (D5, D10, D14) must meet the following conditions)

[0061] Abundance of *Fusobacterium nucleatum* stably colonizing the intestine of C57BL / 6J mice: relative abundance 1%-5%, absolute abundance 1×10⁻⁶. 5 -1×10 7 A population with a coefficient of variation of <30% and a per-g of feces can be considered stably established.

[0062] 4. PCOS phenotypic monitoring

[0063] 1) Monitoring of the estrous cycle

[0064] Starting from day 7 after bacterial culture transfer, vaginal exfoliated cell smears were performed daily from 08:00 to 10:00 for 15 consecutive days; ovulation disorders were defined as ≥60% of the days spent in the interestrus or continuous estrus period.

[0065] 2) Metabolic and reproductive phenotypic endpoints

[0066] The following tests were performed on day 15 after the last transplant (i.e., day 21 of the experiment):

[0067] a) Fasting plasma glucose (FPG) and fasting insulin (FINS), calculate HOMA-IR;

[0068] b) Serum total testosterone (TT), free testosterone (FT), LH, FSH, and calculate LH / FSH;

[0069] c) Ovarian histology: Fixed with 4% paraformaldehyde, serially sectioned in paraffin at 5μm, stained with HE, and counted the number of antral follicles, cystic follicles and corpus luteum;

[0070] d) Intestinal permeability: Blood was collected from the orbital vein 4 hours after FITC-glucan (600 mg / kg) was administered by gavage, and the serum FITC concentration was measured using a fluorescence microplate reader;

[0071] e) 16S rDNA sequencing: Cecal contents were collected to verify the relative abundance of Fn (1%–5%) and absolute abundance (1×10⁻⁶). 5 –1×10 7 The number of copies / g of feces and the coefficient of variation <30% indicate a decrease in α-diversity.

[0072] 5. Results

[0073] 1) After the modeling process was completed, the estrous cycle of mice in each group was further monitored, and the results were as follows: Figure 1 As shown in the figure. The results showed that the estrous cycle disorder (absence of cyclical estrous cycles) was observed in mice in the model group constructed by transplantation of Fusobacterium nucleatum (PCOS), while the estrous cycle was normal in the control mice.

[0074] 2) After the mouse model was established, venous blood was collected for sex hormone level measurement. The results are as follows: Figure 2 As shown in the figure. The results showed that the mice in the model group constructed with Fusobacterium nucleatum transplantation exhibited significant changes in PCOS characteristic hormone levels: LH level, LH / FSH ratio, and total T level were all significantly increased.

[0075] 6. Pathological changes in mouse ovarian tissue after modeling

[0076] After successful modeling, ovarian tissue sections were taken from both the model group and the control group. HE staining results are as follows: Figure 3 As shown in the figure. The results showed that multiple vesicle structures were observed in the ovaries of mice in the model group constructed by transplanting Fusobacterium nucleatum, but no mature follicles or corpora lutea were observed.

[0077] The above results indicate that Fusobacterium nucleatum transplantation can successfully construct a PCOS mouse model.

[0078] 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, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of polycystic ovary syndrome, characterized in that, A polycystic ovary syndrome (PCOS) animal model was obtained by gavage administration of a single suspension of Fusobacterium nucleatum into the intestines of mice at least once a day for several days until stable colonization was achieved; the mice underwent endogenous gut microbiota elimination before receiving gavage.

2. The construction method according to claim 1, characterized in that, The abundance of the stably established plants meets the requirements of a relative abundance of 1-5% and an absolute abundance of 1×10⁻⁶. 5 -1×10 7 Copy / g feces.

3. The construction method according to claim 1 or 3, characterized in that, The coefficient of variation for stable colonization is <30%.

4. The construction method according to claim 1, characterized in that, The concentration of a single suspension of *Fusobacterium nucleatum* was 1 × 10⁻⁶. 9 The dose was CFU / mL, and the gavage was administered once daily.

5. The construction method according to claim 1 or 4, characterized in that, The gavage was administered for 14 days.

6. The construction method according to claim 1, characterized in that, The elimination of the endogenous flora was achieved using an antibiotic cocktail formulation.

7. The construction method according to claim 1, characterized in that, The mice were fasted for 1-5 hours before gavage.

8. The construction method according to claim 1, characterized in that, The mice were an inbred strain.

9. An animal model of polycystic ovary syndrome, characterized in that, It is constructed by the construction method described in claim 1.

10. The application of the polycystic ovary syndrome animal model according to claim 9, characterized in that, The applications include screening drugs for the treatment of polycystic ovary syndrome (PCOS) or experimental studies of PCOS.

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